Firefighter willingness to participate in a stem cell clinical trial for burns: A mixed methods study.

PubMed

Horch, Jenny D; Carr, Eloise C J; Harasym, Patricia; Burnett, Lindsay; Biernaskie, Jeff; Gabriel, Vincent

2016-12-01

Adult stem cells represent a potentially renewable and autologous source of cells to regenerate skin and improve wound healing. Firefighters are at risk of sustaining a burn and potentially benefiting from a split thickness skin graft (STSG). This mixed methods study examined firefighter willingness to participate in a future stem cell clinical trial, outcome priorities and factors associated with this decision. A sequential explanatory mixed methods design was used. The quantitative phase (online questionnaire) was followed by the qualitative phase (semi-structured interviews). A sample of 149 firefighters completed the online survey, and a purposeful sample of 15 firefighters was interviewed. A majority (74%) reported they would participate in a future stem cell clinical trial if they experienced burn benefiting from STSG. Hypothetical concerns related to receiving a STSG were pain, itch, scarring/redness and skin durability. Participants indicated willingness to undergo stem cell therapy if the risk of no improvement was 43% or less. Risk tolerance was predicted by perceived social support and having children. Interviews revealed four main themes: a desire to help others, improving clinical outcomes, trusting relationships, and a belief in scientific investigation. Many participants admitted lacking sufficient knowledge to make an informed decision regarding stem cell therapies. Firefighters indicated they were largely willing to participate in a stem cell clinical trial but also indicated a lack of knowledge upon which to make a decision. Public education of the role of stem cells in STSG will be increasingly important as clinical trials are developed. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

Histone h1 depletion impairs embryonic stem cell differentiation.

PubMed

Zhang, Yunzhe; Cooke, Marissa; Panjwani, Shiraj; Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T; Pan, Chenyi; McDevitt, Todd C; Fan, Yuhong

2012-01-01

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

Advances and Prospects in Stem Cells for Cartilage Regeneration

PubMed Central

Wang, Mingjie; Yuan, Zhiguo; Ma, Ning; Hao, Chunxiang; Guo, Weimin; Zou, Gengyi; Zhang, Yu; Chen, Mingxue; Gao, Shuang; Wang, Aiyuan; Wang, Yu; Sui, Xiang; Xu, Wenjing; Lu, Shibi

2017-01-01

The histological features of cartilage call attention to the fact that cartilage has a little capacity to repair itself owing to the lack of a blood supply, nerves, or lymphangion. Stem cells have emerged as a promising option in the field of cartilage tissue engineering and regenerative medicine and could lead to cartilage repair. Much research has examined cartilage regeneration utilizing stem cells. However, both the potential and the limitations of this procedure remain controversial. This review presents a summary of emerging trends with regard to using stem cells in cartilage tissue engineering and regenerative medicine. In particular, it focuses on the characterization of cartilage stem cells, the chondrogenic differentiation of stem cells, and the various strategies and approaches involving stem cells that have been used in cartilage repair and clinical studies. Based on the research into chondrocyte and stem cell technologies, this review discusses the damage and repair of cartilage and the clinical application of stem cells, with a view to increasing our systematic understanding of the application of stem cells in cartilage regeneration; additionally, several advanced strategies for cartilage repair are discussed. PMID:28246531

Epithelial-mesenchymal transition transcription factors control pluripotent adult stem cell migration in vivo in planarians

PubMed Central

Abnave, Prasad; Aboukhatwa, Ellen; Kosaka, Nobuyoshi; Thompson, James; Hill, Mark A.

2017-01-01

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1, snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum. Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo. PMID:28893948

Stem Cell-Based Therapies for Polyglutamine Diseases.

PubMed

Mendonça, Liliana S; Onofre, Isabel; Miranda, Catarina Oliveira; Perfeito, Rita; Nóbrega, Clévio; de Almeida, Luís Pereira

2018-01-01

Polyglutamine (polyQ) diseases are a family of neurodegenerative disorders with very heterogeneous clinical presentations, although with common features such as progressive neuronal death. Thus, at the time of diagnosis patients might present an extensive and irreversible neuronal death demanding cell replacement or support provided by cell-based therapies. For this purpose stem cells, which include diverse populations ranging from embryonic stem cells (ESCs), to fetal stem cells, mesenchymal stromal cells (MSCs) or induced pluripotent stem cells (iPSCs) have remarkable potential to promote extensive brain regeneration and recovery in neurodegenerative disorders. This regenerative potential has been demonstrated in exciting pre and clinical assays. However, despite these promising results, several drawbacks are hampering their successful clinical implementation. Problems related to ethical issues, quality control of the cells used and the lack of reliable models for the efficacy assessment of human stem cells. In this chapter the main advantages and disadvantages of the available sources of stem cells as well as their efficacy and potential to improve disease outcomes are discussed.

Training stem cells for treatment of malignant brain tumors

PubMed Central

Li, Shengwen Calvin; Kabeer, Mustafa H; Vu, Long T; Keschrumrus, Vic; Yin, Hong Zhen; Dethlefs, Brent A; Zhong, Jiang F; Weiss, John H; Loudon, William G

2014-01-01

The treatment of malignant brain tumors remains a challenge. Stem cell technology has been applied in the treatment of brain tumors largely because of the ability of some stem cells to infiltrate into regions within the brain where tumor cells migrate as shown in preclinical studies. However, not all of these efforts can translate in the effective treatment that improves the quality of life for patients. Here, we perform a literature review to identify the problems in the field. Given the lack of efficacy of most stem cell-based agents used in the treatment of malignant brain tumors, we found that stem cell distribution (i.e., only a fraction of stem cells applied capable of targeting tumors) are among the limiting factors. We provide guidelines for potential improvements in stem cell distribution. Specifically, we use an engineered tissue graft platform that replicates the in vivo microenvironment, and provide our data to validate that this culture platform is viable for producing stem cells that have better stem cell distribution than with the Petri dish culture system. PMID:25258664

Are stem cells drugs? The regulation of stem cell research and development.

PubMed

Rosen, Michael R

2006-10-31

Stem cell research and its clinical application have become political, social, and medical lightning rods, polarizing opinion among members of the lay community and among medical/scientific professionals. A potpourri of opinion, near-anecdotal observation, and scientifically sound data has sown confusion in ways rarely seen in the medical arts and sciences. A major issue is regulation, with different aspects of stem cell research falling within the purview of different government agencies and local offices. An overarching clearinghouse to review the field and recommend policy is lacking. In the following pages, I touch on the societal framework for regulation, the known and potential risks and benefits of cardiovascular stem cell therapies, whether stem cells should be regulated as drugs or in analogy to drugs, and if there is to be regulation, then by whom. In so doing, I refer to the stem cell literature only as it relates to the discussion of regulation because this is not a review of stem cell research; it is an opinion regarding regulation.

Which bank? A guardian model for regulation of embryonic stem cell research in Australia.

PubMed

McLennan, A

2007-08-01

In late 2005 the Legislation Review: Prohibition of Human Cloning Act 2002 (Cth) and the Research Involving Human Embryos Act 2002 (Cth) recommended the establishment of an Australian stem cell bank. This article aims to address a lack of discussion of issues surrounding stem cell banking by suggesting possible answers to the questions of whether Australia should establish a stem cell bank and what its underlying philosophy and functions should be. Answers are developed through an analysis of regulatory, scientific and intellectual property issues relating to embryonic stem cell research in the United Kingdom, United States and Australia. This includes a detailed analysis of the United Kingdom Stem Cell Bank. It is argued that a "guardian" model stem cell bank should be established in Australia. This bank would aim to promote the maximum public benefit from human embryonic stem cell research by providing careful regulatory oversight and addressing ethical issues, while also facilitating research by addressing practical scientific concerns and intellectual property issues.

Oscillatory Protein Expression Dynamics Endows Stem Cells with Robust Differentiation Potential

PubMed Central

Kaneko, Kunihiko

2011-01-01

The lack of understanding of stem cell differentiation and proliferation is a fundamental problem in developmental biology. Although gene regulatory networks (GRNs) for stem cell differentiation have been partially identified, the nature of differentiation dynamics and their regulation leading to robust development remain unclear. Herein, using a dynamical system modeling cell approach, we performed simulations of the developmental process using all possible GRNs with a few genes, and screened GRNs that could generate cell type diversity through cell-cell interactions. We found that model stem cells that both proliferated and differentiated always exhibited oscillatory expression dynamics, and the differentiation frequency of such stem cells was regulated, resulting in a robust number distribution. Moreover, we uncovered the common regulatory motifs for stem cell differentiation, in which a combination of regulatory motifs that generated oscillatory expression dynamics and stabilized distinct cellular states played an essential role. These findings may explain the recently observed heterogeneity and dynamic equilibrium in cellular states of stem cells, and can be used to predict regulatory networks responsible for differentiation in stem cell systems. PMID:22073296

Indispensable role of Notch ligand-dependent signaling in the proliferation and stem cell niche maintenance of APC-deficient intestinal tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakata, Toru; Shimizu, Hiromichi; Department of Medicine, University of California, San Francisco, San Francisco, CA

Ligand-dependent activation of Notch signaling is required to maintain the stem-cell niche of normal intestinal epithelium. However, the precise role of Notch signaling in the maintenance of the intestinal tumor stem cell niche and the importance of the RBPJ-independent non-canonical pathway in intestinal tumors remains unknown. Here we show that Notch signaling was activated in LGR5{sup +ve} cells of APC-deficient mice intestinal tumors. Accordingly, Notch ligands, including Jag1, Dll1, and Dll4, were expressed in these tumors. In vitro studies using tumor-derived organoids confirmed the intrinsic Notch activity-dependent growth of tumor cells. Surprisingly, the targeted deletion of Jag1 but not RBPJ inmore » LGR5{sup +ve} tumor-initiating cells resulted in the silencing of Hes1 expression, disruption of the tumor stem cell niche, and dramatic reduction in the proliferation activity of APC-deficient intestinal tumors in vivo. Thus, our results highlight the importance of ligand-dependent non-canonical Notch signaling in the proliferation and maintenance of the tumor stem cell niche in APC-deficient intestinal adenomas. - Highlights: • Notch signaling is activated in LGR5{sup +ve} cells of APC-deficient intestinal tumors. • Lack of Jag1 but not RBPJ disrupts stem cell niche formation in those tumors. • Lack of Jag1 reduces the proliferation activity of APC-deficient intestinal tumors.« less

Cancer stem cells, cancer cell plasticity and radiation therapy.

PubMed

Vlashi, Erina; Pajonk, Frank

2015-04-01

Since the first prospective identification of cancer stem cells in solid cancers the cancer stem cell hypothesis has reemerged as a research topic of increasing interest. It postulates that solid cancers are organized hierarchically with a small number of cancer stem cells driving tumor growth, repopulation after injury and metastasis. They give rise to differentiated progeny, which lack these features. The model predicts that for any therapy to provide cure, all cancer stem cells have to be eliminated while the survival of differentiated progeny is less critical. In this review we discuss recent reports challenging the idea of a unidirectional differentiation of cancer cells. These reports provide evidence supporting the idea that non-stem cancer cells exhibit a remarkable degree of plasticity that allows them to re-acquire cancer stem cell traits, especially in the context of radiation therapy. We summarize conditions under which differentiation is reversed and discuss the current knowledge of the underlying mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

The regulatory sciences for stem cell-based medicinal products.

PubMed

Yuan, Bao-Zhu; Wang, Junzhi

2014-06-01

Over the past few years, several new achievements have been made from stem cell studies, many of which have moved up from preclinical stages to early, or from early to middle or late, stages thanks to relatively safe profile and preliminary evidence of effectiveness. Moreover, some stem cell-based products have been approved for marketing by different national regulatory authorities. However, many critical issues associated mainly with incomplete understanding of stem cell biology and the relevant risk factors, and lack of effective regulations still exist and need to be urgently addressed, especially in countries where establishment of appropriate regulatory system just commenced. More relevantly, the stem cell regulatory sciences need to be established or improved to more effectively evaluate quality, safety and efficacy of stem cell products, and for building up the appropriate regulatory framework. In this review, we summarize some new achievements in stem cell studies, especially the preclinical and clinical studies, the existing regulations, and the associated challenges, and we then propose some considerations for improving stem cell regulatory sciences with a goal of promoting the steadfast growth of the well-regulated stem cell therapies abreast of evolvement of stem cell sciences and technologies.

Epithelial-mesenchymal transition transcription factors control pluripotent adult stem cell migration in vivo in planarians.

PubMed

Abnave, Prasad; Aboukhatwa, Ellen; Kosaka, Nobuyoshi; Thompson, James; Hill, Mark A; Aboobaker, A Aziz

2017-10-01

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1 , snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo . © 2017. Published by The Company of Biologists Ltd.

Advances in translational inner ear stem cell research.

PubMed

Warnecke, Athanasia; Mellott, Adam J; Römer, Ariane; Lenarz, Thomas; Staecker, Hinrich

2017-09-01

Stem cell research is expanding our understanding of developmental biology as well as promising the development of new therapies for a range of different diseases. Within hearing research, the use of stem cells has focused mainly on cell replacement. Stem cells however have a broad range of other potential applications that are just beginning to be explored in the ear. Mesenchymal stem cells are an adult derived stem cell population that have been shown to produce growth factors, modulate the immune system and can differentiate into a wide variety of tissue types. Potential advantages of mesenchymal/adult stem cells are that they have no ethical constraints on their use. However, appropriate regulatory oversight seems necessary in order to protect patients from side effects. Disadvantages may be the lack of efficacy in many preclinical studies. But if proven safe and efficacious, they are easily translatable to clinical trials. The current review will focus on the potential application on mesenchymal stem cells for the treatment of inner ear disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

PubMed

Sakib, Sadman; Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2016-01-01

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-01-01

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation. PMID:20133835

MicroRNA let-7b regulates neural stem cell proliferation and differentiation by targeting nuclear receptor TLX signaling.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Lang, Ming-Fei; Yang, Su; Li, Wendong; Shi, Yanhong

2010-02-02

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation.

CD133(+)/CD44(+)/Oct4(+)/Nestin(+) stem-like cells isolated from Panc-1 cell line may contribute to multi-resistance and metastasis of pancreatic cancer.

PubMed

Wang, Dongqing; Zhu, Haitao; Zhu, Ying; Liu, Yanfang; Shen, Huiling; Yin, Ruigen; Zhang, Zhijian; Su, Zhaoliang

2013-05-01

Pancreatic cancer is an aggressive malignant disease. Owing to the lack of early symptoms, accompanied by extensive metastasis and high resistance to chemotherapy, pancreatic adenocarcinoma becomes the fourth leading cause of cancer-related deaths. In this study, we identified a subpopulation of cells isolated from the Panc-1 cell line and named pancreatic cancer stem-like cells. These Panc-1 stem-like cells expressed high levels of CD133/CD44/Oct4/Nestin. Compared to Panc-1 cells, Panc-1 stem-like cells were resistant to gemcitabine and expressed high levels of MDR1; furthermore, Panc-1 stem-like cells have high anti-apoptotic, but weak proliferative potential. These results indicated that Panc-1 stem-like cells, as a novel group, may be a potential major cause of pancreatic cancer multidrug resistance and extensive metastasis. Copyright © 2012 Elsevier GmbH. All rights reserved.

A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Li, Shengxiu; Shi, Yanhong

2009-01-01

Summary MicroRNAs are important players in stem cell biology. Among them, microRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain. Whether miR-9 plays a role in neural stem cell self-renewal and differentiation is unknown. We showed previously that nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector lacking the miR-9 recognition site rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses miR-9 pri-miRNA expression. MiR-9, by forming a negative regulatory loop with TLX, establishes a model for controlling the balance between neural stem cell proliferation and differentiation. PMID:19330006

[Characterization of stem cells derived from the neonatal auditory sensory epithelium].

PubMed

Diensthuber, M; Heller, S

2010-11-01

In contrast to regenerating hair cell-bearing organs of nonmammalian vertebrates the adult mammalian organ of Corti appears to have lost its ability to maintain stem cells. The result is a lack of regenerative ability and irreversible hearing loss following auditory hair cell death. Unexpectedly, the neonatal auditory sensory epithelium has recently been shown to harbor cells with stem cell features. The origin of these cells within the cochlea's sensory epithelium is unknown. We applied a modified neurosphere assay to identify stem cells within distinct subregions of the neonatal mouse auditory sensory epithelium. Sphere cells were characterized by multiple markers and morphologic techniques. Our data reveal that both the greater and the lesser epithelial ridge contribute to the sphere-forming stem cell population derived from the auditory sensory epithelium. These self-renewing sphere cells express a variety of markers for neural and otic progenitor cells and mature inner ear cell types. Stem cells can be isolated from specific regions of the auditory sensory epithelium. The distinct features of these cells imply a potential application in the development of a cell replacement therapy to regenerate the damaged sensory epithelium.

Fraudsters operate and officialdom turns a blind eye: a proposal for controlling stem cell therapy in China.

PubMed

Jiang, Li; Dong, Bing He

2016-09-01

Stem cell tourism-the flow of patients from home countries to destination countries to obtain stem cell treatment-is a growing business in China. Many concerns have been raised regarding fraudsters that operate unsafe stem cell therapies and an officialdom that turns a blind eye to the questionable technology. The Chinese regulatory approach to stem cell research is based on Guidelines and Administrative Measures, rather than legislation, and may have no binding force on certain institutions, such as military hospitals. There is no liability and traceability system and no visible set of penalties for non-compliance in the stem cell legal framework. In addition to the lack of safety and efficacy systems in the regulations, no specific expert authority has been established to monitor stem cell therapy to date. Recognizing the global nature of stem cell tourism, this article argues that resolving stem cell tourism issues may require not only the Chinese government but also an international mechanism for transparency and ethical oversight. A stringent set of international regulations that govern stem cell therapies can encourage China to improve stem cell regulation and enforcement to fulfill its obligations. Through an international consensus, a minimum standard for clinical stem cell research and a central enforcement system will be provided. As a result, rogue clinics that conduct unauthorized stem cell therapies can be penalized, and countries that are reluctant to implement the reconciled regulations should be sanctioned.

The gap between law and ethics in human embryonic stem cell research: overcoming the effect of U.S. federal policy on research advances and public benefit.

PubMed

Taylor, Patrick L

2005-10-01

Key ethical issues arise in association with the conduct of stem cell research by research institutions in the United States. These ethical issues, summarized in detail, receive no adequate translation into federal laws or regulations, also described in this article. U.S. Federal policy takes a passive approach to these ethical issues, translating them simply into limitations on taxpayer funding, and foregoes scientific and ethical leadership while protecting intellectual property interests through a laissez faire approach to stem cell patents and licenses. Those patents and licenses, far from being scientifically and ethically neutral in effect, virtually prohibit commercially sponsored research that could otherwise be a realistic alternative to the federal funding gap. The lack of federal funding and related data-sharing principles, combined with the effect of U.S. patent policy, the lack of key agency guidance, and the proliferation of divergent state laws arising from the lack of Federal leadership, significantly impede ethical stem cell research in the United States, without coherently supporting any consensus ethical vision. Research institutions must themselves implement steps, described in the article, to integrate addressing ethical review with the many legal compliance issues U.S. federal and state laws create.

Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

PubMed

Shah, Prajay T; Stratton, Jo A; Stykel, Morgan Gail; Abbasi, Sepideh; Sharma, Sandeep; Mayr, Kyle A; Koblinger, Kathrin; Whelan, Patrick J; Biernaskie, Jeff

2018-05-03

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche. Copyright © 2018 Elsevier Inc. All rights reserved.

76 FR 70520 - RMD Technologies, Inc., Rockwall Holdings, Inc., Southmark Corp., Stargold Mines, Inc., Stelax...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-14

... SECURITIES AND EXCHANGE COMMISSION [File No. 500-1] RMD Technologies, Inc., Rockwall Holdings, Inc., Southmark Corp., Stargold Mines, Inc., Stelax Industries, Ltd., Stem Cell Innovations, Inc., and Surfect... there is a lack of current and accurate information concerning the securities of Stem Cell Innovations...

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

PubMed Central

Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

2010-01-01

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

PubMed

Lei, Lei; Spradling, Allan C

2013-05-21

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

PubMed

Jackson, Hartland W; Waterhouse, Paul; Sinha, Ankit; Kislinger, Thomas; Berman, Hal K; Khokha, Rama

2015-03-01

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.

Personalized nanomedicine advancements for stem cell tracking☆

PubMed Central

Janowski, Mirek; Bulte, Jeff W.M.; Walczak, Piotr

2012-01-01

Recent technological developments in biomedicine have facilitated the generation of data on the anatomical, physiological and molecular level for individual patients and thus introduces opportunity for therapy to be personalized in an unprecedented fashion. Generation of patient-specific stem cells exemplifies the efforts toward this new approach. Cell-based therapy is a highly promising treatment paradigm; however, due to the lack of consistent and unbiased data about the fate of stem cells in vivo, interpretation of therapeutic remains challenging hampering the progress in this field. The advent of nanotechnology with a wide palette of inorganic and organic nanostructures has expanded the arsenal of methods for tracking transplanted stem cells. The diversity of nanomaterials has revolutionized personalized nanomedicine and enables individualized tailoring of stem cell labeling materials for the specific needs of each patient. The successful implementation of stem cell tracking will likely be a significant driving force that will contribute to the further development of nanotheranostics. The purpose of this review is to emphasize the role of cell tracking using currently available nanoparticles. PMID:22820528

The Potential of Stem Cells in Treatment of Traumatic Brain Injury.

PubMed

Weston, Nicole M; Sun, Dong

2018-01-25

Traumatic brain injury (TBI) is a global public health concern, with limited treatment options available. Despite improving survival rate after TBI, treatment is lacking for brain functional recovery and structural repair in clinic. Recent studies have suggested that the mature brain harbors neural stem cells which have regenerative capacity following brain insults. Much progress has been made in preclinical TBI model studies in understanding the behaviors, functions, and regulatory mechanisms of neural stem cells in the injured brain. Different strategies targeting these cell population have been assessed in TBI models. In parallel, cell transplantation strategy using a wide range of stem cells has been explored for TBI treatment in pre-clinical studies and some in clinical trials. This review summarized strategies which have been explored to enhance endogenous neural stem cell-mediated regeneration and recent development in cell transplantation studies for post-TBI brain repair. Thus far, neural regeneration through neural stem cells either by modulating endogenous neural stem cells or by stem cell transplantation has attracted much attention. It is highly speculated that targeting neural stem cells could be a potential strategy to repair and regenerate the injured brain. Neuroprotection and neuroregeneration are major aspects for TBI therapeutic development. With technique advancement, it is hoped that stem cell-based therapy targeting neuroregeneration will be able to translate to clinic in not so far future.

A natural stem cell therapy? How novel findings and biotechnology clarify the ethics of stem cell research

PubMed Central

Patel, P

2006-01-01

The natural replacement of damaged cells by stem cells occurs actively and often in adult tissues, especially rapidly dividing cells such as blood cells. An exciting case in Boston, however, posits a kind of natural stem cell therapy provided to a mother by her fetus—long after the fetus is born. Because there is a profound lack of medical intervention, this therapy seems natural enough and is unlikely to be morally suspect. Nevertheless, we feel morally uncertain when we consider giving this type of therapy to patients who would not naturally receive it. Much has been written about the ethics of stem cell research and therapy; this paper will focus on how recent advances in biotechnology and biological understandings of development narrow the debate. Here, the author briefly reviews current stem cell research practices, revisits the natural stem cell therapy case for moral evaluation, and ultimately demonstrates the importance of permissible stem cell research and therapy, even absent an agreement about the definition of when embryonic life begins. Although one promising technology, blighted ovum utilisation, uses fertilised but developmentally bankrupt eggs, it is argued that utilisation of unfertilised eggs to derive totipotent stem cells obviates the moral debate over when life begins. There are two existing technologies that fulfil this criterion: somatic cell nuclear transfer and parthenogenic stem cell derivation. Although these technologies are far from therapeutic, concerns over the morality of embryonic stem cell derivation should not hinder their advancement. PMID:16574879

Adult subventricular zone neural stem cells as a potential source of dopaminergic replacement neurons

PubMed Central

Cave, John W.; Wang, Meng; Baker, Harriet

2014-01-01

Clinical trials engrafting human fetal ventral mesencephalic tissue have demonstrated, in principle, that cell replacement therapy provides substantial long-lasting improvement of motor impairments generated by Parkinson's Disease (PD). The use of fetal tissue is not practical for widespread clinical implementation of this therapy, but stem cells are a promising alternative source for obtaining replacement cells. The ideal stem cell source has yet to be established and, in this review, we discuss the potential of neural stem cells in the adult subventricular zone (SVZ) as an autologous source of replacement cells. We identify three key challenges for further developing this potential source of replacement cells: (1) improving survival of transplanted cells, (2) suppressing glial progenitor proliferation and survival, and (3) developing methods to efficiently produce dopaminergic neurons. Subventricular neural stem cells naturally produce a dopaminergic interneuron phenotype that has an apparent lack of vulnerability to PD-mediated degeneration. We also discuss whether olfactory bulb dopaminergic neurons derived from adult SVZ neural stem cells are a suitable source for cell replacement strategies. PMID:24574954

Comparative studies of mesenchymal stem cells derived from different cord tissue compartments - The influence of cryopreservation and growth media.

PubMed

Dulugiac, Magda; Moldovan, Lucia; Zarnescu, Otilia

2015-10-01

We have identified some critical aspects concerning umbilical cord tissue mesenchymal stem cells: the lack of standards for cell isolation, expansion and cryopreservation, the lack of unanimous opinions upon their multilineage differentiation potential and the existence of very few results related to the functional characterization of the cells isolated from cryopreserved umbilical cord tissue. Umbilical cord tissue cryopreservation appears to be the optimal solution for umbilical cord tissue mesenchymal stem cells storage for future clinical use. Umbilical cord tissue cryopreservation allows mesenchymal stem cells isolation before expected use, according with the specific clinical applications, by different customized isolation and expansion protocols agreed by cell therapy institutions. Using an optimized protocol for umbilical cord tissue cryopreservation in autologous cord blood plasma, isolation explant method and growth media supplemented with FBS or human serum, we performed comparative studies with respect to the characteristics of mesenchymal stem cells (MSC) isolated from different compartments of the same umbilical cord tissue such as Wharton's jelly, vein, arteries, before cryopreservation (pre freeze) and after cryopreservation (post thaw). Expression of histochemical and immunohistochemical markers as well as electron microscopy observations revealed similar adipogenic, chondrogenic and osteogenic differentiation capacity for cells isolated from pre freeze and corresponding post thaw tissue fragments of Wharton's jelly, vein or arteries of the same umbilical cord tissue, regardless growth media used for cells isolation and expansion. Our efficient umbilical cord tissue cryopreservation protocol is reliable for clinical applicability of mesenchymal stem cells that could next be isolated and expanded in compliance with future accepted standards. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mannitol-Enhanced Delivery of Stem Cells and Their Growth Factors Across the Blood–Brain Barrier

PubMed Central

Gonzales-Portillo, Gabriel S.; Sanberg, Paul R.; Franzblau, Max; Gonzales-Portillo, Chiara; Diamandis, Theo; Staples, Meaghan; Sanberg, Cyndy D.; Borlongan, Cesar V.

2014-01-01

Ischemic brain injury in adults and neonates is a significant clinical problem with limited therapeutic interventions. Currently, clinicians have only tPA available for stroke treatment and hypothermia for cerebral palsy. Owing to the lack of treatment options, there is a need for novel treatments such as stem cell therapy. Various stem cells including cells from embryo, fetus, perinatal, and adult tissues have proved effective in preclinical and small clinical trials. However, a limiting factor in the success of these treatments is the delivery of the cells and their by-products (neurotrophic factors) into the injured brain. We have demonstrated that mannitol, a drug with the potential to transiently open the blood–brain barrier and facilitate the entry of stem cells and trophic factors, as a solution to the delivery problem. The combination of stem cell therapy and mannitol may improve therapeutic outcomes in adult stroke and neonatal cerebral palsy. PMID:24480552

Biomaterials and Stem Cells for Tissue Engineering

PubMed Central

Zhang, Zhanpeng; Gupte, Melanie J.; Ma, Peter X.

2013-01-01

Importance of the field Organ failure and tissue loss are challenging health issues due to widespread injury, the lack of organs for transplantation, and limitations of conventional artificial implants. The field of tissue engineering aims to provide alternative living substitutes that restore, maintain or improve tissue function. Areas covered in this review In this paper, a wide range of porous scaffolds are reviewed, with an emphasis on phase separation techniques that generate advantageous nanofibrous 3D scaffolds for stem cell-based tissue engineering applications. In addition, methods for presentation and delivery of bioactive molecules to mimic the properties of stem cell niche are summarized. Recent progress in using these bio-instructive scaffolds to support stem cell differentiation and tissue regeneration is also presented. What the reader will gain Stem cells have great clinical potential because of their capability to differentiate into multiple cell types. Biomaterials have served as artificial extracellular environments to regulate stem cell behavior. Biomaterials with various physical, mechanical, and chemical properties can be designed to control stem cell development for regeneration. Take home message The research at the interface of stem cell biology and biomaterials has made and will continue to make exciting advances in tissue engineering. PMID:23327471

Isolation and purification of rabbit mesenchymal stem cells using an optimized protocol.

PubMed

Lin, Chunbo; Shen, Maorong; Chen, Weiping; Li, Xiaofeng; Luo, Daoming; Cai, Jinhong; Yang, Yuan

2015-11-01

Mesenchymal stem cells were first isolated and grown in vitro by Friedenstein over 40 yr ago; however, their isolation remains challenging as they lack unique markers for identification and are present in very small quantities in mesenchymal tissues and bone marrow. Using whole marrow samples, common methods for mesenchymal stem cell isolation are the adhesion method and density gradient fractionation. The whole marrow sample adhesion method still results in the nonspecific isolation of mononuclear cells, and activation and/or potential loss of target cells. Density gradient fractionation methods are complicated, and may result in contamination with toxic substances that affect cell viability. In the present study, we developed an optimized protocol for the isolation and purification of mesenchymal stem cells based on the principles of hypotonic lysis and natural sedimentation.

Differential Properties of Human ALP+ Periodontal Ligament Stem Cells vs Their ALP- Counterparts

PubMed Central

Tran, Quynh T; El-Ayachi, Ikbale; Bhatti, Fazal-Ur-Rehman; Bahabri, Rayan; Al-Habib, Mey; Huang, George TJ

2015-01-01

Characterizing subpopulations of stem cells is important to understand stem cell properties. Tissue-nonspecific alkaline phosphatase (ALP) is associated with mineral tissue forming cells as well as stem cells. Information regarding ALP subpopulation of human periodontal ligament stem cells (hPDLSCs) is limited. In the present study, we examined ALP+ and ALP− hPDLSC subpopulations, their surface markers STRO-1 and CD146, and the expression of stemness genes at various cell passages. We found that ALP+ subpopulation had higher levels of STRO-1 (30.6 ± 5.6%) and CD146 (90.4 ± 3.3%) compared to ALP− (STRO-1: 0.5 ± 0.1%; CD146: 75.3 ± 7.2%). ALP+ cells expressed significantly higher levels of stemness associated genes, NANOG, OCT4 and SOX than ALP− cells at low cell passages of 2-3 (p<0.05). ALP+ and ALP− cells had similar osteogenic, chondrogenic and neurogenic potential while ALP−, not ALP+ cells, lacked adipogenic potential. Upon continuous culturing and passaging, ALP+ continued to express higher stemness genes and STRO-1 and CD146 than ALP− cells at ≥passage 19. Under conditions (over-confluence and vitamin C treatment) when ALP+ subpopulation was increased, the stemness gene levels of ALP+ was no longer significantly higher than those in ALP− cells. In conclusion, ALP+ hPDLSCs possess differential properties from their ALP− counterparts. PMID:26807329

Stem cell plasticity.

PubMed

Lakshmipathy, Uma; Verfaillie, Catherine

2005-01-01

The central dogma in stem cell biology has been that cells isolated from a particular tissue can renew and differentiate into lineages of the tissue it resides in. Several studies have challenged this idea by demonstrating that tissue specific cell have considerable plasticity and can cross-lineage restriction boundary and give rise to cell types of other lineages. However, the lack of a clear definition for plasticity has led to confusion with several reports failing to demonstrate that a single cell can indeed differentiate into multiple lineages at significant levels. Further, differences between results obtained in different labs has cast doubt on some results and several studies still await independent confirmation. In this review, we critically evaluate studies that report stem cell plasticity using three rigid criteria to define stem cell plasticity; differentiation of a single cell into multiple cell lineages, functionality of differentiated cells in vitro and in vivo, robust and persistent engraft of transplanted cells.

Stem/Progenitor Cell Proteoglycans Decorated with 7-D-4, 4-C-3 and 3-B-3(-) Chondroitin Sulphate Motifs Are Morphogenetic Markers Of Tissue Development.

PubMed

Hayes, Anthony J; Smith, Susan M; Caterson, Bruce; Melrose, James

2018-06-11

This study reviewed the occurrence of chondroitin sulphate (CS) motifs 4-C-3, 7-D-4 and 3-B-3(-) which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulphation motifs 7-D-4, 4-C-3 and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

The emergence and popularisation of autologous somatic cellular therapies in Australia: therapeutic innovation or regulatory failure?

PubMed

McLean, Alison K; Stewart, Cameron; Kerridge, Ian

2014-09-01

Private stem cell clinics throughout Australia are providing autologous stem cell therapies for a range of chronic and debilitating illnesses despite the lack of published literature to support the clinical application of these therapies. The Therapeutic Goods Administration has excluded autologous stem cell therapies from its regulatory domain leaving such therapies to be regulated by the same mechanisms that regulate research, such as the National Health and Medical Research Council Research Ethics Guidelines, and clinical practice, such as the Australian Health Practitioner Regulation Agency. However, the provision of these stem cell therapies does not follow the established pathways for legitimate medical advance--therapeutic innovation or research. The current regulatory framework is failing to achieve its aims of protecting vulnerable patients and ensuring the proper conduct of medical practitioners in the private stem cell industry.

MtDNA depleted PC3 cells exhibit Warburg effect and cancer stem cell features

PubMed Central

Li, Xiaoran; Zhong, Yali; Lu, Jie; Axcrona, Karol; Eide, Lars; Syljuåsen, Randi G.; Peng, Qian; Wang, Junbai; Zhang, Hongquan; Goscinski, Mariusz Adam; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2016-01-01

Reducing mtDNA content was considered as a critical step in the metabolism restructuring for cell stemness restoration and further neoplastic development. However, the connections between mtDNA depletion and metabolism reprograming-based cancer cell stemness in prostate cancers are still lack of studies. Here, we demonstrated that human CRPC cell line PC3 tolerated high concentration of the mtDNA replication inhibitor ethidium bromide (EtBr) and the mtDNA depletion triggered a universal metabolic remodeling process. Failure in completing that process caused lethal consequences. The mtDNA depleted (MtDP) PC3 cells could be steadily maintained in the special medium in slow cycling status. The MtDP PC3 cells contained immature mitochondria and exhibited Warburg effect. Furthermore, the MtDP PC3 cells were resistant to therapeutic treatments and contained greater cancer stem cell-like subpopulations: CD44+, ABCG2+, side-population and ALDHbright. In conclusion, these results highlight the association of mtDNA content, mitochondrial function and cancer cell stemness features. PMID:27248169

Predicting gene regulatory networks by combining spatial and temporal gene expression data in Arabidopsis root stem cells

PubMed Central

de Luis Balaguer, Maria Angels; Fisher, Adam P.; Clark, Natalie M.; Fernandez-Espinosa, Maria Guadalupe; Möller, Barbara K.; Weijers, Dolf; Williams, Cranos; Lorenzo, Oscar; Sozzani, Rosangela

2017-01-01

Identifying the transcription factors (TFs) and associated networks involved in stem cell regulation is essential for understanding the initiation and growth of plant tissues and organs. Although many TFs have been shown to have a role in the Arabidopsis root stem cells, a comprehensive view of the transcriptional signature of the stem cells is lacking. In this work, we used spatial and temporal transcriptomic data to predict interactions among the genes involved in stem cell regulation. To accomplish this, we transcriptionally profiled several stem cell populations and developed a gene regulatory network inference algorithm that combines clustering with dynamic Bayesian network inference. We leveraged the topology of our networks to infer potential major regulators. Specifically, through mathematical modeling and experimental validation, we identified PERIANTHIA (PAN) as an important molecular regulator of quiescent center function. The results presented in this work show that our combination of molecular biology, computational biology, and mathematical modeling is an efficient approach to identify candidate factors that function in the stem cells. PMID:28827319

Embryonic Stem Cell Therapy of Heart Failure in Genetic Cardiomyopathy

PubMed Central

Yamada, Satsuki; Nelson, Timothy J.; Crespo-Diaz, Ruben J.; Perez-Terzic, Carmen; Liu, Xiao-Ke; Miki, Takashi; Seino, Susumu; Behfar, Atta; Terzic, Andre

2009-01-01

Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K+ (KATP) channel sub-units. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional KATP channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. PMID:18669912

Embryonic stem cell therapy of heart failure in genetic cardiomyopathy.

PubMed

Yamada, Satsuki; Nelson, Timothy J; Crespo-Diaz, Ruben J; Perez-Terzic, Carmen; Liu, Xiao-Ke; Miki, Takashi; Seino, Susumu; Behfar, Atta; Terzic, Andre

2008-10-01

Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K(+) (K(ATP)) channel subunits. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional K(ATP) channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. Disclosure of potential conflicts of interest is found at the end of this article.

Variable methylation of the imprinted gene, SNRPN, supports a relationship between intracranial germ cell tumours and neural stem cells.

PubMed

Lee, Shih-Han; Appleby, Vanessa; Jeyapalan, Jennie N; Palmer, Roger D; Nicholson, James C; Sottile, Virginie; Gao, Erning; Coleman, Nicholas; Scotting, Paul J

2011-02-01

Germ cell tumours (GCTs) are a diverse group of neoplasms all of which are generally believed to arise from germ cell progenitors (PGCs). Even those that form in the nervous system are likewise believed to be PGC-derived, despite being found a great distance from the normal location of germ cells. The primary evidence in favour of this model for the origins of intracranial GCTs is that they share molecular features with other GCTs. Those features include shared gene expression and a lack of methylation of imprinted genes, including SNRPN. Contrary to this model, we have proposed that endogenous neural stem cells of the brain are a more likely origin for these tumours. We show here that the lack of methylation of SNRPN that has previously been taken to indicate an origin for GCTs from PGCs is also seen in neural stem cells of mice and humans. We believe that, in the light of these and other recent observations, endogenous neural precursors of the brain are a more plausible origin for intracranial GCTs than are misplaced PGCs.

Expression of Epithelial Mesenchymal Transition and Cancer Stem Cell Markers in Circulating Tumor Cells.

PubMed

Werner, Stefan; Stenzl, Arnulf; Pantel, Klaus; Todenhöfer, Tilman

2017-01-01

The characterization of circulating tumor cells (CTC) has the potential not only to provide important insights into molecular alterations of advanced tumor disease but also to facilitate risk prediction. Epithelial mesenchymal transition (EMT) has been discovered as important process for the development of metastases and the dissemination of tumor cells into the blood stream. In different tumor types, CTC with a mesenchymal phenotype have been reported that have presumably underwent EMT. Moreover, CTC with stem-cell like characteristics have been postulated as important drivers of tumor progression. Different platforms have been introduced to allow CTC enrichment independent of expression of epithelial antigens, as these may be downregulated in EMT- or stem-cell-like CTC. Both for CTCs with EMT- or stem-cell features different markers have been proposed. However, there is still a lack of evidence on the association of these markers with functional features and characteristics for stem cells and cells undergoing EMT.

Increasing magnetite contents of polymeric magnetic particles dramatically improves labeling of neural stem cell transplant populations.

PubMed

Adams, Christopher F; Rai, Ahmad; Sneddon, Gregor; Yiu, Humphrey H P; Polyak, Boris; Chari, Divya M

2015-01-01

Safe and efficient delivery of therapeutic cells to sites of injury/disease in the central nervous system is a key goal for the translation of clinical cell transplantation therapies. Recently, 'magnetic cell localization strategies' have emerged as a promising and safe approach for targeted delivery of magnetic particle (MP) labeled stem cells to pathology sites. For neuroregenerative applications, this approach is limited by the lack of available neurocompatible MPs, and low cell labeling achieved in neural stem/precursor populations. We demonstrate that high magnetite content, self-sedimenting polymeric MPs [unfunctionalized poly(lactic acid) coated, without a transfecting component] achieve efficient labeling (≥90%) of primary neural stem cells (NSCs)-a 'hard-to-label' transplant population of major clinical relevance. Our protocols showed high safety with respect to key stem cell regenerative parameters. Critically, labeled cells were effectively localized in an in vitro flow system by magnetic force highlighting the translational potential of the methods used. Copyright © 2015 Elsevier Inc. All rights reserved.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Stem Cell Therapy: A Promising Therapeutic Method for Intracerebral Hemorrhage.

PubMed

Gao, Liansheng; Xu, Weilin; Li, Tao; Chen, Jingyin; Shao, Anwen; Yan, Feng; Chen, Gao

2018-01-01

Spontaneous intracerebral hemorrhage (ICH) is one type of the most devastating cerebrovascular diseases worldwide, which causes high morbidity and mortality. However, efficient treatment is still lacking. Stem cell therapy has shown good neuroprotective and neurorestorative effect in ICH and is a promising treatment. In this study, our aim was to review the therapeutic effects, strategies, related mechanisms and safety issues of various types of stem cell for ICH treatment. Numerous studies had demonstrated the therapeutic effects of diverse stem cell types in ICH. The potential mechanisms include tissue repair and replacement, neurotrophy, promotion of neurogenesis and angiogenesis, anti-apoptosis, immunoregulation and anti-inflammation and so forth. The microenvironment of the central nervous system (CNS) can also influence the effects of stem cell therapy. The detailed therapeutic strategies for ICH treatment such as cell type, the number of cells, time window, and the routes of medication delivery, varied greatly among different studies and had not been determined. Moreover, the safety issues of stem cell therapy for ICH should not be ignored. Stem cell therapy showed good therapeutic effect in ICH, making it a promising treatment. However, safety should be carefully evaluated, and more clinical trials are required before stem cell therapy can be extensively applied to clinical use.

Designing the stem cell microenvironment for guided connective tissue regeneration.

PubMed

Bogdanowicz, Danielle R; Lu, Helen H

2017-12-01

Adult mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine because of their ability to self-renew and their capacity for multilineage differentiation and tissue regeneration. For connective tissues, such as ligaments or tendons, MSCs are vital to the modulation of the inflammatory response following acute injury while also interacting with resident fibroblasts to promote cell proliferation and matrix synthesis. To date, MSC injection for connective tissue repair has yielded mixed results in vivo, likely due to a lack of appropriate environmental cues to effectively control MSC response and promote tissue healing instead of scar formation. In healthy tissues, stem cells reside within a complex microenvironment comprising cellular, structural, and signaling cues that collectively maintain stemness and modulate tissue homeostasis. Changes to the microenvironment following injury regulate stem cell differentiation, trophic signaling, and tissue healing. Here, we focus on models of the stem cell microenvironment that are used to elucidate the mechanisms of stem cell regulation and inspire functional approaches to tissue regeneration. Recent studies in this frontier area are highlighted, focusing on how microenvironmental cues modulate MSC response following connective tissue injury and, more importantly, how this unique cell environment can be programmed for stem cell-guided tissue regeneration. © 2017 New York Academy of Sciences.

Medical innovation versus stem cell tourism.

PubMed

Lindvall, Olle; Hyun, Insoo

2009-06-26

Stem cell tourism is criticized on grounds of consumer fraud, blatant lack of scientific justification, and patient safety. However, the issues are complex because they invoke questions concerning the limits of acceptable medical innovation and medical travel. Here we discuss these issues and articulate conditions under which "unproven" therapies may be offered to patients outside of regular clinical trials.

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

PubMed

Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

2017-03-15

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Stem cells in degenerative orthopaedic pathologies: effects of aging on therapeutic potential.

PubMed

Atesok, Kivanc; Fu, Freddie H; Sekiya, Ichiro; Stolzing, Alexandra; Ochi, Mitsuo; Rodeo, Scott A

2017-02-01

The purpose of this study was to summarize the current evidence on the use of stem cells in the elderly population with degenerative orthopaedic pathologies and to highlight the pathophysiologic mechanisms behind today's therapeutic challenges in stem cell-based regeneration of destructed tissues in the elderly patients with osteoarthritis (OA), degenerative disc disease (DDD), and tendinopathies. Clinical and basic science studies that report the use of stem cells in the elderly patients with OA, DDD, and tendinopathies were identified using a PubMed search. The studies published in English have been assessed, and the best and most recent evidence was included in the current study. Evidence suggests that, although short-term results regarding the effects of stem cell therapy in degenerative orthopaedic pathologies can be promising, stem cell therapies do not appear to reverse age-related tissue degeneration. Causes of suboptimal outcomes can be attributed to the decrease in the therapeutic potential of aged stem cell populations and the regenerative capacity of these cells, which might be negatively influenced in an aged microenvironment within the degenerated tissues of elderly patients with OA, DDD, and tendinopathies. Clinical protocols guiding the use of stem cells in the elderly patient population are still under development, and high-level randomized controlled trials with long-term outcomes are lacking. Understanding the consequences of age-related changes in stem cell function and responsiveness of the in vivo microenvironment to stem cells is critical when designing cell-based therapies for elderly patients with degenerative orthopaedic pathologies.

Mammary stem cells: angels or demons in mammary gland?

PubMed

Chen, Xueman; Liu, Qiang; Song, Erwei

2017-01-01

A highly dynamic development process exits within the epithelia of mammary gland, featuring morphogenetic variation during puberty, pregnancy, lactation, and regression. The identification of mammary stem cells (MaSCs) via lineage-tracing studies has substantiated a hierarchical organization of the mammary epithelia. A single MaSC is capable of reconstituting the entirely functional mammary gland upon orthotopic transplantation. Although different mammary cell subpopulations can be candidate cells-of-origin for distinct breast tumor subtypes, it still lacks experimental proofs whether MaSCs, the most primitive cells, are the 'seeds' of malignant transformation during most, if not all, tumorigenesis in the breast. Here, we review current knowledge of mammary epithelial hierarchy, highlighting the roles of mammary stem/progenitor cells and breast cancer stem cells (BCSCs) along with their key molecular regulators in organ development and cancer evolution. Clarifying these issues will pave the way for developing novel interventions toward stem/progenitor cells in either prevention or treatment of breast cancer (BrCa).

Mammary stem cells: angels or demons in mammary gland?

PubMed Central

Chen, Xueman; Liu, Qiang; Song, Erwei

2017-01-01

A highly dynamic development process exits within the epithelia of mammary gland, featuring morphogenetic variation during puberty, pregnancy, lactation, and regression. The identification of mammary stem cells (MaSCs) via lineage-tracing studies has substantiated a hierarchical organization of the mammary epithelia. A single MaSC is capable of reconstituting the entirely functional mammary gland upon orthotopic transplantation. Although different mammary cell subpopulations can be candidate cells-of-origin for distinct breast tumor subtypes, it still lacks experimental proofs whether MaSCs, the most primitive cells, are the ‘seeds’ of malignant transformation during most, if not all, tumorigenesis in the breast. Here, we review current knowledge of mammary epithelial hierarchy, highlighting the roles of mammary stem/progenitor cells and breast cancer stem cells (BCSCs) along with their key molecular regulators in organ development and cancer evolution. Clarifying these issues will pave the way for developing novel interventions toward stem/progenitor cells in either prevention or treatment of breast cancer (BrCa). PMID:29263909

Governing stem cell therapy in India: regulatory vacuum or jurisdictional ambiguity?

PubMed Central

Tiwari, Shashank S.; Raman, Sujatha

2014-01-01

Stem cell treatments are being offered in Indian clinics although preclinical evidence of their efficacy and safety is lacking. This is attributed to a governance vacuum created by the lack of legally binding research guidelines. By contrast, this paper highlights jurisdictional ambiguities arising from trying to regulate stem cell therapy under the auspices of research guidelines when treatments are offered in a private market disconnected from clinical trials. While statutory laws have been strengthened in 2014, prospects for their implementation remain weak, given embedded challenges of putting healthcare laws and professional codes into practice. Finally, attending to the capacities of consumer law and civil society activism to remedy the problem of unregulated treatments, the paper finds that the very definition of a governance vacuum needs to be reframed to clarify whose rights to health care are threatened by the proliferation of commercial treatments and individualized negligence-based remedies for grievances. PMID:25431534

Generation of a transplantable erythropoietin-producer derived from human mesenchymal stem cells.

PubMed

Yokoo, Takashi; Fukui, Akira; Matsumoto, Kei; Ohashi, Toya; Sado, Yoshikazu; Suzuki, Hideaki; Kawamura, Tetsuya; Okabe, Masataka; Hosoya, Tatsuo; Kobayashi, Eiji

2008-06-15

Differentiation of autologous stem cells into functional transplantable tissue for organ regeneration is a promising regenerative therapeutic approach for cancer, diabetes, and many human diseases. Yet to be established, however, is differentiation into tissue capable of producing erythropoietin (EPO), which has a critical function in anemia. We report a novel EPO-producing organ-like structure (organoid) derived from human mesenchymal stem cells. Using our previously established relay culture system, a human mesenchymal stem cell-derived, human EPO-competent organoid was established in rat omentum. The organoid-derived levels of human EPO increased in response to anemia induced by rapid blood withdrawal. In addition, the presence of an organoid in rats suppressed for native (rat) EPO production enhanced recovery from anemia when compared with control animals lacking the organoid. Together these results confirmed the generation of a stem cell-derived organoid that is capable of producing EPO and sensitive to physiological regulation.

THE POTENTIAL ROLE OF ENDOGENOUS STEM CELLS IN REGENERATION OF THE INNER EAR

PubMed Central

Martinez-Monedero, Rodrigo; Oshima, Kazuo; Heller, Stefan; Edge, Albert S.B.

2007-01-01

Stem cells in various mammalian tissues retain the capacity to renew themselves and may be able to restore damaged tissue. Their existence has been proven by genetic tracer studies that demonstrate their differentiation into multiple tissue types and by their ability to self-renew through proliferation. Stem cells from the adult nervous system proliferate to form clonal floating colonies called spheres in vitro, and recent studies have demonstrated sphere formation by cells in the cochlea in addition to the vestibular system and the auditory ganglia, indicating that these tissues contain cells with stem cell properties. The presence of stem cells in the inner ear raises the hope of regeneration of mammalian inner ear cells but is difficult to correlate with the lack spontaneous regeneration seen in the inner ear after tissue damage. Loss of stem cells postnatally in the cochlea may correlate with the loss of regenerative capacity and may limit our ability to stimulate regeneration. Retention of sphere forming capacity in adult vestibular tissues suggests that the limited capacity for repair may be attributed to the continued presence of progenitor cells. Future strategies for regeneration must consider the distribution of endogenous stem cells in the inner ear and whether cells with the capacity for regeneration are retained. PMID:17321086

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells

PubMed Central

Roth, Therese M.; Chiang, C.-Y. Ason; Inaba, Mayu; Yuan, Hebao; Salzmann, Viktoria; Roth, Caitlin E.; Yamashita, Yukiko M.

2012-01-01

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions. PMID:22357619

Reviewing and Updating the Major Molecular Markers for Stem Cells

PubMed Central

Calloni, Raquel; Cordero, Elvira Alicia Aparicio; Henriques, João Antonio Pêgas

2013-01-01

Stem cells (SC) are able to self-renew and to differentiate into many types of committed cells, making SCs interesting for cellular therapy. However, the pool of SCs in vivo and in vitro consists of a mix of cells at several stages of differentiation, making it difficult to obtain a homogeneous population of SCs for research. Therefore, it is important to isolate and characterize unambiguous molecular markers that can be applied to SCs. Here, we review classical and new candidate molecular markers that have been established to show a molecular profile for human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs). The commonly cited markers for embryonic ESCs are Nanog, Oct-4, Sox-2, Rex-1, Dnmt3b, Lin-28, Tdgf1, FoxD3, Tert, Utf-1, Gal, Cx43, Gdf3, Gtcm1, Terf1, Terf2, Lefty A, and Lefty B. MSCs are primarily identified by the expression of CD13, CD29, CD44, CD49e, CD54, CD71, CD73, CD90, CD105, CD106, CD166, and HLA-ABC and lack CD14, CD31, CD34, CD45, CD62E, CD62L, CD62P, and HLA-DR expression. HSCs are mainly isolated based on the expression of CD34, but the combination of this marker with CD133 and CD90, together with a lack of CD38 and other lineage markers, provides the most homogeneous pool of SCs. Here, we present new and alternative markers for SCs, along with microRNA profiles, for these cells. PMID:23336433

RGD-conjugated rod-like viral nanoparticles on 2D scaffold improved bone differentiation of mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Wang, Qian; Pongkwan, Sitasuwan; Lee, L.; Li, Kai; Nguyen, Huong

2014-05-01

Viral nanoparticles have uniform and well-defined nano-structures and can be produced in large quantities. Several plant viral nanoparticles have been tested in biomedical applications due to the lack of mammalian cell infectivity. We are particularly interested in using Tobacco mosaic virus (TMV), which has been demonstrated to enhance bone tissue regeneration, as a tuneable nanoscale building block for biomaterials development. Unmodified TMV particles have been shown to accelerate osteogenic differentiation of adult stem cells by synergistically upregulating BMP2 and IBSP expression with dexamethasone. However, the lack of affinity to mammalian cell surface resulted in low initial cell adhesion. In this study, to increase cell binding capacity of TMV based material the chemical functionalization of TMV with arginine-glycine-aspartic acid (RGD) peptide was explored. An azide-derivatized RGD peptide was “clicked” to tyrosine residues on TMV outer surface via an efficient copper(I) catalysed azide-alkyne cycloaddition reaction. The ligand spacing is calculated to be 2-4 nm, which could offer a polyvalent ligand clustering effect for enhanced cell receptor signalling, further promoting the proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells.

Mismatched related hematopoietic stem cell transplantation in primary immunodeficiency.

PubMed

Wahadneh, Adel M; Bin Dahman, Haifa A; Abu Shukear, Mohammed E; Habahbeh, Zeyad M; Ajarmeh, Mohammad A; Zyood, Raed M; Habashneh, Mueen S

2013-11-01

Hematopoietic stem cell transplantation (HSCT) is the definitive therapy for a variety of primary immunodeficiency syndromes (PIDs). However, no more than 30% of the patients will have a human leukocyte antigen (HLA)-identical sibling. We retrospectively analyzed our results of ten patients with PID; severe combined immunodeficiency (SCID) (n = 7), hyper IgM (HIgM) (n = 1) and combined immunodeficiency (CID) (n = 2), who lacked a fully matched donor and underwent mismatched related HSCT during the period from 2008 to 2010. The median age at the time of transplantation ranged between 3 and 84 months (median 6.5 months). Peripheral blood stem cells (PBSC) were used in all HSCTs. The mean value of the peripheral CD34+ cells infused was 9.19 × 10 (6) /kg recipient weight. Patients received different conditioning protocols. All patients received anti graft versus host disease (GVHD) prophylaxis and all were engrafted. Mixed chimerism (5-55%) was noticed. GVHD was observed in 50% of the patients. Post-transplant follow-up ranged from 3 weeks to 36 months (median 15 months). Five patients are still alive while one patient developed engraftment syndrome followed by graft slippage for which a second transplant with CD34+ stem cells 5.8 × 10 (6) /kg recipient's weight was infused. The others died from sepsis and transplant-related complications. Immune reconstitution was noticed in four patients. In conclusion, HLA-haploidentical stem cell transplantation may be feasible, with appropriate GVHD prophylaxis, for patients with PID who lack a fully matched donor.

A review of human pluripotent stem cell-derived cardiomyocytes for high-throughput drug discovery, cardiotoxicity screening, and publication standards.

PubMed

Mordwinkin, Nicholas M; Burridge, Paul W; Wu, Joseph C

2013-02-01

Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.

A putative mesenchymal stem cells population isolated from adult human testes.

PubMed

Gonzalez, R; Griparic, L; Vargas, V; Burgee, K; Santacruz, P; Anderson, R; Schiewe, M; Silva, F; Patel, A

2009-08-07

Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we isolated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications.

Stem cell transplantation in the context of HIV--how can we cure HIV infection?

PubMed

Bauer, Gerhard; Anderson, Joseph S

2014-01-01

All HIV target cells are derived from hematopoietic stem cells. More than two decades ago, a hypothesis was postulated that a cure for HIV may be possible by performing a transplant with HIV-resistant hematopoietic stem cells that would allow for an HIV-resistant immune system to arise. HIV-resistant stem cells could be generated by genetically modifying them with gene therapy vectors transferring anti-HIV genes. First attempts of stem cell gene therapy for HIV were carried out in the USA in the 1990s demonstrating safety, but also little efficacy at that time. The first demonstration that the postulated hypothesis was correct was the cure of an HIV-infected individual in Berlin in 2009 who received an allogeneic bone marrow transplant from a donor who lacked the CCR5 chemokine receptor, a naturally arising mutation rendering HIV target cells resistant to infection with macrophage tropic strains of HIV. In 2013, reports were published about a possible cure of HIV-infected individuals who received allogeneic bone marrow transplants with cells not resistant to HIV. We will review these stem cell transplant procedures and discuss their utility to provide a cure for HIV infection, including efficacious future stem cell gene therapy applications.

A Functional Role of RB-Dependent Pathway in the Control of Quiescence in Adult Epidermal Stem Cells Revealed by Genomic Profiling

PubMed Central

Lorz, Corina; García-Escudero, Ramón; Segrelles, Carmen; Garín, Marina I.; Ariza, José M.; Santos, Mirentxu; Ruiz, Sergio; Lara, María F.; Martínez-Cruz, Ana B.; Costa, Clotilde; Buitrago-Pérez, Águeda; Saiz-Ladera, Cristina; Dueñas, Marta

2010-01-01

Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis. Electronic supplementary material The online version of this article (doi:10.1007/s12015-010-9139-0) contains supplementary material, which is available to authorized users. PMID:20376578

Concise review: reprogramming strategies for cardiovascular regenerative medicine: from induced pluripotent stem cells to direct reprogramming.

PubMed

Budniatzky, Inbar; Gepstein, Lior

2014-04-01

Myocardial cell-replacement therapies are emerging as novel therapeutic paradigms for myocardial repair but are hampered by the lack of sources of autologous human cardiomyocytes. The recent advances in stem cell biology and in transcription factor-based reprogramming strategies may provide exciting solutions to this problem. In the current review, we describe the different reprogramming strategies that can give rise to cardiomyocytes for regenerative medicine purposes. Initially, we describe induced pluripotent stem cell technology, a method by which adult somatic cells can be reprogrammed to yield pluripotent stem cells that could later be coaxed ex vivo to differentiate into cardiomyocytes. The generated induced pluripotent stem cell-derived cardiomyocytes could then be used for myocardial cell transplantation and tissue engineering strategies. We also describe the more recent direct reprogramming approaches that aim to directly convert the phenotype of one mature cell type (fibroblast) to another (cardiomyocyte) without going through a pluripotent intermediate cell type. The advantages and shortcomings of each strategy for cardiac regeneration are discussed, along with the hurdles that need to be overcome on the road to clinical translation.

[German stem-cell law: content and critique].

PubMed

Simon, Jürgen

2002-01-01

The author analyzes in this work the new German Stem Cells Act, approved by the German Parliament on 10 of May 2002. In this law it is prohibited with general character the investigation with stem cells of embryonic origin, although it is allowed under certain very strict requirements. The conflict that arises among a very strict protection of the embryo, such and like it happens in the present law, and the constitutional right to scientific investigation is also analyzed, a reference to the derivative problems of the lack of clarity of some articles of the law is also made.

Non-canonical microRNAs miR-320 and miR-702 promote proliferation in Dgcr8-deficient embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Byeong-Moo; Department of Medicine, Harvard Medical School, Boston, MA 02115; Choi, Michael Y., E-mail: mchoi@partners.org

2012-09-21

Highlights: Black-Right-Pointing-Pointer Embryonic stem cells (ESCs) lacking non-canonical miRNAs proliferate slower. Black-Right-Pointing-Pointer miR-320 and miR-702 are two non-canonical miRNAs expressed in ESCs. Black-Right-Pointing-Pointer miR-320 and miR-702 promote proliferation of Dgcr8-deficient ESCs. Black-Right-Pointing-Pointer miR-320 targets p57 and helps to release Dgcr8-deficient ESCs from G1 arrest. Black-Right-Pointing-Pointer miR-702 targets p21 and helps to release Dgcr8-deficient ESCs from G1 arrest. -- Abstract: MicroRNAs are known to contribute significantly to stem cell phenotype by post-transcriptionally regulating gene expression. Most of our knowledge of microRNAs comes from the study of canonical microRNAs that require two sequential cleavages by the Drosha/Dgcr8 heterodimer and Dicer to generatemore » mature products. In contrast, non-canonical microRNAs bypass the cleavage by the Drosha/Dgcr8 heterodimer within the nucleus but still require cytoplasmic cleavage by Dicer. The function of non-canonical microRNAs in embryonic stem cells (ESCs) remains obscure. It has been hypothesized that non-canonical microRNAs have important roles in ESCs based upon the phenotypes of ESC lines that lack these specific classes of microRNAs; Dicer-deficient ESCs lacking both canonical and non-canonical microRNAs have much more severe proliferation defect than Dgcr8-deficient ESCs lacking only canonical microRNAs. Using these cell lines, we identified two non-canonical microRNAs, miR-320 and miR-702, that promote proliferation of Dgcr8-deficient ESCs by releasing them from G1 arrest. This is accomplished by targeting the 3 Prime -untranslated regions of the cell cycle inhibitors p57 and p21 and thereby inhibiting their expression. This is the first report of the crucial role of non-canonical microRNAs in ESCs.« less

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

PubMed

Bose, Bipasha; Shenoy P, Sudheer

2016-02-01

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas.

PubMed

Würth, Roberto; Barbieri, Federica; Pattarozzi, Alessandra; Gaudenzi, Germano; Gatto, Federico; Fiaschi, Pietro; Ravetti, Jean-Louis; Zona, Gianluigi; Daga, Antonio; Persani, Luca; Ferone, Diego; Vitale, Giovanni; Florio, Tullio

2017-09-01

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.

ERECTA-family receptor kinases regulate stem cell homeostasis via buffering its cytokinin responsiveness in the shoot apical meristem.

PubMed

Uchida, Naoyuki; Shimada, Masanori; Tasaka, Masao

2013-03-01

Shoot apical meristems (SAMs), which are maintained at the tips of stems, are indeterminate structures and sources of stem cells from which all aerial organs are ultimately derived. Although mechanisms that regulate the homeostasis of the stem cells have been extensively investigated, identification of further unknown regulators should provide better understanding of the regulation. Here, we report that members of the Arabidopsis ERECTA (ER) receptor kinase family redundantly play a significant role in the regulation of stem cell homeostasis. In wild-type seedlings, the expression of WUSCHEL (WUS), a central regulator of the stem cell population, is stimulated by cytokinin. Interestingly, however, the SAM morphology and the expression of CLAVATA3 (CLV3), which is expressed in stem cells and therefore serves as a stem cell marker, are relatively stable against cytokinin treatment regardless of increased WUS expression. These findings indicate the presence of a mechanism to buffer stem cell homeostasis against an increase in cytokinin. Mutant seedlings lacking all ER-family members, which are expressed in the SAM, show an increase in the stem cell population and also the up-regulation of a cytokinin-responsive gene in the SAM. In this mutant, WUS expression is stimulated by cytokinin treatment as efficiently as in wild-type plants. However, in contrast to wild-type plants, SAM morphology and CLV3 expression respond drastically to cytokinin treatment, suggesting that the buffering mechanism to maintain stem cell homeostasis against an increase in cytokinin is severely impaired in this mutant. We suggest that the ER family regulates stem cell homeostasis via buffering its cytokinin responsiveness in the SAM.

Current Therapeutic Strategies for Stem Cell-Based Cartilage Regeneration

PubMed Central

Nam, Yoojun; Lee, Jennifer

2018-01-01

The process of cartilage destruction in the diarthrodial joint is progressive and irreversible. This destruction is extremely difficult to manage and frustrates researchers, clinicians, and patients. Patients often take medication to control their pain. Surgery is usually performed when pain becomes uncontrollable or joint function completely fails. There is an unmet clinical need for a regenerative strategy to treat cartilage defect without surgery due to the lack of a suitable regenerative strategy. Clinicians and scientists have tried to address this using stem cells, which have a regenerative potential in various tissues. Cartilage may be an ideal target for stem cell treatment because it has a notoriously poor regenerative potential. In this review, we describe past, present, and future strategies to regenerate cartilage in patients. Specifically, this review compares a surgical regenerative technique (microfracture) and cell therapy, cell therapy with and without a scaffold, and therapy with nonaggregated and aggregated cells. We also review the chondrogenic potential of cells according to their origin, including autologous chondrocytes, mesenchymal stem cells, and induced pluripotent stem cells. PMID:29765426

De novo generation of HSCs from somatic and pluripotent stem cell sources

PubMed Central

Vo, Linda T.

2015-01-01

Generating human hematopoietic stem cells (HSCs) from autologous tissues, when coupled with genome editing technologies, is a promising approach for cellular transplantation therapy and for in vitro disease modeling, drug discovery, and toxicology studies. Human pluripotent stem cells (hPSCs) represent a potentially inexhaustible supply of autologous tissue; however, to date, directed differentiation from hPSCs has yielded hematopoietic cells that lack robust and sustained multilineage potential. Cellular reprogramming technologies represent an alternative platform for the de novo generation of HSCs via direct conversion from heterologous cell types. In this review, we discuss the latest advancements in HSC generation by directed differentiation from hPSCs or direct conversion from somatic cells, and highlight their applications in research and prospects for therapy. PMID:25762177

Concise review: tailoring bioengineered scaffolds for stem cell applications in tissue engineering and regenerative medicine.

PubMed

Cosson, Steffen; Otte, Ellen A; Hezaveh, Hadi; Cooper-White, Justin J

2015-02-01

The potential for the clinical application of stem cells in tissue regeneration is clearly significant. However, this potential has remained largely unrealized owing to the persistent challenges in reproducibly, with tight quality criteria, and expanding and controlling the fate of stem cells in vitro and in vivo. Tissue engineering approaches that rely on reformatting traditional Food and Drug Administration-approved biomedical polymers from fixation devices to porous scaffolds have been shown to lack the complexity required for in vitro stem cell culture models or translation to in vivo applications with high efficacy. This realization has spurred the development of advanced mimetic biomaterials and scaffolds to increasingly enhance our ability to control the cellular microenvironment and, consequently, stem cell fate. New insights into the biology of stem cells are expected to eventuate from these advances in material science, in particular, from synthetic hydrogels that display physicochemical properties reminiscent of the natural cell microenvironment and that can be engineered to display or encode essential biological cues. Merging these advanced biomaterials with high-throughput methods to systematically, and in an unbiased manner, probe the role of scaffold biophysical and biochemical elements on stem cell fate will permit the identification of novel key stem cell behavioral effectors, allow improved in vitro replication of requisite in vivo niche functions, and, ultimately, have a profound impact on our understanding of stem cell biology and unlock their clinical potential in tissue engineering and regenerative medicine. ©AlphaMed Press.

Concise Review: Conceptualizing Paralogous Stem-Cell Niches and Unfolding Bone Marrow Progenitor Cell Identities.

PubMed

Chen, Kevin G; Johnson, Kory R; McKay, Ronald D G; Robey, Pamela G

2018-01-01

Lineage commitment and differentiation of skeletal stem cells/bone marrow stromal cells (SSCs/BMSCs, often called bone marrow-derived "mesenchymal stem/stromal" cells) offer an important opportunity to study skeletal and hematopoietic diseases, and for tissue engineering and regenerative medicine. Currently, many studies in this field have relied on cell lineage tracing methods in mouse models, which have provided a significant advancement in our knowledge of skeletal and hematopoietic stem-cell niches in bone marrow (BM). However, there is a lack of agreement in numerous fundamental areas, including origins of various BM stem-cell niches, cell identities, and their physiological roles in the BM. In order to resolve these issues, we propose a new hypothesis of "paralogous" stem-cell niches (PSNs); that is, progressively altered parallel niches within an individual species throughout the life span of the organism. A putative PSN code seems to be plausible based on analysis of transcriptional signatures in two representative genes that encode Nes-GFP and leptin receptors, which are frequently used to monitor SSC lineage development in BM. Furthermore, we suggest a dynamic paralogous BM niche (PBMN) model that elucidates the coupling and uncoupling mechanisms between BM stem-cell niches and their zones of active regeneration during different developmental stages. Elucidation of these PBMNs would enable us to resolve the existing controversies, thus paving the way to achieving precision regenerative medicine and pharmaceutical applications based on these BM cell resources. Stem Cells 2018;36:11-21. © 2017 AlphaMed Press.

Arrhythmia in Stem Cell Transplantation

PubMed Central

Almeida, Shone O.; Skelton, Rhys J.; Adigopula, Sasikanth; Ardehali, Reza

2015-01-01

Synopsis Stem cell regenerative therapies hold promise for treating diseases across the spectrum of medicine. Recent clinical trials have confirmed the safety of stem cell delivery to the heart with promising but variable results. While significant progress has been made in the preclinical stages, the clinical application of cardiac cell therapy is limited by technical challenges, including inability to isolate a pure population of cardiac-specific progenitors capable of robust engraftment and regeneration, lack of appropriate pre-clinical animal models, uncertainty about the best mode of delivery, paucity of adequate imaging modalities, and lack of knowledge about the fate of transplanted cells. The inability of transplanted cells to structurally and functionally integrate into the host myocardium may pose arrhythmogenic risk to patients. This is in part dependent on the type of cell transplanted, where the expression of gap junctions such as connexin-43 is essential not only for electromechanical integration, but has also been found to be protective against electrical instability post-transplant. Additionally, certain methods of cell delivery, such as intramyocardial injection, carry a higher rate of arrhythmias. Other potential contributors to the arrhythmogenicity of cell transplantation include re-entrant pathways due to heterogeneity in conduction velocities between graft and host as well as graft automaticity. In this paper, we discuss the arrhythmogenic potential of cell delivery to the heart. PMID:26002399

Generation of functional human pancreatic β cells in vitro

PubMed Central

Pagliuca, Felicia W.; Millman, Jeffrey R.; Gürtler, Mads; Segel, Michael; Van Dervort, Alana; Ryu, Jennifer Hyoje; Peterson, Quinn P.; Greiner, Dale; Melton, Douglas A.

2015-01-01

Summary The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem cell derived β cells (SC-β) express markers found in mature β cells, flux Ca2+ in response to glucose, package insulin into secretory granules and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice. PMID:25303535

The Fountain of Stem Cell-Based Youth? Online Portrayals of Anti-Aging Stem Cell Technologies.

PubMed

Rachul, Christen M; Percec, Ivona; Caulfield, Timothy

2015-08-01

The hype surrounding stem cell science has created a market opportunity for the cosmetic industry. Cosmetic and anti-aging products and treatments that make claims regarding stem cell technology are increasingly popular, despite a lack of evidence for safety and efficacy of such products. This study explores how stem cell-based products and services are portrayed to the public through online sources, in order to gain insight into the key messages available to consumers. A content analysis of 100 web pages was conducted to examine the portrayals of stem cell-based cosmetic and anti-aging products and treatments. A qualitative discourse analysis of one web page further examined how language contributes to the portrayals of these products and treatments to public audiences. The majority of web pages portrayed stem cell-based products as ready for public use. Very few web pages substantiated claims with scientific evidence, and even fewer mentioned any risks or limitations associated with stem cell science. The discourse analysis revealed that the framing and use of metaphor obscures the certainty of the efficacy of and length of time for stem cell-based anti-aging technology to be publicly available. This study highlights the need to educate patients and the public on the current limits of stem cell applications in this context. In addition, generating scientific evidence for stem cell-based anti-aging and aesthetic applications is needed for optimizing benefits and minimizing adverse effects for the public. Having more evidence on efficacy and risks will help to protect patients who are eagerly seeking out these treatments. © 2015 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

The "Growing" Reality of the Neurological Complications of Global "Stem Cell Tourism".

PubMed

Julian, Katie; Yuhasz, Nick; Hollingsworth, Ethan; Imitola, Jaime

2018-04-01

"Stem cell tourism" is defined as the unethical practice of offering unproven cellular preparations to patients suffering from various medical conditions. This phenomenon is rising in the field of neurology as patients are requesting information and opportunities for treatment with stem cells for incurable conditions such as multiple sclerosis and amyotrophic lateral sclerosis, despite their clinical research and experimental designation. Here, we review the recent trends in "stem cell tourism" in both the United States and abroad, and discuss the recent reports of neurological complications from these activities. Finally, we frame critical questions for the field of neurology regarding training in the ethical, legal, and societal issues of the global "stem cell tourism," as well as suggest strategies to alleviate this problem. Although there are ongoing legitimate clinical trials with stem cells for neurological diseases, procedures offered by "stem cell clinics" cannot be defined as clinical research. They lack the experimental and state-of-the-art framework defined by peers and the FDA that focus on human research that safeguard the protection of human subjects against economical exploitation, unwanted side effects, and futility of unproven procedures. "Stem cell tourism" ultimately exploits therapeutic hope of patients and families with incurable neurological diseases and can put in danger the legitimacy of stem cell research as a whole. We posit that an improvement in education, regulation, legislation, and involvement of authorities in global health in neurology and neurosurgery is required. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors.

PubMed

Almutawaa, Walaa; Rohani, Leili; Rancourt, Derrick E

2016-01-01

Human induced pluripotent stem cells (hiPSCs) hold great promise as a cell source for therapeutic applications and regenerative medicine. Traditionally, hiPSCs are expanded in two-dimensional static culture as colonies in the presence or absence of feeder cells. However, this expansion procedure is associated with lack of reproducibility and low cell yields. To fulfill the large cell number demand for clinical use, robust large-scale production of these cells under defined conditions is needed. Herein, we describe a scalable, low-cost protocol for expanding hiPSCs as aggregates in a lab-scale bioreactor.

Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.

PubMed

Phondeechareon, Tanapol; Wattanapanitch, Methichit; U-Pratya, Yaowalak; Damkham, Chanapa; Klincumhom, Nuttha; Lorthongpanich, Chanchao; Kheolamai, Pakpoom; Laowtammathron, Chuti; Issaragrisil, Surapol

2016-10-01

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH, however, lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore, other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs), characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming, and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation, the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients.

In vitro long-term development of cultured inner ear stem cells of newborn rat.

PubMed

Carricondo, Francisco; Iglesias, Mari Cruz; Rodríguez, Fernando; Poch-Broto, Joaquin; Gil-Loyzaga, Pablo

2010-10-01

The adult mammalian auditory receptor lacks any ability to repair and/or regenerate after injury. However, the late developing cochlea still contains some stem-cell-like elements that might be used to regenerate damaged neurons and/or cells of the organ of Corti. Before their use in any application, stem cell numbers need to be amplified because they are usually rare in late developing and adult tissues. The numerous re-explant cultures required for the progressive amplification process can result in a spontaneous differentiation process. This aspect has been implicated in the tumorigenicity of stem cells when transplanted into a tissue. The aim of this study has been to determine whether cochlear stem cells can proliferate and differentiate spontaneously in long-term cultures without the addition of any factor that might influence these processes. Cochlear stem cells, which express nestin protein, were cultured in monolayers and fed with DMEM containing 5% FBS. They quickly organized themselves into typical spheres exhibiting a high proliferation rate, self-renewal property, and differentiation ability. Secondary cultures of these stem cell spheres spontaneously differentiated into neuroectodermal-like cells. The expression of nestin, glial-fibrillary-acidic protein, vimentin, and neurofilaments was evaluated to identify early differentiation. Nestin expression appeared in primary and secondary cultures. Other markers were also identified in differentiating cells. Further research might demonstrate the spontaneous differentiation of cochlear stem cells and their teratogenic probability when they are used for transplantation.

The unregulated commercialization of stem cell treatments: a global perspective.

PubMed

Sipp, Douglas

2011-12-01

Research into the biological properties and clinical potential of stem cells has spurred strong public investment, industry development, media coverage, and patient interest in recent years. To date, however, few clinical applications of demonstrated safety and efficacy have been developed with the exception of uses of hematopoietic stem cells in the treatment of diseases of the blood and immune systems. This lack of an evidence basis notwithstanding, hundreds of companies and private clinics around the world now sell putative stem cell treatments for an enormously broad range of medical and quality-of-life conditions. This represents a major challenge for legitimate scientists working in the field, for authorities seeking to protect their constituencies, and for patients and consumers targeted by such companies' marketing strategies. In this review, I provide an overview of the global industry in pseudomedical stem cell treatments, with an investigation of claims in a single disease area (amyotrophic lateral sclerosis), and make recommendations for the introduction and enforcement of appropriate regulatory responses to this problem.

Comparative evaluation of the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin scaffold.

PubMed

Khurana, Rohit; Kudva, Praveen Bhasker; Husain, Syed Yawer

2017-01-01

The present study aims to comparatively evaluate the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin (PRF) scaffold. A total of 15 systemically healthy individuals between the age group of 15-25 years requiring third molar or orthodontic premolar extractions. Teeth were extracted atraumatically and transported to the laboratory. Stem cells were isolated from dental pulp and periodontal ligament. After attaining more than 90% confluency by the 7 th day, these cells were tested for their viability and characterization. Stem cells were also incubated with PRF and viability was assessed on the 7 th day. The mean number of cell for dental pulp stem cells (DPSCs) and periodontal ligament stem cell (PDLSC) was statistically insignificant ( P > 0.05). The mean live cell viability was compared between DPSC (98.07%) and PDLSC (98%). Both DPSC and PDLSC showed a high percentage of expression of CD73 markers, 30.40% and 29.80%, respectively. However, DPSCs and PDLSCs lacked expression of CD34 expressing only 3.47% and 3.53%, respectively. PRF membrane as a scaffold exhibited no cytotoxic effects on DPCS's or PDLSC's. The cell viability of cells cultured with PRF was statistically insignificant ( P > 0.05) when compared to the cells cultured with culture media. The study thus indicates that dental pulp and periodontal ligament are both rich sources of mesenchymal stem cells and can be successfully used for obtaining stem cells. PRF exhibits no cytotoxic effects on the cells and can be used in conjunction with dental stem cells.

Spiral Ganglion Stem Cells Can Be Propagated and Differentiated Into Neurons and Glia

PubMed Central

Zecha, Veronika; Wagenblast, Jens; Arnhold, Stefan; Edge, Albert S. B.; Stöver, Timo

2014-01-01

Abstract The spiral ganglion is an essential functional component of the peripheral auditory system. Most types of hearing loss are associated with spiral ganglion cell degeneration which is irreversible due to the inner ear's lack of regenerative capacity. Recent studies revealed the existence of stem cells in the postnatal spiral ganglion, which gives rise to the hope that these cells might be useful for regenerative inner ear therapies. Here, we provide an in-depth analysis of sphere-forming stem cells isolated from the spiral ganglion of postnatal mice. We show that spiral ganglion spheres have characteristics similar to neurospheres isolated from the brain. Importantly, spiral ganglion sphere cells maintain their major stem cell characteristics after repeated propagation, which enables the culture of spheres for an extended period of time. In this work, we also demonstrate that differentiated sphere-derived cell populations not only adopt the immunophenotype of mature spiral ganglion cells but also develop distinct ultrastructural features of neurons and glial cells. Thus, our work provides further evidence that self-renewing spiral ganglion stem cells might serve as a promising source for the regeneration of lost auditory neurons. PMID:24940560

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.

PubMed

Sviderskaya, Elena V; Easty, David J; Lawrence, Mark A; Sánchez, Daniel P; Negulyaev, Yuri A; Patel, Ricken H; Anand, Praveen; Korchev, Yuri E; Bennett, Dorothy C

2009-09-01

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells

PubMed Central

Pomeroy, Jordan E.; Nguyen, Hung X.; Hoffman, Brenton D.; Bursac, Nenad

2017-01-01

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells. PMID:28912894

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells

PubMed Central

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2012-01-01

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 plays an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that histone demethylase LSD1, a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development. PMID:22068596

Self-assembled dual-modality contrast agents for non-invasive stem cell tracking via near-infrared fluorescence and magnetic resonance imaging.

PubMed

Liu, Hong; Tan, Yan; Xie, Lisi; Yang, Lei; Zhao, Jing; Bai, Jingxuan; Huang, Ping; Zhan, Wugen; Wan, Qian; Zou, Chao; Han, Yali; Wang, Zhiyong

2016-09-15

Stem cells hold great promise for treating various diseases. However, one of the main drawbacks of stem cell therapy is the lack of non-invasive image-tracking technologies. Although magnetic resonance imaging (MRI) and near-infrared fluorescence (NIRF) imaging have been employed to analyse cellular and subcellular events via the assistance of contrast agents, the sensitivity and temporal resolution of MRI and the spatial resolution of NIRF are still shortcomings. In this study, superparamagnetic iron oxide nanocrystals and IR-780 dyes were co-encapsulated in stearic acid-modified polyethylenimine to form a dual-modality contrast agent with nano-size and positive charge. These resulting agents efficiently labelled stem cells and did not influence the cellular viability and differentiation. Moreover, the labelled cells showed the advantages of dual-modality imaging in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

The transcriptional landscape of hematopoietic stem cell ontogeny

PubMed Central

McKinney-Freeman, Shannon; Cahan, Patrick; Li, Hu; Lacadie, Scott A.; Huang, Hsuan-Ting; Curran, Matthew; Loewer, Sabine; Naveiras, Olaia; Kathrein, Katie L.; Konantz, Martina; Langdon, Erin M.; Lengerke, Claudia; Zon, Leonard I.; Collins, James J.; Daley, George Q.

2012-01-01

Transcriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSC purified from >2500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSC, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource (http://hsc.hms.harvard.edu) will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification. PMID:23122293

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Meng-Yu; Nestvold, Janne, E-mail: j.m.nestvold@medisin.uio.no; Rekdal, Øystein

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cellmore » marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.« less

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions.

PubMed

Guitart, Amelie V; Panagopoulou, Theano I; Villacreces, Arnaud; Vukovic, Milica; Sepulveda, Catarina; Allen, Lewis; Carter, Roderick N; van de Lagemaat, Louie N; Morgan, Marcos; Giles, Peter; Sas, Zuzanna; Gonzalez, Marta Vila; Lawson, Hannah; Paris, Jasmin; Edwards-Hicks, Joy; Schaak, Katrin; Subramani, Chithra; Gezer, Deniz; Armesilla-Diaz, Alejandro; Wills, Jimi; Easterbrook, Aaron; Coman, David; So, Chi Wai Eric; O'Carroll, Donal; Vernimmen, Douglas; Rodrigues, Neil P; Pollard, Patrick J; Morton, Nicholas M; Finch, Andrew; Kranc, Kamil R

2017-03-06

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1 / Hoxa9 -driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation. © 2017 Guitart et al.

Requirement of Zinc Transporter SLC39A7/ZIP7 for Dermal Development to Fine-Tune Endoplasmic Reticulum Function by Regulating Protein Disulfide Isomerase.

PubMed

Bin, Bum-Ho; Bhin, Jinhyuk; Seo, Juyeon; Kim, Se-Young; Lee, Eunyoung; Park, Kyuhee; Choi, Dong-Hwa; Takagishi, Teruhisa; Hara, Takafumi; Hwang, Daehee; Koseki, Haruhiko; Asada, Yoshinobu; Shimoda, Shinji; Mishima, Kenji; Fukada, Toshiyuki

2017-08-01

Skin is the first area that manifests zinc deficiency. However, the molecular mechanisms by which zinc homeostasis affects skin development remain largely unknown. Here, we show that zinc-regulation transporter-/iron-regulation transporter-like protein 7 (ZIP7) localized to the endoplasmic reticulum plays critical roles in connective tissue development. Mice lacking the Slc39a7/Zip7 gene in collagen 1-expressing tissue exhibited dermal dysplasia. Ablation of ZIP7 in mesenchymal stem cells inhibited cell proliferation thereby preventing proper dermis formation, indicating that ZIP7 is required for dermal development. We also found that mesenchymal stem cells lacking ZIP7 accumulated zinc in the endoplasmic reticulum, which triggered zinc-dependent aggregation and inhibition of protein disulfide isomerase, leading to endoplasmic reticulum dysfunction. These results suggest that ZIP7 is necessary for endoplasmic reticulum function in mesenchymal stem cells and, as such, is essential for dermal development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

New advances in stem cell research: practical implications for regenerative medicine.

PubMed

Ratajczak, Mariusz Z; Jadczyk, Tomasz; Pędziwiatr, Daniel; Wojakowski, Wojciech

2014-01-01

Regenerative medicine is searching for stem cells that can be safely and efficiently employed for regeneration of damaged solid organs (e.g., the heart, brain, or liver). Ideal for this purpose would be pluripotent stem cells, which, according to their definition, have broad potential to differentiate into all types of adult cells. For almost 20 years, there have been unsuccessful attempts to harness controversial embryonic stem cells (ESCs) isolated from embryos. Induced pluripotent stem cells (iPSCs), generated by genetic modification of adult somatic cells, are a more promising source. However, both iPSC and ESCs are associated with a risk of teratoma formation. At the same time, various types of more‑differentiated adult stem and progenitor cells derived from the bone marrow, umbilical cord blood, mobilized peripheral blood, or fat tissue are being employed in clinical trials to regenerate damaged solid organs. However, for most of these cells, there is a lack of convincing documentation for successful regeneration of the treated organs. Beneficial effects of those cells might be explained by paracrine effects of growth factors, cytokines, chemokines, bioactive lipids, and extracellular microvesicles, which are released from the cells and have trophic, antiapoptotic, and angiopoietic effects. Nevertheless, there is evidence that adult tissues harbor a promising population of very rare dormant stem cells with broad differentiation potential. In this review, we will discuss various potential sources of stem cells for regenerative medicine and the mechanisms that explain some of their beneficial effects as well as highlight the results of the first clinical trials.  

A Review of Human Pluripotent Stem Cell-Derived Cardiomyocytes for High-Throughput Drug Discovery, Cardiotoxicity Screening and Publication Standards

PubMed Central

Mordwinkin, Nicholas M.; Burridge, Paul W.; Wu, Joseph C.

2013-01-01

Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results, and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach. PMID:23229562

Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells.

PubMed

Pournasr, Behshad; Duncan, Stephen A

2017-11-01

Inborn errors of hepatic metabolism are because of deficiencies commonly within a single enzyme as a consequence of heritable mutations in the genome. Individually such diseases are rare, but collectively they are common. Advances in genome-wide association studies and DNA sequencing have helped researchers identify the underlying genetic basis of such diseases. Unfortunately, cellular and animal models that accurately recapitulate these inborn errors of hepatic metabolism in the laboratory have been lacking. Recently, investigators have exploited molecular techniques to generate induced pluripotent stem cells from patients' somatic cells. Induced pluripotent stem cells can differentiate into a wide variety of cell types, including hepatocytes, thereby offering an innovative approach to unravel the mechanisms underlying inborn errors of hepatic metabolism. Moreover, such cell models could potentially provide a platform for the discovery of therapeutics. In this mini-review, we present a brief overview of the state-of-the-art in using pluripotent stem cells for such studies. © 2017 American Heart Association, Inc.

Impact of autologous and allogeneic stem cell transplantation in peripheral T-cell lymphomas.

PubMed

Reimer, Peter

2010-01-01

Peripheral T/NK-cell lymphomas (PTCLs) are rare malignancies characterized by poor prognosis. So far, no standard therapy has been established, due to the lack of randomised studies. High-dose therapy and autologous stem cell transplantation (HDT-autoSCT) have shown good feasibility with low toxicity in retrospective studies. In relapsing and refractory PTCL several comparison analyses suggest similar efficacy for PTCL when compared with aggressive B-cell lymphoma. In the upfront setting, prospective data show promising results with a long-lasting overall survival in a relevant subset of patients. Achieving a complete remission at transplantation seems to be the most important prognostic factor. Allogeneic stem cell transplantation (alloSCT) has been investigated only as salvage treatment. Especially when using reduced intensity conditioning regimen, eligible patients seem to benefit from this approach. To define the role for upfront stem cell transplantation a randomised trial by the German High-Grade Non-Hodgkin Lymphoma Study Group comparing HDT-autoSCT and alloSCT will be initiated this year.

[Stem cell-based cardiac regeneration after myocardial infarction].

PubMed

Reinsch, M; Weinberger, F

2018-03-01

Myocardial infarction leads to an irreversible loss of vital myocardial cells. The transplantation of new cardiomyocytes into the heart was first described over 20 years ago and represents a straightforward approach to remuscularize a damaged heart. Due to the lack of human cells a clinical application seemed ambitious; however, dramatic progress in stem cell biology over the last two decades has paved the way towards a clinical application. This is especially important as the prognosis for patients with terminal heart failure is still poor. The transplantation of either cardiomyocytes or engineered heart tissue derived from pluripotent stem cells (either embryonic stem cells or induced pluripotent stem cells) might represent a new regenerative approach. Transplantation of either cells or tissue constructs has now been evaluated in several preclinical models, which have demonstrated that an injured heart can be (partially) remuscularized; however, major hurdles towards a clinical application are the transplantation-related occurrence of arrhythmia, the potential tumorigenicity of pluripotent cells and the required immunosuppression. Several groups are working hard to solve these problems and we are optimistic that the first clinical studies will take place within the next few years.

Prospects of Pluripotent and Adult Stem Cells for Rare Diseases.

PubMed

García-Castro, Javier; Singeç, Ilyas

2017-01-01

Rare diseases are highly diverse and complex regarding molecular underpinning and clinical manifestation and afflict millions of patients worldwide. The lack of appropriate model systems with face and construct validity and the limited availability of live tissues and cells from patients has largely hampered the understanding of underlying disease mechanisms. As a consequence, there are no adequate treatment options available for the vast majority of rare diseases. Over the last decade, remarkable progress in pluripotent and adult stem cell biology and the advent of powerful genomic technologies opened up exciting new avenues for the investigation, diagnosis, and personalized therapy of intractable human diseases. Utilizing the entire range of available stem cell types will continue to cross-fertilize different research areas and leverage the investigation of rare diseases based on evidence-based medicine. Standardized cell engineering and manufacturing from inexhaustible stem cell sources should lay the foundation for next-generation drug discovery and cell therapies that are broadly applicable in regenerative medicine. In this chapter we discuss how patient- and disease-specific iPS cells as well as adult stem cells are changing the pace of biomedical research and the translational landscape.

Clinical and molecular insights into adenoid cystic carcinoma: Neural crest‐like stemness as a target

PubMed Central

Panaccione, Alexander; Chang, Michael T.; Ivanov, Sergey V.

2016-01-01

Objectives This review surveys trialed therapies and molecular defects in adenoid cystic carcinoma (ACC), with an emphasis on neural crest‐like stemness characteristics of newly discovered cancer stem cells (CSCs) and therapies that may target these CSCs. Data Sources Articles available on Pubmed or OVID MEDLINE databases and unpublished data. Review Methods Systematic review of articles pertaining to ACC and neural crest‐like stem cells. Results Adenoid cystic carcinoma of the salivary gland is a slowly growing but relentless cancer that is prone to nerve invasion and metastases. A lack of understanding of molecular etiology and absence of targetable drivers has limited therapy for patients with ACC to surgery and radiation. Currently, no curative treatments are available for patients with metastatic disease, which highlights the need for effective new therapies. Research in this area has been inhibited by the lack of validated cell lines and a paucity of clinically useful markers. The ACC research environment has recently improved, thanks to the introduction of novel tools, technologies, approaches, and models. Improved understanding of ACC suggests that neural crest‐like stemness is a major target in this rare tumor. New cell culture techniques and patient‐derived xenografts provide tools for preclinical testing. Conclusion Preclinical research has not identified effective targets in ACC, as confirmed by the large number of failed clinical trials. New molecular data suggest that drivers of neural crest‐like stemness may be required for maintenance of ACC; as such, CSCs are a target for therapy of ACC. PMID:28894804

Mammalian Gcm genes induce Hes5 expression by active DNA demethylation and induce neural stem cells.

PubMed

Hitoshi, Seiji; Ishino, Yugo; Kumar, Akhilesh; Jasmine, Salma; Tanaka, Kenji F; Kondo, Takeshi; Kato, Shigeaki; Hosoya, Toshihiko; Hotta, Yoshiki; Ikenaka, Kazuhiro

2011-07-17

Signaling mediated by Notch receptors is crucial for the development of many organs and the maintenance of various stem cell populations. The activation of Notch signaling is first detectable by the expression of an effector gene, Hes5, in the neuroepithelium of mouse embryos at embryonic day (E) 8.0-8.5, and this activation is indispensable for the generation of neural stem cells. However, the molecular mechanism by which Hes5 expression is initiated in stem-producing cells remains unknown. We found that mammalian Gcm1 and Gcm2 (glial cells missing 1 and 2) are involved in the epigenetic regulation of Hes5 transcription by DNA demethylation independently of DNA replication. Loss of both Gcm genes and subsequent lack of Hes5 upregulation in the neuroepithelium of E7.5-8.5 Gcm1(-/-); Gcm2(-/-) mice resulted in the impaired induction of neural stem cells. Our data suggest that Hes5 expression is serially activated first by Gcms and later by the canonical Notch pathway.

Craniopharyngiomas express embryonic stem cell markers (SOX2, OCT4, KLF4, and SOX9) as pituitary stem cells but do not coexpress RET/GFRA3 receptors.

PubMed

Garcia-Lavandeira, Montserrat; Saez, Carmen; Diaz-Rodriguez, Esther; Perez-Romero, Sihara; Senra, Ana; Dieguez, Carlos; Japon, Miguel A; Alvarez, Clara V

2012-01-01

Adult stem cells maintain some markers expressed by embryonic stem cells and express other specific markers depending on the organ where they reside. Recently, stem/progenitor cells in the rodent and human pituitary have been characterized as expressing GFRA2/RET, PROP1, and stem cell markers such as SOX2 and OCT4 (GPS cells). Our objective was to detect other specific markers of the pituitary stem cells and to investigate whether craniopharyngiomas (CRF), a tumor potentially derived from Rathke's pouch remnants, express similar markers as normal pituitary stem cells. We conducted mRNA and Western blot studies in pituitary extracts, and immunohistochemistry and immunofluorescence on sections from normal rat and human pituitaries and 20 CRF (18 adamantinomatous and two papillary). Normal pituitary GPS stem cells localized in the marginal zone (MZ) express three key embryonic stem cell markers, SOX2, OCT4, and KLF4, in addition to SOX9 and PROP1 and β-catenin overexpression. They express the RET receptor and its GFRA2 coreceptor but also express the coreceptor GFRA3 that could be detected in the MZ of paraffin pituitary sections. CRF maintain the expression of SOX2, OCT4, KLF4, SOX9, and β-catenin. However, RET and GFRA3 expression was altered in CRF. In 25% (five of 20), both RET and GFRA3 were detected but not colocalized in the same cells. The other 75% (15 of 20) lose the expression of RET, GFRA3, or both proteins simultaneously. Human pituitary adult stem/progenitor cells (GPS) located in the MZ are characterized by expression of embryonic stem cell markers SOX2, OCT4, and KLF4 plus the specific pituitary embryonic factor PROP1 and the RET system. Redundancy in RET coreceptor expression (GFRA2 and GFRA3) suggest an important systematic function in their physiological behavior. CRF share the stem cell markers suggesting a common origin with GPS. However, the lack of expression of the RET/GFRA system could be related to the cell mislocation and deregulated growth of CRF.

SCF increases in utero-labeled stem cells migration and improves wound healing.

PubMed

Zgheib, Carlos; Xu, Junwang; Mallette, Andrew C; Caskey, Robert C; Zhang, Liping; Hu, Junyi; Liechty, Kenneth W

2015-01-01

Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF-1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)-labeled skin stem cells via in utero intra-amniotic injection of lenti-RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin. © 2015 by the Wound Healing Society.

Stem Cell Therapies for Treating Diabetes: Progress and Remaining Challenges.

PubMed

Sneddon, Julie B; Tang, Qizhi; Stock, Peter; Bluestone, Jeffrey A; Roy, Shuvo; Desai, Tejal; Hebrok, Matthias

2018-06-01

Restoration of insulin independence and normoglycemia has been the overarching goal in diabetes research and therapy. While whole-organ and islet transplantation have become gold-standard procedures in achieving glucose control in diabetic patients, the profound lack of suitable donor tissues severely hampers the broad application of these therapies. Here, we describe current efforts aimed at generating a sustainable source of functional human stem cell-derived insulin-producing islet cells for cell transplantation and present state-of-the-art efforts to protect such cells via immune modulation and encapsulation strategies. Copyright © 2018. Published by Elsevier Inc.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells

PubMed Central

Sviderskaya, Elena V.; Easty, David J.; Lawrence, Mark A.; Sánchez, Daniel P.; Negulyaev, Yuri A.; Patel, Ricken H.; Anand, Praveen; Korchev, Yuri E.; Bennett, Dorothy C.

2009-01-01

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.—Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sánchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells. PMID:19447881

Human mesenchymal stem cells - current trends and future prospective

PubMed Central

Ullah, Imran; Subbarao, Raghavendra Baregundi; Rho, Gyu Jin

2015-01-01

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials. PMID:25797907

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells.

PubMed

Sluch, Valentin M; Chamling, Xitiz; Liu, Melissa M; Berlinicke, Cynthia A; Cheng, Jie; Mitchell, Katherine L; Welsbie, Derek S; Zack, Donald J

2017-11-01

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972-1986. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Ablation of Coactivator Med1 Switches the Cell Fate of Dental Epithelia to That Generating Hair

PubMed Central

Nguyen, Thai; Sakai, Kiyoshi; He, Bing; Fong, Chak; Oda, Yuko

2014-01-01

Cell fates are determined by specific transcriptional programs. Here we provide evidence that the transcriptional coactivator, Mediator 1 (Med1), is essential for the cell fate determination of ectodermal epithelia. Conditional deletion of Med1 in vivo converted dental epithelia into epidermal epithelia, causing defects in enamel organ development while promoting hair formation in the incisors. We identified multiple processes by which hairs are generated in Med1 deficient incisors: 1) dental epithelial stem cells lacking Med 1 fail to commit to the dental lineage, 2) Sox2-expressing stem cells extend into the differentiation zone and remain multi-potent due to reduced Notch1 signaling, and 3) epidermal fate is induced by calcium as demonstrated in dental epithelial cell cultures. These results demonstrate that Med1 is a master regulator in adult stem cells to govern epithelial cell fate. PMID:24949995

Using the cost-effectiveness of allogeneic islet transplantation to inform induced pluripotent stem cell-derived β-cell therapy reimbursement.

PubMed

Archibald, Peter R T; Williams, David J

2015-11-01

In the present study a cost-effectiveness analysis of allogeneic islet transplantation was performed and the financial feasibility of a human induced pluripotent stem cell-derived β-cell therapy was explored. Previously published cost and health benefit data for islet transplantation were utilized to perform the cost-effectiveness and sensitivity analyses. It was determined that, over a 9-year time horizon, islet transplantation would become cost saving and 'dominate' the comparator. Over a 20-year time horizon, islet transplantation would incur significant cost savings over the comparator (GB£59,000). Finally, assuming a similar cost of goods to islet transplantation and a lack of requirement for immunosuppression, a human induced pluripotent stem cell-derived β-cell therapy would dominate the comparator over an 8-year time horizon.

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7.

PubMed

Jung, Peter; Sommer, Christian; Barriga, Francisco M; Buczacki, Simon J; Hernando-Momblona, Xavier; Sevillano, Marta; Duran-Frigola, Miquel; Aloy, Patrick; Selbach, Matthias; Winton, Douglas J; Batlle, Eduard

2015-12-08

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs). Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Do ray cells provide a pathway for radial water movement in the stems of conifer trees?

Treesearch

David M. Barnard; Barbara Lachenbruch; Katherine A. McCulloh; Peter Kitin; Frederick C. Meinzer

2013-01-01

The pathway of radial water movement in tree stems presents an unknown with respect to whole-tree hydraulics. Radial profiles have shown substantial axial sap flow in deeper layers of sapwood (that may lack direct connection to transpiring leaves), which suggests the existence of a radial pathway for water movement. Rays in tree stems include ray tracheids and/or ray...

“Just one animal among many?” Existential phenomenology, ethics, and stem cell research

PubMed Central

2010-01-01

Stem cell research and associated or derivative biotechnologies are proceeding at a pace that has left bioethics behind as a discipline that is more or less reactionary to their developments. Further, much of the available ethical deliberation remains determined by the conceptual framework of late modern metaphysics and the correlative ethical theories of utilitarianism and deontology. Lacking, to any meaningful extent, is a sustained engagement with ontological and epistemological critiques, such as with “postmodern” thinking like that of Heidegger’s existential phenomenology. Some basic “Heideggerian” conceptual strategies are reviewed here as a way of remedying this deficiency and adding to ethical deliberation about current stem cell research practices. PMID:20521117

Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.

PubMed

Liu, Chang Ching; Ma, Dong Liang; Yan, Ting-Dong; Fan, XiuBo; Poon, Zhiyong; Poon, Lai-Fong; Goh, Su-Ann; Rozen, Steve G; Hwang, William Ying Khee; Tergaonkar, Vinay; Tan, Patrick; Ghosh, Sujoy; Virshup, David M; Goh, Eyleen L K; Li, Shang

2016-10-01

In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484. © 2016 AlphaMed Press.

Stem cells for regenerative medicine: advances in the engineering of tissues and organs

NASA Astrophysics Data System (ADS)

Ringe, Jochen; Kaps, Christian; Burmester, Gerd-Rüdiger; Sittinger, Michael

2002-07-01

The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as mesenchymal stem or mesenchymal progenitor cells (MSC). These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, MSC have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma, which suggest these cells as an attractive cell source for tissue engineering approaches. The interest in modern biological technologies such as tissue engineering has dramatically increased since it is feasible to isolate living, healthy cells from the body, expand them under cell culture conditions, combine them with biocompatible carrier materials and retransplant them into patients. Therefore, tissue engineering gives the opportunity to generate living substitutes for tissues and organs, which may overcome the drawbacks of classical tissue reconstruction: lacking quality and quantity of autologous grafts, immunogenicity of allogenic grafts and loosening of alloplastic implants. Due to the prerequisite for tissue engineering to ensure a sufficient number of tissue specific cells without donor site morbidity, much attention has been drawn to multipotential progenitor cells such as embryonic stem cells, periosteal cells and mesenchymal stem cells. In this report we review the state of the art in tissue engineering with mesenchymal stem and mesenchymal progenitor cells with emphasis on bone and cartilage reconstruction. Furthermore, several issues of importance, especially with regard to the clinical application of mesenchymal stem cells, are discussed.

Directing stem cell trafficking via GPS.

PubMed

Sackstein, Robert

2010-01-01

The success of stem-cell-based regenerative therapeutics critically hinges on delivering relevant stem/progenitor cells to sites of tissue injury. To achieve adequate parenchymal infiltration following intravascular administration, it is first necessary that circulating cells bind to target tissue endothelium with sufficient strength to overcome the prevailing forces of hemodynamic shear. The principal mediators of these shear-resistant binding interactions consist of a family of C-type lectins known as "selectins" that bind discrete sialofucosylated glycans on their respective ligands. One member of this family, E-selectin, is an endothelial molecule that is inducibly expressed on postcapillary venules at all sites of tissue injury, but is also constitutively expressed on the luminal surface of bone marrow and dermal microvascular endothelium. Most stem/progenitor cells express high levels of CD44, and, in particular, human hematopoietic stem cells express a specialized sialofucosylated glycoform of CD44 known as "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a potent E-selectin ligand. This chapter describes a method called "glycosyltransferase-programmed stereosubstitution" (GPS) for custom-modifying CD44 glycans to create HCELL on the surface of living cells that natively lack HCELL. Ex vivo glycan engineering of HCELL via GPS licenses trafficking of infused cells to endothelial beds that express E-selectin, thereby enabling efficient vascular delivery of stem/progenitor cells to sites where they are needed. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle.

PubMed

Lapidos, Karen A; Chen, Yiyin E; Earley, Judy U; Heydemann, Ahlke; Huber, Jill M; Chien, Marcia; Ma, Averil; McNally, Elizabeth M

2004-12-01

Pluripotent bone marrow-derived side population (BM-SP) stem cells have been shown to repopulate the hematopoietic system and to contribute to skeletal and cardiac muscle regeneration after transplantation. We tested BM-SP cells for their ability to regenerate heart and skeletal muscle using a model of cardiomyopathy and muscular dystrophy that lacks delta-sarcoglycan. The absence of delta-sarcoglycan produces microinfarcts in heart and skeletal muscle that should recruit regenerative stem cells. Additionally, sarcoglycan expression after transplantation should mark successful stem cell maturation into cardiac and skeletal muscle lineages. BM-SP cells from normal male mice were transplanted into female delta-sarcoglycan-null mice. We detected engraftment of donor-derived stem cells into skeletal muscle, with the majority of donor-derived cells incorporated within myofibers. In the heart, donor-derived nuclei were detected inside cardiomyocytes. Skeletal muscle myofibers containing donor-derived nuclei generally failed to express sarcoglycan, with only 2 sarcoglycan-positive fibers detected in the quadriceps muscle from all 14 mice analyzed. Moreover, all cardiomyocytes with donor-derived nuclei were sarcoglycan-negative. The absence of sarcoglycan expression in cardiomyocytes and skeletal myofibers after transplantation indicates impaired differentiation and/or maturation of bone marrow-derived stem cells. The inability of BM-SP cells to express this protein severely limits their utility for cardiac and skeletal muscle regeneration.

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Curbing stem cell tourism in South Africa.

PubMed

Meissner-Roloff, Madelein; Pepper, Michael S

2013-12-01

Stem cells have received much attention globally due in part to the immense therapeutic potential they harbor. Unfortunately, malpractice and exploitation (financial and emotional) of vulnerable patients have also drawn attention to this field as a result of the detrimental consequences experienced by some individuals that have undergone unproven stem cell therapies. South Africa has had limited exposure to stem cells and their applications and, while any exploitation is detrimental to the field of stem cells, South Africa is particularly vulnerable in this regard. The current absence of adequate legislation and the inability to enforce existing legislation, coupled to the sea of misinformation available on the Internet could lead to an increase in illegitimate stem cell practices in South Africa. Circumstances are already precarious because of a lack of understanding of concepts involved in stem cell applications. What is more, credible and easily accessible information is not available to the public. This in turn cultivates fears born out of existing superstitions, cultural beliefs, rituals and practices. Certain cultural or religious concerns could potentially hinder the effective application of stem cell therapies in South Africa and novel ways of addressing these concerns are necessary. Understanding how scientific progress and its implementation will affect each individual and, consequently, the community, will be of cardinal importance to the success of the fields of stem cell therapy and regenerative medicine in South Africa. A failure to understand the ethical, cultural or moral ramifications when new scientific concepts are introduced could hinder the efficacy and speed of bringing discoveries to the patient. Neglecting proper procedure for establishing the field would lead to long delays in gaining public support in South Africa. Understanding the dangers of stem cell tourism - where vulnerable patients are subjected to unproven stem cell therapies that have not undergone peer review or been registered with the relevant local authorities - becomes imperative so that strategies to overcome this threat can be implemented.

miR-137 forms a regulatory loop with nuclear receptor TLX and LSD1 in neural stem cells.

PubMed

Sun, GuoQiang; Ye, Peng; Murai, Kiyohito; Lang, Ming-Fei; Li, Shengxiu; Zhang, Heying; Li, Wendong; Fu, Chelsea; Yin, Jason; Wang, Allen; Ma, Xiaoxiao; Shi, Yanhong

2011-11-08

miR-137 is a brain-enriched microRNA. Its role in neural development remains unknown. Here we show that miR-137 has an essential role in controlling embryonic neural stem cell fate determination. miR-137 negatively regulates cell proliferation and accelerates neural differentiation of embryonic neural stem cells. In addition, we show that the histone lysine-specific demethylase 1 (LSD1), a transcriptional co-repressor of nuclear receptor TLX, is a downstream target of miR-137. In utero electroporation of miR-137 in embryonic mouse brains led to premature differentiation and outward migration of the transfected cells. Introducing a LSD1 expression vector lacking the miR-137 recognition site rescued miR-137-induced precocious differentiation. Furthermore, we demonstrate that TLX, an essential regulator of neural stem cell self-renewal, represses the expression of miR-137 by recruiting LSD1 to the genomic regions of miR-137. Thus, miR-137 forms a feedback regulatory loop with TLX and LSD1 to control the dynamics between neural stem cell proliferation and differentiation during neural development.

Morphological description of limbal epithelium: searching for stem cells crypts in the dog, cat, pig, cow, sheep and horse.

PubMed

Patruno, M; Perazzi, A; Martinello, T; Blaseotto, A; Di Iorio, E; Iacopetti, I

2017-06-01

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.

Can civil lawsuits stem the tide of direct-to-consumer marketing of unproven stem cell interventions.

PubMed

Horner, Claire; Tenenbaum, Evelyn; Sipp, Douglas; Master, Zubin

2018-01-01

The sale of unproven stem cell interventions (SCIs) by commercial entities has proliferated in highly developed countries, most notably in the USA. Yet, there have been few criminal prosecutions and regulatory enforcement actions against providers who have violated laws and best practice standards due to the lack of resources and legal ambiguity. While the stem cell research community has invested much in protecting patients and preventing the growth of this industry, some patients are seeking remedies under civil law to hold stem cell clinics responsible for fraudulent practices. Several patients have filed lawsuits against providers demanding compensation for physical injuries caused by unproven treatments and financial losses due to fraud and false advertising. Lawsuits can be used as a tool not only to compensate plaintiffs but also to achieve positive public health and policy outcomes. In this paper, we explore the value of a public health litigation strategy as a countermeasure against the exploitative practices of the unproven SCI industry by analyzing stem cell lawsuits and comparing them with other major public health litigation efforts. We argue that stem cell lawsuits complement other approaches to reining in unsafe practices. In particular, stem cell lawsuits could intensify publicity and raise awareness of the harms of unproven treatments, set legal precedent, reshape the media narrative from one focused on the right to try or practice to one highlighting the need for adequate safety and efficacy standards, and encourage authorities to turn their attention to policy reform and enforcement.

Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue

PubMed Central

Yee, Karen K.; Li, Yan; Redding, Kevin M.; Iwatsuki, Ken; Margolskee, Robert F.; Jiang, Peihua

2013-01-01

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knock-in allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of circumvallate and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. PMID:23377989

Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

PubMed

Yee, Karen K; Li, Yan; Redding, Kevin M; Iwatsuki, Ken; Margolskee, Robert F; Jiang, Peihua

2013-05-01

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. Copyright © 2013 AlphaMed Press.

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors.

PubMed

Sambi, Manpreet; Chow, Theresa; Whiteley, Jennifer; Li, Mira; Chua, Shawn; Raileanu, Vanessa; Rogers, Ian M

2017-08-01

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Comparative evaluation of the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin scaffold

PubMed Central

Khurana, Rohit; Kudva, Praveen Bhasker; Husain, Syed Yawer

2017-01-01

Background: The present study aims to comparatively evaluate the isolation and quantification of stem cells derived from dental pulp and periodontal ligament of a permanent tooth and to assess their viability and proliferation on a platelet-rich fibrin (PRF) scaffold. Materials and Methods: A total of 15 systemically healthy individuals between the age group of 15–25 years requiring third molar or orthodontic premolar extractions. Teeth were extracted atraumatically and transported to the laboratory. Stem cells were isolated from dental pulp and periodontal ligament. After attaining more than 90% confluency by the 7th day, these cells were tested for their viability and characterization. Stem cells were also incubated with PRF and viability was assessed on the 7th day. Results: The mean number of cell for dental pulp stem cells (DPSCs) and periodontal ligament stem cell (PDLSC) was statistically insignificant (P > 0.05). The mean live cell viability was compared between DPSC (98.07%) and PDLSC (98%). Both DPSC and PDLSC showed a high percentage of expression of CD73 markers, 30.40% and 29.80%, respectively. However, DPSCs and PDLSCs lacked expression of CD34 expressing only 3.47% and 3.53%, respectively. PRF membrane as a scaffold exhibited no cytotoxic effects on DPCS's or PDLSC's. The cell viability of cells cultured with PRF was statistically insignificant (P > 0.05) when compared to the cells cultured with culture media. Conclusion: The study thus indicates that dental pulp and periodontal ligament are both rich sources of mesenchymal stem cells and can be successfully used for obtaining stem cells. PRF exhibits no cytotoxic effects on the cells and can be used in conjunction with dental stem cells. PMID:29386795

Sino-Canadian collaborations in stem cell research: a scientometric analysis.

PubMed

Ali-Khan, Sarah E; Ray, Monali; McMahon, Dominique S; Thorsteinsdóttir, Halla

2013-01-01

International collaboration (IC) is essential for the advance of stem cell research, a field characterized by marked asymmetries in knowledge and capacity between nations. China is emerging as a global leader in the stem cell field. However, knowledge on the extent and characteristics of IC in stem cell science, particularly China's collaboration with developed economies, is lacking. We provide a scientometric analysis of the China-Canada collaboration in stem cell research, placing this in the context of other leading producers in the field. We analyze stem cell research published from 2006 to 2010 from the Scopus database, using co-authored papers as a proxy for collaboration. We examine IC levels, collaboration preferences, scientific impact, the collaborating institutions in China and Canada, areas of mutual interest, and funding sources. Our analysis shows rapid global expansion of the field with 48% increase in papers from 2006 to 2010. China now ranks second globally after the United States. China has the lowest IC rate of countries examined, while Canada has one of the highest. China-Canada collaboration is rising steadily, more than doubling during 2006-2010. China-Canada collaboration enhances impact compared to papers authored solely by China-based researchers This difference remained significant even when comparing only papers published in English. While China is increasingly courted in IC by developed countries as a partner in stem cell research, it is clear that it has reached its status in the field largely through domestic publications. Nevertheless, IC enhances the impact of stem cell research in China, and in the field in general. This study establishes an objective baseline for comparison with future studies, setting the stage for in-depth exploration of the dynamics and genesis of IC in stem cell research.

Sino-Canadian Collaborations in Stem Cell Research: A Scientometric Analysis

PubMed Central

Ali-Khan, Sarah E.; Ray, Monali; McMahon, Dominique S.; Thorsteinsdóttir, Halla

2013-01-01

Background International collaboration (IC) is essential for the advance of stem cell research, a field characterized by marked asymmetries in knowledge and capacity between nations. China is emerging as a global leader in the stem cell field. However, knowledge on the extent and characteristics of IC in stem cell science, particularly China’s collaboration with developed economies, is lacking. Methods and Findings We provide a scientometric analysis of the China–Canada collaboration in stem cell research, placing this in the context of other leading producers in the field. We analyze stem cell research published from 2006 to 2010 from the Scopus database, using co-authored papers as a proxy for collaboration. We examine IC levels, collaboration preferences, scientific impact, the collaborating institutions in China and Canada, areas of mutual interest, and funding sources. Our analysis shows rapid global expansion of the field with 48% increase in papers from 2006 to 2010. China now ranks second globally after the United States. China has the lowest IC rate of countries examined, while Canada has one of the highest. China–Canada collaboration is rising steadily, more than doubling during 2006–2010. China–Canada collaboration enhances impact compared to papers authored solely by China-based researchers This difference remained significant even when comparing only papers published in English. Conclusions While China is increasingly courted in IC by developed countries as a partner in stem cell research, it is clear that it has reached its status in the field largely through domestic publications. Nevertheless, IC enhances the impact of stem cell research in China, and in the field in general. This study establishes an objective baseline for comparison with future studies, setting the stage for in-depth exploration of the dynamics and genesis of IC in stem cell research. PMID:23468927

Metformin selectively affects human glioblastoma tumor-initiating cell viability

PubMed Central

Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirana; Corsaro, Alessandro; Parodi, Alessia; Sirito, Rodolfo; Massollo, Michela; Marini, Cecilia; Zona, Gianluigi; Fenoglio, Daniela; Sambuceti, Gianmario; Filaci, Gilberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2013-01-01

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect. PMID:23255107

Induced pluripotent stem (iPS) cells: a new source for cell-based therapeutics?

PubMed

de Lázaro, Irene; Yilmazer, Açelya; Kostarelos, Kostas

2014-07-10

The generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of defined transcription factors has provided the regenerative medicine field with a new tool for cell replacement strategies. The advantages that these pluripotent cells can offer in comparison to other sources of stem cells include the generation of patient-derived cells and the lack of embryonic tissue while maintaining a versatile differentiation potential. The promise of iPS cell derivatives for therapeutic applications is encouraging albeit very early in development, with the first clinical study currently ongoing in Japan. Many challenges are yet to be circumvented before this technology can be clinically translated widely though. The delivery and expression of the reprogramming factors, the genomic instability, epigenetic memory and impact of cell propagation in culture are only some of the concerns. This article aims to critically discuss the potential of iPS cells as a new source of cell therapeutics. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell signaling pathways in the adrenal cortex: Links to stem/progenitor biology and neoplasia.

PubMed

Penny, Morgan K; Finco, Isabella; Hammer, Gary D

2017-04-15

The adrenal cortex is a dynamic tissue responsible for the synthesis of steroid hormones, including mineralocorticoids, glucocorticoids, and androgens in humans. Advances have been made in understanding the role of adrenocortical stem/progenitor cell populations in cortex homeostasis and self-renewal. Recently, large molecular profiling studies of adrenocortical carcinoma (ACC) have given insights into proteins and signaling pathways involved in normal tissue homeostasis that become dysregulated in cancer. These data provide an impetus to examine the cellular pathways implicated in adrenocortical disease and study connections, or lack thereof, between adrenal homeostasis and tumorigenesis, with a particular focus on stem and progenitor cell pathways. In this review, we discuss evidence for stem/progenitor cells in the adrenal cortex, proteins and signaling pathways that may regulate these cells, and the role these proteins play in pathologic and neoplastic conditions. In turn, we also examine common perturbations in adrenocortical tumors (ACT) and how these proteins and pathways may be involved in adrenal homeostasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fanconi anemia genes are highly expressed in primitive CD34+ hematopoietic cells

PubMed Central

Aubé, Michel; Lafrance, Matthieu; Brodeur, Isabelle; Delisle, Marie-Chantal; Carreau, Madeleine

2003-01-01

Background Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. Methods Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. Results We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34- stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. Conclusions These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34+ cells correlates with a role at the CD34+ differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism. PMID:12809565

Applications of Raman micro-spectroscopy to stem cell technology: label-free molecular discrimination and monitoring cell differentiation.

PubMed

Ghita, Adrian; Pascut, Flavius C; Sottile, Virginie; Denning, Chris; Notingher, Ioan

Stem cell therapy is widely acknowledged as a key medical technology of the 21st century which may provide treatments for many currently incurable diseases. These cells have an enormous potential for cell replacement therapies to cure diseases such as Parkinson's disease, diabetes and cardiovascular disorders, as well as in tissue engineering as a reliable cell source for providing grafts to replace and repair diseased tissues. Nevertheless, the progress in this field has been difficult in part because of lack of techniques that can measure non-invasively the molecular properties of cells. Such repeated measurements can be used to evaluate the culture conditions during differentiation, cell quality and phenotype heterogeneity of stem cell progeny. Raman spectroscopy is an optical technique based on inelastic scattering of laser photons by molecular vibrations of cellular molecules and can be used to provide chemical fingerprints of cells or organelles without fixation, lysis or use of labels and other contrast enhancing chemicals. Because differentiated cells are specialized to perform specific functions, these cells produce specific biochemicals that can be detected by Raman micro-spectroscopy. This mini-review paper describes applications of Raman micro-scpectroscopy to measure moleculare properties of stem cells during differentiation in-vitro. The paper focuses on time- and spatially-resolved Raman spectral measurements that allow repeated investigation of live stem cells in-vitro.

Loss of β-catenin triggers oxidative stress and impairs hematopoietic regeneration

PubMed Central

Lento, William; Ito, Takahiro; Zhao, Chen; Harris, Jeffrey R.; Huang, Wei; Jiang, Chen; Owzar, Kouros; Piryani, Sadhna; Racioppi, Luigi; Chao, Nelson; Reya, Tannishtha

2014-01-01

Accidental or deliberate ionizing radiation exposure can be fatal due to widespread hematopoietic destruction. However, little is known about either the course of injury or the molecular pathways that regulate the subsequent regenerative response. Here we show that the Wnt signaling pathway is critically important for regeneration after radiation-induced injury. Using Wnt reporter mice, we show that radiation triggers activation of Wnt signaling in hematopoietic stem and progenitor cells. β-Catenin-deficient mice, which lack the ability to activate canonical Wnt signaling, exhibited impaired hematopoietic stem cell regeneration and bone marrow recovery after radiation. We found that, as part of the mechanism, hematopoietic stem cells lacking β-catenin fail to suppress the generation of reactive oxygen species and cannot resolve DNA double-strand breaks after radiation. Consistent with the impaired response to radiation, β-catenin-deficient mice are also unable to recover effectively after chemotherapy. Collectively, these data indicate that regenerative responses to distinct hematopoietic injuries share a genetic dependence on β-catenin and raise the possibility that modulation of Wnt signaling may be a path to improving bone marrow recovery after damage. PMID:24788518

[The embryonic stem cells research. Example of biotechnology progress under extra-scientific pressure].

PubMed

Gámez Escalona, José Antonio

2013-01-01

The possibility to isolate, cultivate, preserve, characterize and differentiate Human Embryonic Stem Cells (ES) discovered by James Thomson and his colleagues in 1998 was a milestone in the history of Stem Cell Research. Immediately after this discovery many speculations were made about the therapeutic possibilities of ES, motivated by ideological, political and economic aspects. The episode made clear the lack of scientific rationality and ethics when assessing realities as meaningful as those of human embryos obtained by in vitro fertilization techniques (IVF) or human eggs. Therapeutic Cloning as a promise to produce ″tailored″ Stem Cells reported by Hwang and his team in 2004, ended up being a scandal within the scientific community. The technical difficulties and ethical controversies that arose from obtaining ES were insurmountable. In 2010 only two clinical trials were reported using these cells. Those trials were abandoned in late 2011 arguing financial reasons. The discovery of Induced Pluripotent Stem Cell (iPS) in 2006 in mice and in 2007 in humans, represented the possibility of obtaining pluripotent stem cells without the need to destroy embryos. Today, the absence of clinical trials using ES, caused by financial difficulties as a result of its ineffectiveness, anticipates that the use of ES will be limited to certain experimental controls. Probably, the main contribution of Embryonic Stem Cells will be the understanding that biomedical research should follow an ethically and rationally based rigorous method that cannot be ignore.

Development of autologous blood cell therapies

PubMed Central

Kim, Ah Ram; Sankaran, Vijay G.

2016-01-01

Allogeneic hematopoietic stem cell transplantation and blood cell transfusions are commonly performed in patients with a variety of blood disorders. Unfortunately, these donor-derived cell therapies are constrained due to limited supplies, infectious risk factors, a lack of appropriately matched donors, and the risk of immunologic complications from such products. The use of autologous cell therapies has been proposed to overcome these shortcomings. One can derive such therapies directly from hematopoietic stem and progenitor cells of individuals, which can then be manipulated ex vivo to produce desired modifications or differentiated to produce a particular target population. Alternatively, pluripotent stem cells, which have a theoretically unlimited self-renewal capacity and an ability to differentiate into any desired cell type, can be used as an autologous starting source for such manipulation and differentiation approaches. In addition, such cell products can also be used as a delivery vehicle for therapeutics. In this review, we highlight recent advances and discuss ongoing challenges for the in vitro generation of autologous hematopoietic cells that can be used for cell therapy. PMID:27345108

Efficacy of periodontal stem cell transplantation in the treatment of advanced periodontitis.

PubMed

Park, Joo-Young; Jeon, Soung Hoo; Choung, Pill-Hoon

2011-01-01

Periodontitis is the most common cause for tooth loss in adults and advanced types affect 10-15% of adults worldwide. The attempts to save tooth and regenerate the periodontal apparatus including cementum, periodontal ligament, and alveolar bone reach to the dental tissue-derived stem cell therapy. Although there have been several periodontitis models suggested, the apical involvement of tooth root is especially challenging to be regenerated and dental stem cell therapy for the state has never been investigated. Three kinds of dental tissue-derived adult stem cells (aDSCs) were obtained from the extracted immature molars of beagle dogs (n = 8), and ex vivo expanded periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and periapical follicular stem cells (PAFSCs) were transplanted into the apical involvement defect. As for the lack of cementum-specific markers, anti-human cementum protein 1 (rhCEMP1) antibody was fabricated and the aDSCs and the regenerated tissues were immunostained with anti-CEMP1 antibody. Autologous PDLSCs showed the best regenerating capacity of periodontal ligament, alveolar bone, and cementum as well as peripheral nerve and blood vessel, which were evaluated by conventional and immune histology, 3D micro-CT, and clinical index. The rhCEMP1 was expressed strongest in PDLSCs and in the regenerated periodontal ligament space. We suggest here the PDLSCs as the most favorable candidate for the clinical application among the three dental stem cells and can be used for treatment of advanced periodontitis where tooth removal was indicated in the clinical cases. © 2011 Cognizant Comm. Corp.

In utero hematopoietic stem cell transfer: current status and future strategies.

PubMed

Surbek, D V; Gratwohl, A; Holzgreve, W

1999-07-01

Successful prenatal treatment of severe immunodeficiencies by allogeneic hematopoietic stem cell transplantation in utero has been reported. Though other diseases like hemoglobinopathies or storage diseases are potentially amenable to this novel therapeutic approach, no success has yet been achieved in recipients without severe immunodeficiency. Graft rejection by the developing fetus and/or lack of selective, competitive advantage of donor versus host stem cells preventing stable engraftment seem to be the major obstacles. Several strategies to overcome these hurdles are being explored in preclinical settings, including timing and repeated dosing of stem cell administration to the fetus, ex vivo modification of the transplant, using different fetal compartments as targets for early stem cell transfer, or inducing microchimerism for postnatal transplantation from the same donor. In addition, the exact definition of the basic concept of early fetal immunologic naivete and the understanding of the molecular basics of migration and homing in fetal hematopoiesis system seem mandatory for a successful approach. Gene therapy using ex vivo transduced autologous cord blood cells or direct gene targeting in utero are other potential means to correct hematopoietic and immunologic single gene disorders in utero, though this approach is still away from the stage of clinical trials.

Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

PubMed

Sun, Zhao; Yu, Wenjie; Sanz Navarro, Maria; Sweat, Mason; Eliason, Steven; Sharp, Thad; Liu, Huan; Seidel, Kerstin; Zhang, Li; Moreno, Myriam; Lynch, Thomas; Holton, Nathan E; Rogers, Laura; Neff, Traci; Goodheart, Michael J; Michon, Frederic; Klein, Ophir D; Chai, Yang; Dupuy, Adam; Engelhardt, John F; Chen, Zhi; Amendt, Brad A

2016-11-15

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development. © 2016. Published by The Company of Biologists Ltd.

Adipose-Derived Stem Cells in Aesthetic Surgery: A Mixed Methods Evaluation of the Current Clinical Trial, Intellectual Property, and Regulatory Landscape.

PubMed

Arshad, Zeeshaan; Halioua-Haubold, Celine-Lea; Roberts, Mackenna; Urso-Baiarda, Fulvio; Branford, Oliver A; Brindley, David A; Davies, Benjamin M; Pettitt, David

2018-02-17

Adipose tissue, which can be readily harvested via a number of liposuction techniques, offers an easily accessible and abundant source of adipose-derived stem cells (ASCs). Consequently, ASCs have become an increasingly popular reconstructive option and a novel means of aesthetic soft tissue augmentation. This paper examines recent advances in the aesthetic surgery field, extending beyond traditional review formats to incorporate a comprehensive analysis of current clinical trials, adoption status, and the commercialization pathway. Keyword searches were carried out on clinical trial databases to search for trials using ASCs for aesthetic indications. An intellectual property landscape was created using commercial software (Thomson Reuters Thomson Innovation, New York, NY). Analysis of who is claiming what in respect of ASC use in aesthetic surgery for commercial purposes was analyzed by reviewing the patent landscape in relation to these techniques. Key international regulatory guidelines were also summarized. Completed clinical trials lacked robust controls, employed small sample sizes, and lacked long-term follow-up data. Ongoing clinical trials still do not address such issues. In recent years, claims to intellectual property ownership have increased in the "aesthetic stem cell" domain, reflecting commercial interest in the area. However, significant translational barriers remain including regulatory challenges and ethical considerations. Further rigorous randomized controlled trials are required to delineate long-term clinical efficacy and safety. Providers should consider the introduction of patient reported outcome metrics to facilitate clinical adoption. Robust regulatory and ethical policies concerning stem cells and aesthetic surgery should be devised to discourage further growth of "stem cell tourism." © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

Osteoarthritis and stem cell therapy in humans: a systematic review.

PubMed

Jevotovsky, D S; Alfonso, A R; Einhorn, T A; Chiu, E S

2018-06-01

Osteoarthritis (OA) is a leading cause of disability in the world. Mesenchymal stem cells (MSCs) have been studied to treat OA. This review was performed to systematically assess the quality of literature and compare the procedural specifics surrounding MSC therapy for osteoarthritis. PubMed, CINAHL, EMBASE and Cochrane Central Register of Controlled Trials were searched for studies using MSCs for OA treatment (final search December 2017). Outcomes of interest included study evidence level, patient demographics, MSC protocol, treatment results and adverse events. Level I and II evidence articles were further analyzed. Sixty-one of 3,172 articles were identified. These studies treated 2,390 patients with osteoarthritis. Most used adipose-derived stem cells (ADSCs) (n = 29) or bone marrow-derived stem cells (BMSCs) (n = 30) though the preparation varied within group. 57% of the sixty-one studies were level IV evidence, leaving five level I and nine level II studies containing 288 patients to be further analyzed. Eight studies used BMSCs, five ADSCs and one peripheral blood stem cells (PBSCs). The risk of bias in these studies showed five level I studies at low risk with seven level II at moderate and two at high risk. While studies support the notion that MSC therapy has a positive effect on OA patients, there is limited high quality evidence and long-term follow-up. The present study summarizes the specifics of high level evidence studies and identifies a lack of consistency, including a diversity of MSC preparations, and thus a lack of reproducibility amongst these articles' methods. Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Long-Term Hematopoietic Engraftment of Congenic Amniotic Fluid Stem Cells After in Utero Intraperitoneal Transplantation to Immune Competent Mice

PubMed Central

Shangaris, Panicos; Loukogeorgakis, Stavros P.; Blundell, Michael P.; Petra, Eleni; Shaw, Steven W.; Ramachandra, Durrgah L.; Maghsoudlou, Panagiotis; Urbani, Luca; Thrasher, Adrian J.

2018-01-01

Clinical success of in utero transplantation (IUT) using allogeneic hematopoietic stem cells (HSCs) has been limited to fetuses that lack an immune response to allogeneic cells due to severe immunological defects, and where transplanted genetically normal cells have a proliferative or survival advantage. Amniotic fluid (AF) is an autologous source of stem cells with hematopoietic potential that could be used to treat congenital blood disorders. We compared the ability of congenic and allogeneic mouse AF stem cells (AFSC) to engraft the hematopoietic system of time-mated C57BL/6J mice (E13.5). At 4 and 16 weeks of age, multilineage donor engraftment was higher in congenic versus allogeneic animals. In vitro mixed lymphocyte reaction confirmed an immune response in the allogeneic group with higher CD4 and CD8 cell counts and increased proliferation of stimulated lymphocytes. IUT with congenic cells resulted in 100% of donor animals having chimerism of around 8% and successful hematopoietic long-term engraftment in immune-competent mice when compared with IUT with allogeneic cells. AFSCs may be useful for autologous cell/gene therapy approaches in fetuses diagnosed with congenital hematopoietic disorders. PMID:29482456

Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls.

PubMed

MacMillan, Colleen P; Birke, Hannah; Chuah, Aaron; Brill, Elizabeth; Tsuji, Yukiko; Ralph, John; Dennis, Elizabeth S; Llewellyn, Danny; Pettolino, Filomena A

2017-07-18

Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a 'typical' lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre. Transcriptome comparisons from cotton xylem and pith as well as from a developmental series of seed fibres revealed tissue-specific and developmentally regulated expression of several NAC transcription factors some of which are likely to be important as top tier regulators of SCW formation in xylem and/or seed fibre. A so far undescribed hierarchy was identified between the top tier NAC transcription factors SND1-like and NST1/2 in cotton. Key SCW MYB transcription factors, homologs of Arabidopsis MYB46/83, were practically absent in cotton stem xylem. Lack of expression of other lignin-specific MYBs in seed fibre relative to xylem could account for the lack of lignin deposition in seed fibre. Expression of a MYB103 homolog correlated with temporal expression of SCW CesAs and cellulose synthesis in seed fibres. FLAs were highly expressed and may be important structural components of seed fibre SCWs. Finally, we made the unexpected observation that cell walls in the pith of cotton stems contained lignin and had a higher S:G ratio than in xylem, despite that tissue's lacking many of the gene transcripts normally associated with lignin biosynthesis. Our study in cotton confirmed some features of the currently accepted gene regulatory cascade for 'typical' plant SCWs, but also revealed substantial differences, especially with key downstream NACs and MYBs. The lignocellulosic SCW of cotton xylem appears to be achieved differently from that in Arabidopsis. Pith cell walls in cotton stems are compositionally very different from that reported for other plant species, including Arabidopsis. The current definition of a 'typical' primary or secondary cell wall might not be applicable to all cell types in all plant species.

High-resolution Myogenic Lineage Mapping by Single-Cell Mass Cytometry

PubMed Central

Porpiglia, Ermelinda; Samusik, Nikolay; Ho, Andrew Tri Van; Cosgrove, Benjamin D.; Mai, Thach; Davis, Kara L.; Jager, Astraea; Nolan, Garry P.; Bendall, Sean C.; Fantl, Wendy J.; Blau, Helen M.

2017-01-01

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force directed layout visualization, defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and aging. PMID:28414312

The Core of Sibling Stem Cell Donation - A Grounded Theory Study.

PubMed

Kisch, Annika M; Forsberg, Anna

2017-01-01

There is a lack of theoretical framework supporting stem cell transplant nurses in their assessment, judgment and caring interventions of sibling stem cell donors. The purpose of this study was to explore sibling stem cell donors' main concerns and how they deal with them before and after donation. Ten healthy sibling donors, 5 men and 5 women, with a median age of 54 years were included in this study when they were due to donate stem cells to a brother or sister. Data were collected prospectively on three occasions (before the donation and three and twelve months after it) through in-depth interviews, which were recorded and transcribed verbatim for analysis by the Grounded Theory method according to Charmaz. This study describes the efforts of the ten donors to fulfil their duty as a sibling by doing what they considered necessary in order to help. Their efforts were summarised in a process wherein the grounded theory generated three main categories; Prepare, Promote and Preserve. A clear path of transition leading to fulfilment is evident, starting before the donation and continuing for one year afterwards. Being a sibling stem cell donor means doing what you have to do to fulfil your duty and if possible, saving the life of a seriously ill brother or sister. The relationship between the siblings is strengthened by the donation process. Sibling stem cell donation appears to be about fulfilment and the theoretical framework may support clinicians in their evaluation and support of donors.

Generation of diverse neuronal subtypes in cloned populations of stem-like cells

PubMed Central

Varga, Balázs V; Hádinger, Nóra; Gócza, Elen; Dulberg, Vered; Demeter, Kornél; Madarász, Emília; Herberth, Balázs

2008-01-01

Background The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material. Results In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons. Conclusion The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification. PMID:18808670

Germ cell tumour growth patterns originating from clear cell carcinomas of the ovary and endometrium: a comparative immunohistochemical study favouring their origin from somatic stem cells.

PubMed

Nogales, Francisco F; Prat, Jaime; Schuldt, Maolly; Cruz-Viruel, Nelly; Kaur, Baljeet; D'Angelo, Emanuela; Matias-Guiu, Xavier; Vidal, August; McCluggage, W Glenn; Oosterhuis, J Wolter

2018-03-01

To report a series of 11 ovarian and one endometrial neoplasm in elderly patients with mixed clear cell tumour and germ cell tumour (GCT) components, to compare their immunohistochemical profiles and demonstrate a putative stem cell population. The clear cell tumours included 11 clear cell carcinomas (CCC) and one borderline clear cell tumour, while the GCT always included glandular yolk sac tumour (YST). In four cases, there were also foci of teratoma with immature neuroepithelial and endodermal tissues and undifferentiated areas showing true embryoids. To distinguish between the clear cell and YST components, the following antibodies were used: HNF1-β, napsin-A, cytokeratin 7 (CK7), PAX8, EMA, AFP, SALL4, villin, glypican-3 (GPC-3), GATA3, HepPar-1, OCT4, CDX2, CD30 and SOX2. HNF1-β, CK7, EMA and GPC-3 were often expressed in both components. Other markers had higher specificity for each cellular lineage; napsin-A and PAX8 were expressed only in CCC, while SALL4, villin, AFP and HepPar-1 were positive in the glandular YST component but negative in the clear cell component. OCT4 expression occurred in six of 10 cases and consistently in teratoma (four of four). There is considerable immunophenotypical overlap between the two components in these mixed neoplasms, and a panel of markers should be used to facilitate the distinction. We propose that OCT4-expressing somatic cancer cells differentiate into GCT and represent spontaneously induced pluripotent stem cells, possibly conditioned by age-related epigenetic factors. These neoplasms have features of prepubertal type GCT showing lack of 12p gain, preponderance of YST and coexistence with immature neuroectoderm. However, there may also be undifferentiated stem cell areas with embryoid bodies, of the type seen in postpubertal testicular GCT, but lacking a complete embryonal carcinoma immunophenotype. © 2017 John Wiley & Sons Ltd.

Making Blood: The Haematopoietic Niche throughout Ontogeny

PubMed Central

Al-Drees, Mohammad A.; Yeo, Jia Hao; Boumelhem, Badwi B.; Antas, Veronica I.; Brigden, Kurt W. L.; Colonne, Chanukya K.; Fraser, Stuart T.

2015-01-01

Approximately one-quarter of all cells in the adult human body are blood cells. The haematopoietic system is therefore massive in scale and requires exquisite regulation to be maintained under homeostatic conditions. It must also be able to respond when needed, such as during infection or following blood loss, to produce more blood cells. Supporting cells serve to maintain haematopoietic stem and progenitor cells during homeostatic and pathological conditions. This coalition of supportive cell types, organised in specific tissues, is termed the haematopoietic niche. Haematopoietic stem and progenitor cells are generated in a number of distinct locations during mammalian embryogenesis. These stem and progenitor cells migrate to a variety of anatomical locations through the conceptus until finally homing to the bone marrow shortly before birth. Under stress, extramedullary haematopoiesis can take place in regions that are typically lacking in blood-producing activity. Our aim in this review is to examine blood production throughout the embryo and adult, under normal and pathological conditions, to identify commonalities and distinctions between each niche. A clearer understanding of the mechanism underlying each haematopoietic niche can be applied to improving ex vivo cultures of haematopoietic stem cells and potentially lead to new directions for transplantation medicine. PMID:26113865

A mesenchymal-like phenotype and expression of CD44 predict lack of apoptotic response to sorafenib in liver tumor cells.

PubMed

Fernando, Joan; Malfettone, Andrea; Cepeda, Edgar B; Vilarrasa-Blasi, Roser; Bertran, Esther; Raimondi, Giulia; Fabra, Àngels; Alvarez-Barrientos, Alberto; Fernández-Salguero, Pedro; Fernández-Rodríguez, Conrado M; Giannelli, Gianluigi; Sancho, Patricia; Fabregat, Isabel

2015-02-15

The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal-like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF-β) and high expression of the stem cell marker CD44 were refractory to sorafenib-induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial-like cells expressing the stem-related proteins EpCAM or CD133 were sensitive to sorafenib-induced apoptosis both in vitro and in vivo. A cross-talk between the TGF-β pathway and the acquisition of a mesenchymal-like phenotype with up-regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock-down in the mesenchymal-like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib-induced apoptosis. However, CD44 effect requires a TGF-β-induced mesenchymal background, since the only overexpression of CD44 in epithelial-like HCC cells is not sufficient to impair sorafenib-induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF-β pathway, may predict lack of response to sorafenib in HCC patients. © 2014 UICC.

Stem Cells, Progenitor Cells, and Lineage Decisions in the Ovary

PubMed Central

Hummitzsch, Katja; Anderson, Richard A.; Wilhelm, Dagmar; Wu, Ji; Telfer, Evelyn E.; Russell, Darryl L.; Robertson, Sarah A.

2015-01-01

Exploring stem cells in the mammalian ovary has unleashed a Pandora's box of new insights and questions. Recent evidence supports the existence of stem cells of a number of the different cell types within the ovary. The evidence for a stem cell model producing mural granulosa cells and cumulus cells is strong, despite a limited number of reports. The recent identification of a precursor granulosa cell, the gonadal ridge epithelial-like cell, is exciting and novel. The identification of female germline (oogonial) stem cells is still very new and is currently limited to just a few species. Their origins and physiological roles, if any, are unknown, and their potential to produce oocytes and contribute to follicle formation in vivo lacks robust evidence. The precursor of thecal cells remains elusive, and more compelling data are needed. Similarly, claims of very small embryonic-like cells are also preliminary. Surface epithelial cells originating from gonadal ridge epithelial-like cells and from the mesonephric epithelium at the hilum of the ovary have also been proposed. Another important issue is the role of the stroma in guiding the formation of the ovary, ovigerous cords, follicles, and surface epithelium. Immune cells may also play key roles in developmental patterning, given their critical roles in corpora lutea formation and regression. Thus, while the cellular biology of the ovary is extremely important for its major endocrine and fertility roles, there is much still to be discovered. This review draws together the current evidence and perspectives on this topic. PMID:25541635

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

PubMed Central

Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

Metformin selectively affects human glioblastoma tumor-initiating cell viability: A role for metformin-induced inhibition of Akt.

PubMed

Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirano; Corsaro, Alessandro; Parodi, Alessia; Sirito, Rodolfo; Massollo, Michela; Marini, Cecilia; Zona, Gianluigi; Fenoglio, Daniela; Sambuceti, Gianmario; Filaci, Gilberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2013-01-01

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect.

Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets.

PubMed

Sun, Lue; Moritake, Takashi; Ito, Kazuya; Matsumoto, Yoshitaka; Yasui, Hironobu; Nakagawa, Hidehiko; Hirayama, Aki; Inanami, Osamu; Tsuboi, Koji

2017-01-01

Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma.

Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets

PubMed Central

Sun, Lue; Moritake, Takashi; Ito, Kazuya; Matsumoto, Yoshitaka; Yasui, Hironobu; Nakagawa, Hidehiko; Hirayama, Aki; Inanami, Osamu; Tsuboi, Koji

2017-01-01

Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs) by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS) production, mitochondria function, oxygen consumption rate (OCR), energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA) resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability) in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma. PMID:28426747

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

PubMed Central

Xiong, Jimin; Menicanin, Danijela; Marino, Victor

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

PubMed

Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Breaking tolerance to self, circulating natural killer cells expressing inhibitory KIR for non-self HLA exhibit effector function after T cell-depleted allogeneic hematopoietic cell transplantation.

PubMed

Yu, Junli; Venstrom, Jeffrey M; Liu, Xiao-Rong; Pring, James; Hasan, Reenat S; O'Reilly, Richard J; Hsu, Katharine C

2009-04-16

Alloreactive natural killer (NK) cells are an important influence on hematopoietic stem cell transplantation (HSCT) outcome. In HLA-mismatched HSCT, alloreactivity occurs when licensed donor NK cells expressing inhibitory killer Ig-like receptors (KIR) for donor MHC class I ligands recognize the lack of the class I ligands in the mismatched recipient ("missing self"). Studies in HLA-matched HSCT, however, have also demonstrated improved outcome in patients lacking class I ligands for donor inhibitory KIR ("missing ligand"), indicating that classically nonlicensed donor NK cells expressing KIR for non-self MHC class I ligands may exhibit functional competence in HSCT. We examined NK function in 16 recipients of T cell-depleted allografts from HLA-identical or KIR-ligand matched donors after myeloablative therapy. After HSCT, nonlicensed NK cells expressing inhibitory KIR for non-self class I exhibit robust intracellular IFN-gamma and cytotoxic response to target cells lacking cognate ligand, gradually becoming tolerized to self by day 100. These findings could not be correlated with cytokine environment or phenotypic markers of NK development, nor could they be attributed to non-KIR receptors such as CD94/NKG2A. These findings confirm that NK alloreactivity can occur in HLA-matched HSCT, where tolerance to self is either acquired by the stem cell-derived NK cell after exiting the bone marrow or where tolerance to self can be temporarily overcome.

An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

PubMed

Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario

2012-03-01

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells

PubMed Central

Taverna, Elena; Mora-Bermúdez, Felipe; Strzyz, Paulina J.; Florio, Marta; Icha, Jaroslav; Haffner, Christiane; Norden, Caren; Wilsch-Bräuninger, Michaela; Huttner, Wieland B.

2016-01-01

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change. PMID:26879757

The analysis of novel microRNA mimic sequences in cancer cells reveals lack of specificity in stem-loop RT-qPCR-based microRNA detection.

PubMed

Winata, Patrick; Williams, Marissa; McGowan, Eileen; Nassif, Najah; van Zandwijk, Nico; Reid, Glen

2017-11-17

MicroRNAs are frequently downregulated in cancer, and restoring expression has tumour suppressive activity in tumour cells. Our recent phase I clinical trial investigated microRNA-based therapy in patients with malignant pleural mesothelioma. Treatment with TargomiRs, microRNA mimics with novel sequence packaged in EGFR antibody-targeted bacterial minicells, revealed clear signs of clinical activity. In order to detect delivery of microRNA mimics to tumour cells in future clinical trials, we tested hydrolysis probe-based assays specific for the sequence of the novel mimics in transfected mesothelioma cell lines using RT-qPCR. The custom assays efficiently and specifically amplified the consensus mimics. However, we found that these assays gave a signal when total RNA from untransfected and control mimic-transfected cells were used as templates. Further investigation revealed that the reverse transcription step using stem-loop primers appeared to introduce substantial non-specific amplification with either total RNA or synthetic RNA templates. This suggests that reverse transcription using stem-loop primers suffers from an intrinsic lack of specificity for the detection of highly similar microRNAs in the same family, especially when analysing total RNA. These results suggest that RT-qPCR is unlikely to be an effective means to detect delivery of microRNA mimic-based drugs to tumour cells in patients.

In silico lineage tracing through single cell transcriptomics identifies a neural stem cell population in planarians.

PubMed

Molinaro, Alyssa M; Pearson, Bret J

2016-04-27

The planarian Schmidtea mediterranea is a master regenerator with a large adult stem cell compartment. The lack of transgenic labeling techniques in this animal has hindered the study of lineage progression and has made understanding the mechanisms of tissue regeneration a challenge. However, recent advances in single-cell transcriptomics and analysis methods allow for the discovery of novel cell lineages as differentiation progresses from stem cell to terminally differentiated cell. Here we apply pseudotime analysis and single-cell transcriptomics to identify adult stem cells belonging to specific cellular lineages and identify novel candidate genes for future in vivo lineage studies. We purify 168 single stem and progeny cells from the planarian head, which were subjected to single-cell RNA sequencing (scRNAseq). Pseudotime analysis with Waterfall and gene set enrichment analysis predicts a molecularly distinct neoblast sub-population with neural character (νNeoblasts) as well as a novel alternative lineage. Using the predicted νNeoblast markers, we demonstrate that a novel proliferative stem cell population exists adjacent to the brain. scRNAseq coupled with in silico lineage analysis offers a new approach for studying lineage progression in planarians. The lineages identified here are extracted from a highly heterogeneous dataset with minimal prior knowledge of planarian lineages, demonstrating that lineage purification by transgenic labeling is not a prerequisite for this approach. The identification of the νNeoblast lineage demonstrates the usefulness of the planarian system for computationally predicting cellular lineages in an adult context coupled with in vivo verification.

From stem cell to embryo without centrioles.

PubMed

Stevens, Naomi R; Raposo, Alexandre A S F; Basto, Renata; St Johnston, Daniel; Raff, Jordan W

2007-09-04

Centrosome asymmetry plays a key role in ensuring the asymmetric division of Drosophila neural stem cells (neuroblasts [NBs]) and male germline stem cells (GSCs) [1-3]. In both cases, one centrosome is anchored close to a specific cortical region during interphase, thus defining the orientation of the spindle during the ensuing mitosis. To test whether asymmetric centrosome behavior is a general feature of stem cells, we have studied female GSCs, which divide asymmetrically, producing another GSC and a cystoblast. The cystoblast then divides and matures into an oocyte, a process in which centrosomes exhibit a series of complex behaviors proposed to play a crucial role in oogenesis [4-6]. We show that the interphase centrosome does not define spindle orientation in female GSCs and that DSas-4 mutant GSCs [7], lacking centrioles and centrosomes, invariably divide asymmetrically to produce cystoblasts that proceed normally through oogenesis-remarkably, oocyte specification, microtubule organization, and mRNA localization are all unperturbed. Mature oocytes can be fertilized, but embryos that cannot support centriole replication arrest very early in development. Thus, centrosomes are dispensable for oogenesis but essential for early embryogenesis. These results reveal that asymmetric centrosome behavior is not an essential feature of stem cell divisions.

Spatiotemporal switching signals for cancer stem cell activation in pediatric origins of adulthood cancer: Towards a watch-and-wait lifetime strategy for cancer treatment.

PubMed

Li, Shengwen Calvin; Kabeer, Mustafa H

2018-02-26

Pediatric origin of cancer stem cell hypothesis holds great promise and potential in adult cancer treatment, however; the road to innovation is full of obstacles as there are plenty of questions left unanswered. First, the key question is to characterize the nature of such stem cells (concept). Second, the quantitative imaging of pediatric stem cells should be implemented (technology). Conceptually, pediatric stem cell origins of adult cancer are based on the notion that plasticity in early life developmental programming evolves local environments to cancer. Technologically, such imaging in children is lacking as all imaging is designed for adult patients. We postulate that the need for quantitative imaging to measure space-time changes of plasticity in early life developmental programming in children may trigger research and development of the imaging technology. Such quantitative imaging of pediatric origin of adulthood cancer will help develop a spatiotemporal monitoring system to determine cancer initiation and progression. Clinical validation of such speculative hypothesis-that cancer originates in a pediatric environment-will help implement a wait-and-watch strategy for cancer treatment.

Advances in cell culture: anchorage dependence

PubMed Central

Merten, Otto-Wilhelm

2015-01-01

Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes. PMID:25533097

Comparison of tumor biology of two distinct cell sub-populations in lung cancer stem cells.

PubMed

Wang, Jianyu; Sun, Zhiwei; Liu, Yongli; Kong, Liangsheng; Zhou, Shixia; Tang, Junlin; Xing, Hongmei Rosie

2017-11-14

Characterization of the stem-like properties of cancer stem cells (CSCs) remain indirect and qualitative, especially the ability of CSCs to undergo asymmetric cell division for self renewal and differentiation, a unique property of cells of stem origin. It is partly due to the lack of stable cellular models of CSCs. In this study, we developed a new approach for CSC isolation and purification to derive a CSC-enriched cell line (LLC-SE). By conducting five consecutive rounds of single cell cloning using the LLC-SE cell line, we obtained two distinct sub-population of cells within the Lewis lung cancer CSCs that employed largely symmetric division for self-renewal (LLC-SD) or underwent asymmetric division for differentiation (LLC-ASD). LLC-SD and LLC-ASD cell lines could be stably passaged in culture and be distinguished by cell morphology, stem cell marker, spheroid formation and subcutaneous tumor initiation efficiency, as well as orthotopic lung tumor growth, progression and survival. The ability LLC-ASD cells to undergo asymmetric division was visualized and quantified by the asymmetric segregation of labeled BrdU and NUMB to one of the two daughter cells in anaphase cell division. The more stem-like LLC-SD cells exhibited higher capacity for tumorigenesis and progression and shorter survival. As few as 10 LLC-SD could initiate subcutaneous tumor growth when transplanted to the athymic mice. Collectively, these observations suggest that the SD-type of cells appear to be on the top of the hierarchical order of the CSCs. Furthermore, they have lead to generated cellular models of CSC self-renewal for future mechanistic investigations.

Stem cell therapies in cardiovascular disease A "realistic" appraisal.

PubMed

Partovian, Chohreh; Simons, Michael

2008-01-01

The possibility of reconstituting the damaged heart has introduced a new paradigm in cardiovascular biology and created the potential for a new therapeutic approach in the cardiovascular field, where there is a compelling need for innovative treatments. While the results of animal and early clinical studies are encouraging, the more direct use of cell-based therapies in patients is still long-reached. Gaps in our basic understanding of mechanisms, lack of important randomized, double blind, and controlled clinical trials, as well as technology development for cell production are among challenges to be overcome before full translation of cell based therapies in clinical arena. This review focuses on summarizing the latest knowledge in stem cell therapy for cardiovascular diseases.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

PRMT7 Preserves Satellite Cell Regenerative Capacity.

PubMed

Blanc, Roméo Sébastien; Vogel, Gillian; Chen, Taiping; Crist, Colin; Richard, Stéphane

2016-02-16

Regeneration of skeletal muscle requires the continued presence of quiescent muscle stem cells (satellite cells), which become activated in response to injury. Here, we report that whole-body protein arginine methyltransferase PRMT7(-/-) adult mice and mice conditionally lacking PRMT7 in satellite cells using Pax7-CreERT2 both display a significant reduction in satellite cell function, leading to defects in regenerative capacity upon muscle injury. We show that PRMT7 is preferentially expressed in activated satellite cells and, interestingly, PRMT7-deficient satellite cells undergo cell-cycle arrest and premature cellular senescence. These defects underlie poor satellite cell stem cell capacity to regenerate muscle and self-renew after injury. PRMT7-deficient satellite cells express elevated levels of the CDK inhibitor p21CIP1 and low levels of its repressor, DNMT3b. Restoration of DNMT3b in PRMT7-deficient cells rescues PRMT7-mediated senescence. Our findings define PRMT7 as a regulator of the DNMT3b/p21 axis required to maintain muscle stem cell regenerative capacity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis

PubMed Central

Rosiak, Kamila; Smolarz, Maciej; Stec, Wojciech J.; Peciak, Joanna; Grzela, Dawid; Winiecka-Klimek, Marta; Stoczynska-Fidelus, Ewelina; Krynska, Barbara; Piaskowski, Sylwester; Rieske, Piotr

2016-01-01

Background The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability. Methods Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot. Results Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells. Conclusions Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells. PMID:27145078

No evidence for neo-oogenesis may link to ovarian senescence in adult monkey.

PubMed

Yuan, Jihong; Zhang, Dongdong; Wang, Lei; Liu, Mengyuan; Mao, Jian; Yin, Yu; Ye, Xiaoying; Liu, Na; Han, Jihong; Gao, Yingdai; Cheng, Tao; Keefe, David L; Liu, Lin

2013-11-01

Female germline or oogonial stem cells transiently residing in fetal ovaries are analogous to the spermatogonial stem cells or germline stem cells (GSCs) in adult testes where GSCs and meiosis continuously renew. Oocytes can be generated in vitro from embryonic stem cells and induced pluripotent stem cells, but the existence of GSCs and neo-oogenesis in adult mammalian ovaries is less clear. Preliminary findings of GSCs and neo-oogenesis in mice and humans have not been consistently reproducible. Monkeys provide the most relevant model of human ovarian biology. We searched for GSCs and neo-meiosis in ovaries of adult monkeys at various ages, and compared them with GSCs from adult monkey testis, which are characterized by cytoplasmic staining for the germ cell marker DAZL and nuclear expression of the proliferative markers PCNA and KI67, and pluripotency-associated genes LIN28 and SOX2, and lack of nuclear LAMIN A, a marker for cell differentiation. Early meiocytes undergo homologous pairing at prophase I distinguished by synaptonemal complex lateral filaments with telomere perinuclear distribution. By exhaustive searching using comprehensive experimental approaches, we show that proliferative GSCs and neo-meiocytes by these specific criteria were undetectable in adult mouse and monkey ovaries. However, we found proliferative nongermline somatic stem cells that do not express LAMIN A and germ cell markers in the adult ovaries, notably in the cortex and granulosa cells of growing follicles. These data support the paradigm that adult ovaries do not undergo germ cell renewal, which may contribute significantly to ovarian senescence that occurs with age. Copyright © 2013 AlphaMed Press.

Phasor Fluorescence Lifetime Microscopy of Free and Protein-Bound NADH Reveals Neural Stem Cell Differentiation Potential

PubMed Central

Stringari, Chiara; Nourse, Jamison L.; Flanagan, Lisa A.; Gratton, Enrico

2012-01-01

In the stem cell field there is a lack of non invasive and fast methods to identify stem cell’s metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors. PMID:23144844

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

PubMed Central

Nguyen, Duong Thi Thuy; Richter, Daniel; Michel, Geert; Mitschka, Sibylle; Kolanus, Waldemar; Cuevas, Elisa; Gregory Wulczyn, F

2017-01-01

Rapidity and specificity are characteristic features of proteolysis mediated by the ubiquitin-proteasome system. Therefore, the UPS is ideally suited for the remodeling of the embryonic stem cell proteome during the transition from pluripotent to differentiated states and its inverse, the generation of inducible pluripotent stem cells. The Trim-NHL family member LIN41 is among the first E3 ubiquitin ligases to be linked to stem cell pluripotency and reprogramming. Initially discovered in C. elegans as a downstream target of the let-7 miRNA, LIN41 is now recognized as a critical regulator of stem cell fates as well as the timing of neurogenesis. Despite being indispensable for embryonic development and neural tube closure in mice, the underlying mechanisms for LIN41 function in these processes are poorly understood. To better understand the specific contributions of the E3 ligase activity for the stem cell functions of LIN41, we characterized global changes in ubiquitin or ubiquitin-like modifications using Lin41-inducible mouse embryonic stem cells. The tumor suppressor protein p53 was among the five most strongly affected proteins in cells undergoing neural differentiation in response to LIN41 induction. We show that LIN41 interacts with p53, controls its abundance by ubiquitination and antagonizes p53-dependent pro-apoptotic and pro-differentiation responses. In vivo, the lack of LIN41 is associated with upregulation of Grhl3 and widespread caspase-3 activation, two downstream effectors of p53 with essential roles in neural tube closure. As Lin41-deficient mice display neural tube closure defects, we conclude that LIN41 is critical for the regulation of p53 functions in cell fate specification and survival during early brain development. PMID:28430184

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

NASA Astrophysics Data System (ADS)

Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

2017-03-01

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Early events in xenograft development from the human embryonic stem cell line HS181--resemblance with an initial multiple epiblast formation.

PubMed

Gertow, Karin; Cedervall, Jessica; Jamil, Seema; Ali, Rouknuddin; Imreh, Marta P; Gulyas, Miklos; Sandstedt, Bengt; Ahrlund-Richter, Lars

2011-01-01

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart.

WDR62 Regulates Early Neural and Glial Progenitor Specification of Human Pluripotent Stem Cells

PubMed Central

Alshawaf, Abdullah J.; Antonic, Ana; Skafidas, Efstratios

2017-01-01

Mutations in WD40-repeat protein 62 (WDR62) are commonly associated with primary microcephaly and other developmental cortical malformations. We used human pluripotent stem cells (hPSC) to examine WDR62 function during human neural differentiation and model early stages of human corticogenesis. Neurospheres lacking WDR62 expression showed decreased expression of intermediate progenitor marker, TBR2, and also glial marker, S100β. In contrast, inhibition of c-Jun N-terminal kinase (JNK) signalling during hPSC neural differentiation induced upregulation of WDR62 with a corresponding increase in neural and glial progenitor markers, PAX6 and EAAT1, respectively. These findings may signify a role of WDR62 in specifying intermediate neural and glial progenitors during human pluripotent stem cell differentiation. PMID:28690640

Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD

PubMed Central

Kim, Dae-Sung; Ross, P. Joel; Zaslavsky, Kirill; Ellis, James

2014-01-01

Autism spectrum disorder (ASD) is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons. PMID:24782713

Stem cells and regenerative medicine: principles, prospects and problems.

PubMed

Gardner, Richard L

2007-01-01

Stem cells have been used routinely for more than three decades to repair tissues and organs damaged by injury or disease, most notably haematopoietic stem cells taken from bone marrow, umbilical cord or, increasingly, from peripheral blood. Other examples, such as grafts of skin to treat severe burns, entail transplantation of stem cells within organized tissue rather than following isolation. The prospect of exploiting stem cells more widely in regenerative medicine was encouraged both by the development of human assisted conception and growing evidence that various adult cells retained greater versatility than had been suspected hitherto. The aim is to employ stem cells as a source of appropriately differentiated cells to replace those lost through physical, chemical or ischaemic injury, or as a result of degenerative disease. This may entail transplantation of just a single type of cell or, more challengingly, require a complex of several different types of cells possessing a defined architecture. Cardiomyocytes, hepatocytes or neuronal cells producing specific transmitters offer promising examples of the former, although how transplanted healthy cells will function in a perturbed tissue environment remains to be established. Recent success in repairing urinary bladder defects with grafts of urothelial and muscle cells seeded on a biodegradable collagen scaffold is an encouraging step towards assembling organs in vitro. Nevertheless, this is still far removed from the level of sophistication required to counter the ever increasing shortfall in supply of kidneys for transplantation. Various problems must be addressed if recent advances in the laboratory are to be translated into clinical practice. In many cases, it has yet to be established that cells derived from adults that retain plasticity are actually stem cells. There is also a pressing need for appropriate assays to ensure that, regardless of source, stem cells maintained in vitro are safe to transplant. Assays currently available for human ES cells are far from ideal. It is, in addition, important to ensure that differentiated cultures are pure and, depending on whether cell renewal is required or to be avoided, retain or lack appropriate stem cells. Neither autografts nor those obtained by so-called 'therapeutic cloning' are options for treating condition with an obvious genetic basis. Moreover, claims that some stem cells are more likely than others to yield successful allografts have yet to be confirmed and explained.

Short-Term Grafting of Human Neural Stem Cells: Electrophysiological Properties and Motor Behavioral Amelioration in Experimental Parkinsons Disease.

PubMed

Martnez-Serrano, Alberto; Pereira, Marta P; Avaliani, Natalia; Nelke, Anna; Kokaia, Merab; Ramos-Moreno, Tania

2016-12-13

Cell replacement therapy in Parkinsons disease (PD) still lacks a study addressing the acquisition of electrophysiological properties of human grafted neural stem cells and their relation with the emergence of behavioral recovery after transplantation in the short term. Here we study the electrophysiological and biochemical profiles of two ventral mesencephalic human neural stem cell (NSC) clonal lines (C30-Bcl-XL and C32-Bcl-XL) that express high levels of Bcl-XL to enhance their neurogenic capacity, after grafting in an in vitro parkinsonian model. Electrophysiological recordings show that the majority of the cells derived from the transplants are not mature at 6 weeks after grafting, but 6.7% of the studied cells showed mature electrophysiological profiles. Nevertheless, parallel in vivo behavioral studies showed a significant motor improvement at 7 weeks postgrafting in the animals receiving C30-Bcl-XL, the cell line producing the highest amount of TH+ cells. Present results show that, at this postgrafting time point, behavioral amelioration highly correlates with the spatial dispersion of the TH+ grafted cells in the caudate putamen. The spatial dispersion, along with a high number of dopaminergic-derived cells, is crucial for behavioral improvements. Our findings have implications for long-term standardization of stem cell-based approaches in Parkinsons disease.

MicroRNA let-7d regulates the TLX/microRNA-9 cascade to control neural cell fate and neurogenesis

PubMed Central

Zhao, Chunnian; Sun, GuoQiang; Ye, Peng; Li, Shengxiu; Shi, Yanhong

2013-01-01

MicroRNAs have important functions in the nervous system through post-transcriptional regulation of neurogenesis genes. Here we show that microRNA let-7d, which has been implicated in cocaine addiction and other neurological disorders, targets the neural stem cell regulator TLX. Overexpression of let-7d in vivo reduced neural stem cell proliferation and promoted premature neuronal differentiation and migration, a phenotype similar to those induced by TLX knockdown or overexpression of its negatively-regulated target, microRNA-9. We found a let-7d binding sequence in the tlx 3′ UTR and demonstrated that let-7d reduced TLX expression levels in neural stem cells, which in turn, up-regulated miR-9 expression. Moreover, co-expression of let-7d and TLX lacking its 3′ UTR in vivo restored neural stem cell proliferation and reversed the premature neuronal differentiation and migration. Therefore, manipulating let-7d and its downstream targets could be a novel strategy to unravel neurogenic signaling pathways and identify potential interventions for relevant neurological disorders. PMID:23435502

MicroRNA let-7d regulates the TLX/microRNA-9 cascade to control neural cell fate and neurogenesis.

PubMed

Zhao, Chunnian; Sun, GuoQiang; Ye, Peng; Li, Shengxiu; Shi, Yanhong

2013-01-01

MicroRNAs have important functions in the nervous system through post-transcriptional regulation of neurogenesis genes. Here we show that microRNA let-7d, which has been implicated in cocaine addiction and other neurological disorders, targets the neural stem cell regulator TLX. Overexpression of let-7d in vivo reduced neural stem cell proliferation and promoted premature neuronal differentiation and migration, a phenotype similar to those induced by TLX knockdown or overexpression of its negatively-regulated target, microRNA-9. We found a let-7d binding sequence in the tlx 3' UTR and demonstrated that let-7d reduced TLX expression levels in neural stem cells, which in turn, up-regulated miR-9 expression. Moreover, co-expression of let-7d and TLX lacking its 3' UTR in vivo restored neural stem cell proliferation and reversed the premature neuronal differentiation and migration. Therefore, manipulating let-7d and its downstream targets could be a novel strategy to unravel neurogenic signaling pathways and identify potential interventions for relevant neurological disorders.

In vitro pathological modelling using patient-specific induced pluripotent stem cells: the case of progeria.

PubMed

Nissan, Xavier; Blondel, Sophie; Peschanski, Marc

2011-12-01

Progeria, also known as HGPS (Hutchinson-Gilford progeria syndrome), is a rare fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (C1804T) of the gene encoding lamins A and C, LMNA, leading to the production of a truncated form of the protein called progerin. Owing to their unique potential to self-renew and to differentiate into any cell types of the organism, pluripotent stem cells offer a unique tool to study molecular and cellular mechanisms related to this global and systemic disease. Recent studies have exploited this potential by generating human induced pluripotent stem cells from HGPS patients' fibroblasts displaying several phenotypic defects characteristic of HGPS such as nuclear abnormalities, progerin expression, altered DNA-repair mechanisms and premature senescence. Altogether, these findings provide new insights on the use of pluripotent stem cells for pathological modelling and may open original therapeutic perspectives for diseases that lack pre-clinical in vitro human models, such as HGPS.

Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells.

PubMed

Morata-Tarifa, Cynthia; Jiménez, Gema; García, María A; Entrena, José M; Griñán-Lisón, Carmen; Aguilera, Margarita; Picon-Ruiz, Manuel; Marchal, Juan A

2016-01-11

Cancer stem cells are responsible for tumor progression, metastasis, therapy resistance and cancer recurrence, doing their identification and isolation of special relevance. Here we show that low adherent breast and colon cancer cells subpopulations have stem-like properties. Our results demonstrate that trypsin-sensitive (TS) breast and colon cancer cells subpopulations show increased ALDH activity, higher ability to exclude Hoechst 33342, enlarged proportion of cells with a cancer stem-like cell phenotype and are enriched in sphere- and colony-forming cells in vitro. Further studies in MDA-MB-231 breast cancer cells reveal that TS subpopulation expresses higher levels of SLUG, SNAIL, VIMENTIN and N-CADHERIN while show a lack of expression of E-CADHERIN and CLAUDIN, being this profile characteristic of the epithelial-to-mesenchymal transition (EMT). The TS subpopulation shows CXCL10, BMI-1 and OCT4 upregulation, differing also in the expression of several miRNAs involved in EMT and/or cell self-renewal such as miR-34a-5p, miR-34c-5p, miR-21-5p, miR-93-5p and miR-100-5p. Furthermore, in vivo studies in immunocompromised mice demonstrate that MDA-MB-231 TS cells form more and bigger xenograft tumors with shorter latency and have higher metastatic potential. In conclusion, this work presents a new, non-aggressive, easy, inexpensive and reproducible methodology to isolate prospectively cancer stem-like cells for subsequent biological and preclinical studies.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

PubMed

Ng, Elizabeth S; Azzola, Lisa; Bruveris, Freya F; Calvanese, Vincenzo; Phipson, Belinda; Vlahos, Katerina; Hirst, Claire; Jokubaitis, Vanta J; Yu, Qing C; Maksimovic, Jovana; Liebscher, Simone; Januar, Vania; Zhang, Zhen; Williams, Brenda; Conscience, Aude; Durnall, Jennifer; Jackson, Steven; Costa, Magdaline; Elliott, David; Haylock, David N; Nilsson, Susan K; Saffery, Richard; Schenke-Layland, Katja; Oshlack, Alicia; Mikkola, Hanna K A; Stanley, Edouard G; Elefanty, Andrew G

2016-11-01

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 + cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 + cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 + hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 + vessels from which RUNX1C + blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 + hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Development of autologous blood cell therapies.

PubMed

Kim, Ah Ram; Sankaran, Vijay G

2016-10-01

Allogeneic hematopoietic stem cell transplantation and blood cell transfusions are performed commonly in patients with a variety of blood disorders. Unfortunately, these donor-derived cell therapies are constrained due to limited supplies, infectious risk factors, a lack of appropriately matched donors, and the risk of immunologic complications from such products. The use of autologous cell therapies has been proposed to overcome these shortcomings. One can derive such therapies directly from hematopoietic stem and progenitor cells of individuals, which can then be manipulated ex vivo to produce the desired modifications or differentiated to produce a particular target population. Alternatively, pluripotent stem cells, which have a theoretically unlimited self-renewal capacity and an ability to differentiate into any desired cell type, can be used as an autologous starting source for such manipulation and differentiation approaches. Such cell products can also be used as a delivery vehicle for therapeutics. In this review, we highlight recent advances and discuss ongoing challenges for the in vitro generation of autologous hematopoietic cells that can be used for cell therapy. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

PubMed Central

Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

2012-01-01

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

Human pluripotent stem cells as tools for neurodegenerative and neurodevelopmental disease modeling and drug discovery.

PubMed

Corti, Stefania; Faravelli, Irene; Cardano, Marina; Conti, Luciano

2015-06-01

Although intensive efforts have been made, effective treatments for neurodegenerative and neurodevelopmental diseases have not been yet discovered. Possible reasons for this include the lack of appropriate disease models of human neurons and a limited understanding of the etiological and neurobiological mechanisms. Recent advances in pluripotent stem cell (PSC) research have now opened the path to the generation of induced pluripotent stem cells (iPSCs) starting from somatic cells, thus offering an unlimited source of patient-specific disease-relevant neuronal cells. In this review, the authors focus on the use of human PSC-derived cells in modeling neurological disorders and discovering of new drugs and provide their expert perspectives on the field. The advent of human iPSC-based disease models has fuelled renewed enthusiasm and enormous expectations for insights of disease mechanisms and identification of more disease-relevant and novel molecular targets. Human PSCs offer a unique tool that is being profitably exploited for high-throughput screening (HTS) platforms. This process can lead to the identification and optimization of molecules/drugs and thus move forward new pharmacological therapies for a wide range of neurodegenerative and neurodevelopmental conditions. It is predicted that improvements in the production of mature neuronal subtypes, from patient-specific human-induced pluripotent stem cells and their adaptation to culture, to HTS platforms will allow the increased exploitation of human pluripotent stem cells in drug discovery programs.

The Core of Sibling Stem Cell Donation – A Grounded Theory Study

PubMed Central

Kisch, Annika M; Forsberg, Anna

2017-01-01

Background: There is a lack of theoretical framework supporting stem cell transplant nurses in their assessment, judgment and caring interventions of sibling stem cell donors. Objective: The purpose of this study was to explore sibling stem cell donors’ main concerns and how they deal with them before and after donation. Method: Ten healthy sibling donors, 5 men and 5 women, with a median age of 54 years were included in this study when they were due to donate stem cells to a brother or sister. Data were collected prospectively on three occasions (before the donation and three and twelve months after it) through in-depth interviews, which were recorded and transcribed verbatim for analysis by the Grounded Theory method according to Charmaz. Results: This study describes the efforts of the ten donors to fulfil their duty as a sibling by doing what they considered necessary in order to help. Their efforts were summarised in a process wherein the grounded theory generated three main categories; Prepare, Promote and Preserve. A clear path of transition leading to fulfilment is evident, starting before the donation and continuing for one year afterwards. Conclusions: Being a sibling stem cell donor means doing what you have to do to fulfil your duty and if possible, saving the life of a seriously ill brother or sister. The relationship between the siblings is strengthened by the donation process. Sibling stem cell donation appears to be about fulfilment and the theoretical framework may support clinicians in their evaluation and support of donors. PMID:28839511

Overcoming the Practical Barriers to Spinal Cord Cell Transplantation for ALS

DTIC Science & Technology

2014-10-01

should not be neglected. Moreover, escalating numbers and volumes of injections seem to be associated with lack of accuracy and reflux . At the same...investigations of intraspinal stem cell therapies are underway for a range of neurological diseases . Originally considered entirely immuno-privileged, the

Peripheral blood stem cell mobilization: the CXCR2 ligand GRObeta rapidly mobilizes hematopoietic stem cells with enhanced engraftment properties.

PubMed

Pelus, Louis M; Fukuda, Seiji

2006-08-01

Chemokines direct the movement of leukocytes, including hematopoietic stem and progenitor cells, and can mobilize hematopoietic cells from marrow to peripheral blood where they can be used for transplantation. In this review, we will discuss the stem cell mobilizing activities and mechanisms of action of GRObeta, a CXC chemokine ligand for the CXCR2 receptor. GRObeta rapidly mobilizes short- and long-term repopulating cells in mice and/or monkeys and synergistically enhances mobilization responses when combined with the widely used clinical mobilizer, granulocyte colony-stimulating factor (G-CSF). The hematopoietic graft mobilized by GRObeta contains significantly more CD34(neg), Sca-1+, c-kit+, lineage(neg) (SKL) cells than the graft mobilized by G-CSF. In mice, stem cells mobilized by GRObeta demonstrate a competitive advantage upon long-term repopulation analysis and restore neutrophil and platelet counts significantly faster than cells mobilized by G-CSF. Even greater advantage in repopulation and restoration of hematopoiesis are observed with stem cells mobilized by the combination of GRObeta and G-CSF. GRObeta-mobilized SKL cells demonstrate enhanced adherence to vascular cell adhesion molecule-1 and VCAM(pos) endothelial cells and home more efficiently to bone marrow in vivo. The marrow homing ability of GRObeta-mobilized cells is less dependent on the CXCR4/SDF-1 axis than cells mobilized by G-CSF. The mechanism of mobilization by GRObeta requires active matrix metalloproteinase-9 (MMP-9), which results from release of pro-MMP-9 from peripheral blood, and marrow neutrophils, which alters the stoichiometry between pro-MMP-9 and its inhibitor tissue inhibitor of metalloproteinase-1, resulting in MMP-9 activation. The efficacy and rapid action of GRObeta and lack of proinflammatory activity make it an attractive agent to supplement mobilization by G-CSF. In addition, GRObeta may also have clinical mobilizing efficacy on its own, reducing the overall time and costs associated with peripheral blood stem cell transplantation.

Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells

PubMed Central

Jackson, Kathyjo A.; Majka, Susan M.; Wang, Hongyu; Pocius, Jennifer; Hartley, Craig J.; Majesky, Mark W.; Entman, Mark L.; Michael, Lloyd H.; Hirschi, Karen K.; Goodell, Margaret A.

2001-01-01

Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34–/low, c-Kit+, Sca-1+) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and α-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction. PMID:11390421

Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey

2007-12-31

Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. Thesemore » cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.« less

Delayed Cutaneous Wound Healing and Aberrant Expression of Hair Follicle Stem Cell Markers in Mice Selectively Lacking Ctip2 in Epidermis

PubMed Central

Bajaj, Gaurav; Guha, Gunjan; Wang, Zhixing; Jang, Hyo-Sang; Leid, Mark; Indra, Arup Kumar; Ganguli-Indra, Gitali

2012-01-01

Background COUP-TF interacting protein 2 [(Ctip2), also known as Bcl11b] is an important regulator of skin homeostasis, and is overexpressed in head and neck cancer. Ctip2ep−/− mice, selectively ablated for Ctip2 in epidermal keratinocytes, exhibited impaired terminal differentiation and delayed epidermal permeability barrier (EPB) establishment during development, similar to what was observed in Ctip2 null (Ctip2−/−) mice. Considering that as an important role of Ctip2, and the fact that molecular networks which underlie cancer progression partially overlap with those responsible for tissue remodeling, we sought to determine the role of Ctip2 during cutaneous wound healing. Methodology/Principal Findings Full thickness excisional wound healing experiments were performed on Ctip2L2/L2 and Ctip2ep−/− animals per time point and used for harvesting samples for histology, immunohistochemistry (IHC) and immunoblotting. Results demonstrated inherent defects in proliferation and migration of Ctip2 lacking keratinocytes during re-epithelialization. Mutant mice exhibited reduced epidermal proliferation, delayed keratinocyte activation, altered cell-cell adhesion and impaired ECM development. Post wounding, Ctip2ep−/− mice wounds displayed lack of E-Cadherin suppression in the migratory tongue, insufficient expression of alpha smooth muscle actin (alpha SMA) in the dermis, and robust induction of K8. Importantly, dysregulated expression of several hair follicle (HF) stem cell markers such as K15, NFATc1, CD133, CD34 and Lrig1 was observed in mutant skin during wound repair. Conclusions/Significance Results confirm a cell autonomous role of keratinocytic Ctip2 to modulate cell migration, proliferation and/or differentiation, and to maintain HF stem cells during cutaneous wounding. Furthermore, Ctip2 in a non-cell autonomous manner regulated granulation tissue formation and tissue contraction during wound closure. PMID:22383956

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Zhenhua; Key Laboratory of Neurodegeneration, Ministry of Education, Beijing; Department of Anatomy, Anhui Medical University, Hefei, 230032

2011-12-10

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristicsmore » of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy. -- Highlights: Black-Right-Pointing-Pointer Spontaneous transformation of cynomolgus monkey MSCs in vitro. Black-Right-Pointing-Pointer Transformed mesenchymal cells lack multipotency. Black-Right-Pointing-Pointer Transformed mesenchymal cells are highly tumorigenic. Black-Right-Pointing-Pointer Transformed mesenchymal cells do not have the characteristics of cancer stem cells.« less

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

PubMed

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

Graft failure after allogeneic hematopoietic stem cell transplantation.

PubMed

Ozdemir, Zehra Narli; Civriz Bozdağ, Sinem

2018-04-18

Graft failure is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) defined as either lack of initial engraftment of donor cells (primary graft failure) or loss of donor cells after initial engraftment (secondary graft failure). Successful transplantation depends on the formation of engrafment, in which donor cells are integrated into the recipient's cell population. In this paper, we distinguish two different entities, graft failure (GF) and poor graft function (PGF), and review the current comprehensions of the interactions between the immune and hematopoietic compartments in these conditions. Factors associated with graft failure include histocompatibility locus antigen (HLA)-mismatched grafts, underlying disease, type of conditioning regimen and stem cell source employed, low stem cell dose, ex vivo T-cell depletion, major ABO incompatibility, female donor grafts for male recipients, disease status at transplantation. Although several approaches have been developed which aimed to prevent graft rejection, establish successful engraftment and treat graft failure, GF remains a major obstacle to the success of allo-HSCT. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) still remains to be the curative treatment option for various non-malignant and malignant hematopoietic diseases. The outcome of allo-HSCT primarily depends on the engraftment of the graft. Graft failure (GF), is a life-threatening complication which needs the preferential therapeutic manipulation. In this paper, we focused on the definitions of graft failure / poor graft function and also we reviewed the current understanding of the pathophysiology, risk factors and treatment approaches for these entities. Copyright © 2018. Published by Elsevier Ltd.

Substantial Differentiation of Human Neural Stem Cells Into Motor Neurons on a Biomimetic Polyureaa

PubMed Central

Yun, Donghwa; Lee, Young M.; Laughter, Melissa R.; Freed, Curt R.

2015-01-01

To find the first restorative treatment for spinal cord injury (SCI), researchers have focused on stem cell therapies. However, one obstacle is the lack of an implantable cell scaffold that can support efficient motor neuron (MN) differentiation and proliferation. We aimed to overcome this through the use of an RGD functionalized novel biomimetic polyurea, optimized to encourage efficient differentiation of MNs. Images taken after 14-days showed increased differentiation (~40%) of hNSCs into MNs as well as increased cell count on the biomimetic polymer compared to PDL-Laminin coating, indicating that the RGD-polyurea provides a favorable microenvironment for hNSC survival, having promising implications for future SCI therapies. PMID:26033933

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chubinskiy-Nadezhdin, Vladislav I., E-mail: vchubinskiy@gmail.com; Vasileva, Valeria Y.; Pugovkina, Natalia A.

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances inmore » hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca{sup 2+} entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells. - Highlights: • Stretch-induced channel activity in human mesenchymal stem cells was analyzed. • Functional expression of SACs and Ca{sup 2+}-sensitive BK and SK channels was shown. • Local Ca{sup 2+} influx via stretch-activated channels triggers BK channel activity. • SK channels are not coupled with SACs despite higher sensitivity to [Ca{sup 2+}]{sub i}. • Functional clustering of SACs and BK channels in stem cell membrane is proposed.« less

Direct transdifferentiation of spermatogonial stem cells to morphological, phenotypic and functional hepatocyte-like cells via the ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E

PubMed Central

2013-01-01

Background Severe shortage of liver donors and hepatocytes highlights urgent requirement of extra-liver and stem cell source of hepatocytes for treating liver-related diseases. Here we hypothesized that spermatogonial stem cells (SSCs) can directly transdifferentiate to hepatic stem-like cells capable of differentiating into mature hepatocyte-like cells in vitro without an intervening pluripotent state. Results SSCs first changed into hepatic stem-like cells since they resembled hepatic oval cells in morphology and expressed Ck8, Ck18, Ck7, Ck19, OV6, and albumin. Importantly, they co-expressed CK8 and CK19 but not ES cell markers. Hepatic stem-like cells derived from SSCs could differentiate into small hepatocytes based upon their morphological features and expression of numerous hepatic cell markers but lacking of bile epithelial cell hallmarks. Small hepatocytes were further coaxed to differentiate into mature hepatocyte-like cells, as identified by their morphological traits and strong expression of Ck8, Ck18, Cyp7a1, Hnf3b, Alb, Tat, Ttr, albumin, and CYP1A2 but not Ck7 or CK19. Notably, these differentiated cells acquired functional attributes of hepatocyte-like cells because they secreted albumin, synthesized urea, and uptake and released indocyanine green. Moreover, phosphorylation of ERK1/2 and Smad2/3 rather than Akt was activated in hepatic stem cells and mature hepatocytes. Additionally, cyclin A, cyclin B and cyclin E transcripts and proteins but not cyclin D1 or CDK1 and CDK2 transcripts or proteins were reduced in mature hepatocyte-like cells or hepatic stem-like cells derived from SSCs compared to SSCs. Conclusions SSCs can transdifferentiate to hepatic stem-like cells capable of differentiating into cells with morphological, phenotypic and functional characteristics of mature hepatocytes via the activation of ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E. This study thus provides an invaluable source of mature hepatocytes for treating liver-related diseases and drug toxicity screening and offers novel insights into mechanisms of liver development and cell reprogramming. PMID:24047406

Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT

PubMed Central

Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

2015-01-01

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle. PMID:25602959

Application of a novel population of multipotent stem cells derived from skin fibroblasts as donor cells in bovine SCNT.

PubMed

Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

2015-01-01

Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4(+) cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4(+) cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4(+) cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4(+) cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4(-) cells. Moreover, blastocysts derived from SSEA-4(+) cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4(-) derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4(+) cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.

Isolation and characterization of human CXCR4-positive pancreatic cells.

PubMed

Koblas, T; Zacharovová, K; Berková, Z; Mindlová, M; Girman, P; Dovolilová, E; Karasová, L; Saudek, F

2007-01-01

The existence of an adult PSC that may be used in the treatment of diabetes is still a matter of scientific debate as conclusive evidence of such a stem cell in the adult pancreas has not yet been presented. The main reason why putative PSC has not yet been identified is the lack of specific markers that may be used to isolate and purify them. In order to increase the list of potential PSC markers we have focused on the human pancreatic cells that express cell surface receptor CXCR4, a marker of stem cells derived from different adult tissues. Here we report that CXCR4-positive pancreatic cells express markers of pancreatic endocrine progenitors (neurogenin-3, nestin) and markers of pluripotent stem cells (Oct-4, Nanog, ABCG2, CD133, CD117). Upon in vitro differentiation, these cells form ILCC and produce key islet hormones including insulin. Based on our results, we assume that CXCR4 marks pancreatic endocrine progenitors and in combination with other cell surface markers may be used in the attempt to identify and isolate PSC.

Metformin repositioning as antitumoral agent: selective antiproliferative effects in human glioblastoma stem cells, via inhibition of CLIC1-mediated ion current

PubMed Central

Barbieri, Federica; Peretti, Marta; Pizzi, Erika; Pattarozzi, Alessandra; Carra, Elisa; Sirito, Rodolfo; Daga, Antonio; Curmi, Paul M.G.; Mazzanti, Michele; Florio, Tullio

2014-01-01

Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown. Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity. The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment. PMID:25361004

Metformin repositioning as antitumoral agent: selective antiproliferative effects in human glioblastoma stem cells, via inhibition of CLIC1-mediated ion current.

PubMed

Gritti, Marta; Würth, Roberto; Angelini, Marina; Barbieri, Federica; Peretti, Marta; Pizzi, Erika; Pattarozzi, Alessandra; Carra, Elisa; Sirito, Rodolfo; Daga, Antonio; Curmi, Paul M G; Mazzanti, Michele; Florio, Tullio

2014-11-30

Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown. Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity. The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment.

Expansion on Stromal Cells Preserves the Undifferentiated State of Human Hematopoietic Stem Cells Despite Compromised Reconstitution Ability

PubMed Central

Magnusson, Mattias; Sierra, Maria I.; Sasidharan, Rajkumar; Prashad, Sacha L.; Romero, Melissa; Saarikoski, Pamela; Van Handel, Ben; Huang, Andy; Li, Xinmin; Mikkola, Hanna K. A.

2013-01-01

Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38−CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the ex vivo expanded CD34+CD38−CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of PBX1 regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful ex vivo expansion of transplantable HSC. PMID:23342037

Patient-Specific Induced Pluripotent Stem Cell Models: Generation and Characterization of Cardiac Cells.

PubMed

Zanella, Fabian; Sheikh, Farah

2016-01-01

The generation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes has been of utmost interest for the study of cardiac development, cardiac disease modeling, and evaluation of cardiotoxic effects of novel candidate drugs. Several protocols have been developed to guide human stem cells toward the cardiogenic path. Pioneering work used serum to promote cardiogenesis; however, low cardiogenic throughputs, lack of chemical definition, and batch-to-batch variability of serum lots constituted a considerable impediment to the implementation of those protocols to large-scale cell biology. Further work focused on the manipulation of pathways that mouse genetics indicated to be fundamental in cardiac development to promote cardiac differentiation in stem cells. Although extremely elegant, those serum-free protocols involved the use of human recombinant cytokines that tend to be quite costly and which can also be variable between lots. The latest generation of cardiogenic protocols aimed for a more cost-effective and reproducible definition of the conditions driving cardiac differentiation, using small molecules to manipulate cardiogenic pathways overriding the need for cytokines. This chapter details methods based on currently available cardiac differentiation protocols for the generation and characterization of robust numbers of hiPSC-derived cardiomyocytes under chemically defined conditions.

Concise Review: Stem Cell Interventions for People With Cerebral Palsy: Systematic Review With Meta-Analysis.

PubMed

Novak, Iona; Walker, Karen; Hunt, Rod W; Wallace, Euan M; Fahey, Michael; Badawi, Nadia

2016-08-01

: Evidence for stem cells as a potential intervention for cerebral palsy is emerging. Our objective was to determine the efficacy and safety of stem cells for improving motor and cognitive function of people with cerebral palsy. Searches were conducted in October 2015 in CENTRAL, EMBASE, MEDLINE, and Cochrane Libraries. Randomized controlled trials and controlled clinical trials of stem cells for cerebral palsy were included. Two authors independently decided upon included trials, extracted data, quality, and risk of bias. The primary outcome was gross motor function. Secondary outcomes were cognitive function and adverse events (AEs). Effects were expressed as standardized mean differences (SMD) with 95% confidence intervals (CI), using a random-effects model. Five trials comprising 328 participants met inclusion criteria. Four cell types were studied: olfactory ensheathing, neural, neural progenitors, and allogeneic umbilical cord blood (UCBs). Transplantation procedures differed from central nervous system neurosurgical transplantation to intravenous/arterial infusion. Participants were followed short-term for only 6 months. Evidence of variable quality indicated a small statistically significant intervention effect from stem cells on gross motor skills (SMD 1.27; 95% CI 0.22, 2.33), with UCBs most effective. There were insufficient and heterogeneous data to compare cognitive effects. Serious AEs were rare (n = 4/135 [3%] stem cells; n = 3/139 [2%] controls). Stem cells appeared to induce short-term improvements in motor skills. Different types of stem cell interventions were compared, meaning the data were heterogeneous and are a study limitation. Further randomized controlled trials are warranted, using rigorous methodologies. Stem cells are emerging as a scientifically plausible treatment and possible cure for cerebral palsy, but are not yet proven. The lack of valid animal models has significantly hampered the scope of clinical trials. Despite the state of current treatment evidence, parents remain optimistic about the potential improvements from stem cell intervention and feel compelled to exhaust all therapeutic options, including stem cell tourism. Receiving unproven therapies from unvalidated sources is potentially dangerous. Thus it is essential that researchers and clinicians stay up to date. A systematic review and meta-analysis summarizing and aggregating current research data may provide more conclusive evidence to inform treatment decision making and help direct future research. ©AlphaMed Press.

Concise Review: Stem Cell Interventions for People With Cerebral Palsy: Systematic Review With Meta-Analysis

PubMed Central

Walker, Karen; Hunt, Rod W.; Wallace, Euan M.; Fahey, Michael; Badawi, Nadia

2016-01-01

Evidence for stem cells as a potential intervention for cerebral palsy is emerging. Our objective was to determine the efficacy and safety of stem cells for improving motor and cognitive function of people with cerebral palsy. Searches were conducted in October 2015 in CENTRAL, EMBASE, MEDLINE, and Cochrane Libraries. Randomized controlled trials and controlled clinical trials of stem cells for cerebral palsy were included. Two authors independently decided upon included trials, extracted data, quality, and risk of bias. The primary outcome was gross motor function. Secondary outcomes were cognitive function and adverse events (AEs). Effects were expressed as standardized mean differences (SMD) with 95% confidence intervals (CI), using a random-effects model. Five trials comprising 328 participants met inclusion criteria. Four cell types were studied: olfactory ensheathing, neural, neural progenitors, and allogeneic umbilical cord blood (UCBs). Transplantation procedures differed from central nervous system neurosurgical transplantation to intravenous/arterial infusion. Participants were followed short-term for only 6 months. Evidence of variable quality indicated a small statistically significant intervention effect from stem cells on gross motor skills (SMD 1.27; 95% CI 0.22, 2.33), with UCBs most effective. There were insufficient and heterogeneous data to compare cognitive effects. Serious AEs were rare (n = 4/135 [3%] stem cells; n = 3/139 [2%] controls). Stem cells appeared to induce short-term improvements in motor skills. Different types of stem cell interventions were compared, meaning the data were heterogeneous and are a study limitation. Further randomized controlled trials are warranted, using rigorous methodologies. Significance Stem cells are emerging as a scientifically plausible treatment and possible cure for cerebral palsy, but are not yet proven. The lack of valid animal models has significantly hampered the scope of clinical trials. Despite the state of current treatment evidence, parents remain optimistic about the potential improvements from stem cell intervention and feel compelled to exhaust all therapeutic options, including stem cell tourism. Receiving unproven therapies from unvalidated sources is potentially dangerous. Thus it is essential that researchers and clinicians stay up to date. A systematic review and meta-analysis summarizing and aggregating current research data may provide more conclusive evidence to inform treatment decision making and help direct future research. PMID:27245364

Biogovernance Beyond the State: The Shaping of Stem Cell Therapy by Patient Organizations in India.

PubMed

Heitmeyer, Carolyn

2017-04-01

Public engagement through government-sponsored "public consultations" in biomedical innovation, specifically stem cell research and therapy, has been relatively limited in India. However, patient groups are drawing upon collaborations with medical practitioners to gain leverage in promoting biomedical research and the conditions under which patients can access experimental treatments. Based on qualitative fieldwork conducted between 2012 and 2015, I examine the ways in which two patient groups engaged with debates around how experimental stem cell therapy should be regulated, given the current lack of legally binding research guidelines. Such processes of engagement can be seen as an alternative form of biomedical governance which responds to the priorities and exigencies of Indian patients, contrasting with the current measures taken by the Indian state which, instead, are primarily directed at the global scientific and corporate world.

Stem cells for the prevention of neonatal lung disease.

PubMed

O'Reilly, Megan; Thébaud, Bernard

2015-01-01

Preterm birth affects approximately 11% of all newborns worldwide and is a major risk factor for infant mortality and morbidity. A common complication of preterm birth is the chronic lung disease of prematurity called bronchopulmonary dysplasia (BPD). Due to the lack of a specific treatment for BPD, preterm infants surviving with BPD face a lifelong risk of poor lung health. The therapeutic potential of stem cells in regenerative medicine is being harnessed for many diseases, including BPD. Compelling preclinical data using stem cells to prevent/repair lung damage in animal models of experimental BPD has built the basis for its translation into the clinic in preterm infants. This review highlights the exciting translation from bench to bedside that will hopefully lead in the near future to improved pulmonary outcomes in preterm infants. © 2015 S. Karger AG, Basel.

Monoamine Oxidase Deficiency Causes Prostate Atrophy and Reduces Prostate Progenitor Cell Activity.

PubMed

Yin, Lijuan; Li, Jingjing; Liao, Chun-Peng; Jason Wu, Boyang

2018-04-10

Monoamine oxidases (MAOs) degrade a number of biogenic and dietary amines, including monoamine neurotransmitters, and play an essential role in many biological processes. Neurotransmitters and related neural events have been shown to participate in the development, differentiation, and maintenance of diverse tissues and organs by regulating the specialized cellular function and morphological structures of innervated organs such as the prostate. Here we show that mice lacking both MAO isoforms, MAOA and MAOB, exhibit smaller prostate mass and develop epithelial atrophy in the ventral and dorsolateral prostates. The cellular composition of prostate epithelium showed reduced CK5 + or p63 + basal cells, accompanied by lower Sca-1 expression in p63 + basal cells, but intact differentiated CK8 + luminal cells in MAOA/B-deficient mouse prostates. MAOA/B ablation also decreased epithelial cell proliferation without affecting cell apoptosis in mouse prostates. Using a human prostate epithelial cell line, we found that stable knockdown of MAOA and MAOB impaired the capacity of prostate stem cells to form spheres, coinciding with a reduced CD133 + /CD44 + /CD24 - stem cell population and less expression of CK5 and select stem cell markers, including ALDH1A1, TROP2, and CD166. Alternative pharmacological inhibition of MAOs also repressed prostate cell stemness. In addition, we found elevated expression of MAOA and MAOB in epithelial and/or stromal components of human prostate hyperplasia samples compared with normal prostate tissues. Taken together, our findings reveal critical roles for MAOs in the regulation of prostate basal progenitor cells and prostate maintenance. Stem Cells 2018. © AlphaMed Press 2018.

Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.

PubMed

Kabiri, Zahra; Greicius, Gediminas; Madan, Babita; Biechele, Steffen; Zhong, Zhendong; Zaribafzadeh, Hamed; Edison; Aliyev, Jamal; Wu, Yonghui; Bunte, Ralph; Williams, Bart O; Rossant, Janet; Virshup, David M

2014-06-01

Wnt/β-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth cells as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (Porcn(Del)/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating that epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of Porcn(Del) organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable and that stromal production of Wnts can fully support normal murine intestinal homeostasis.

The Network of Epithelial-mesenchymal transition: potential new targets for tumor resistance

PubMed Central

Nantajit, Danupon; Lin, Dong; Li, Jian Jian

2014-01-01

Purpose In multiple cell metazoans, the ability of polarized epithelial cells to convert to motile mesenchymal cells in order to relocate to another location is governed by a unique process termed epithelial-mesenchymal transition (EMT). While being an essential process of cellular plasticity for normal tissue and organ developments, EMT is found to be involved in an array of malignant phenotypes of tumor cells including proliferation and invasion, angiogenesis, stemness of cancer cells and resistance to chemo-radiotherapy. Although EMT is being extensively studied and demonstrated to play a key role in tumor metastasis and in sustaining tumor hallmarks, there is a lack of clear picture of the overall EMT signaling network, wavering the potential clinical trials targeting EMT. Methods In this review, we highlight the potential key therapeutic targets of EMT linked with tumor aggressiveness, hypoxia, angiogenesis and cancer stem cells, emphasizing on an emerging EMT-associated NF-κB/HER2/STAT3 pathway in radioresistance of breast cancer stem cells. Results Further definition of cancer stem cell repopulation due to EMT-controlled tumor microenvironment will help to understand how tumors exploit the EMT mechanisms for their survival and expansion advantages. Conclusions The knowledge of EMT will offer more effective targets in clinical trials to treat therapy-resistant metastatic lesions. PMID:25270087

Pregnant women's knowledge and attitudes about stem cells and cord blood banking.

PubMed

Dinç, H; Sahin, N H

2009-06-01

This study was to determine pregnant women's knowledge and attitudes towards stem cells and cord blood banking in Istanbul, Turkey. Stem cell research is one of the most important and, at the same time, the most controversial topics of science and technology today. Nurses need to understand stem cell research so they can enter the debate on this issue. They can become important sources of information in order to help parents understand the issues. This exploratory descriptive study was conducted in two antenatal outpatient clinics in Istanbul. The sample consisted of 334 pregnant women during routine prenatal visits. Data were collected in interviews by using an interview form developed by the researchers according to the literature. The form included demographic characteristics of participants and 20 questions about stem cells, storing cord blood and banking and 10 independent attitude statements. The majority of the participants had a lack of knowledge about stem cells and cord blood banking and wanted more information. Before pregnancy, they received some information through the media (newspaper, Internet, television, etc.), but unintentionally. It was determined that they wanted information before becoming pregnant, more from their obstetrician but also from nurses and midwives. The majority also wanted to store their infants' cord blood and stated that they would be more likely to choose a public cord blood bank. Those giving ante- and perinatal care need to offer accurate and scientific counselling services on this subject to parents who need to be informed.

Harmonizing the international regulation of embryonic stem cell research: possibilities, promises and potential pitfalls.

PubMed

Campbell, Angela; Nycum, Gillian

2005-01-01

Despite near unanimous global opposition to human reproductive cloning, the United Nations has been unable to reach a consensus as to how cloning practices should be regulated at the international level. As a result, the U.N. objective of establishing binding international regulations governing cloning and stem cell research has yet to be achieved. Given the lack of consensus that exists within the global community on this topic, it seems that any attempt to harmonize the international regulation of cloning and stem cell science will face important obstacles. This paper seeks to illuminate the particular challenges to harmonizing international laws and policies related to stem cell research and human cloning, and to investigate potential methods for overcoming these challenges. By drawing on two other areas in which regulatory harmonization has been attempted, namely: environmental and human safety aspects of international trade, and pharmaceutical research and development, we study approaches to global regulatory harmonization. We conclude that while the challenges to harmonization are diverse and important, so too are the benefits of establishing uniformity in approaches to stem cell research worldwide. This paper proposes a model for harmonizing the regulation of stem cell research that focuses on broader norms and principles rather than specific rules. It further recommends that such harmonization should occur through a process initiated and developed by an independent international agency marked by diversity, both in terms of the cultural identities and perspectives represented, and the interdisciplinary expertise of its members.

Being a haematopoietic stem cell donor for a sick sibling: Adult donors' experiences prior to donation.

PubMed

Kisch, Annika; Bolmsjö, Ingrid; Lenhoff, Stig; Bengtsson, Mariette

2015-10-01

There is a lack of knowledge about sibling stem cell donors' experiences pre-donation and the waiting period before the donation might have been long. The donors and their corresponding sibling recipients were simultaneously included in two different interview studies. The results from the recipient study have been presented in a separate paper. The aim was to explore the experiences of being a stem cell donor for a sibling, prior to donation. Ten adult sibling donors were interviewed prior to stem cell donation. The interviews were digitally recorded, transcribed verbatim and subjected to qualitative content analysis. The main theme Being a cog in a big wheel describes the complex process of being a sibling donor prior to donation, covering a mixture of emotions and thoughts. The four subthemes Being available, Being anxious, Being concerned and Being obliged cover the various experiences. The sibling donors' experiences are influenced by the quality of the relationship with the sick sibling. Sibling stem cell donors go through a complex process once they have accidentally got involved in. They have been asked to become a donor; it was not a voluntary choice. In caring for sibling stem cell donors the nurses should be aware of the complexity of the process they experience and take into consideration their personal situation and needs. Providing optimal care for both sibling donors and their corresponding recipients is a challenge, and further improvement and exploration are needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hair cell regeneration

PubMed Central

Edge, Albert SB; Chen, Zheng-Yi

2017-01-01

The mammalian inner ear largely lacks the capacity to regenerate hair cells, the sensory cells required for hearing and balance. Recent studies in both lower vertebrates and mammals have uncovered genes and pathways important in hair cell development and have suggested ways that the sensory epithelia could be manipulated to achieve hair cell regeneration. These approaches include the use of inner ear stem cells, transdifferentiation of nonsensory cells, and induction of a proliferative response in the cells that can become hair cells. PMID:18929656

Lack of a p21waf1/cip -dependent G1/S checkpoint in neural stem and progenitor cells after DNA damage in vivo.

PubMed

Roque, Telma; Haton, Céline; Etienne, Olivier; Chicheportiche, Alexandra; Rousseau, Laure; Martin, Ludovic; Mouthon, Marc-André; Boussin, François D

2012-03-01

The cyclin-dependent kinase inhibitor p21(waf1/cip) mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2'deoxyuridine/5-bromo-2'-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21(waf1/cip) in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21(waf1/cip1) -dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage. Copyright © 2011 AlphaMed Press.

An Adult Mouse Thyroid Side Population Cell Line that Exhibits Enriched Epithelial–Mesenchymal Transition

PubMed Central

Murata, Tsubasa; Iwadate, Manabu; Takizawa, Yoshinori; Miyakoshi, Masaaki; Hayase, Suguru; Yang, Wenjing; Cai, Yan; Yokoyama, Shigetoshi; Nagashima, Kunio; Wakabayashi, Yoshiyuki; Zhu, Jun

2017-01-01

Background: Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. Method: A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. Results: These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial–mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. Conclusion: These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid. PMID:28125936

Application of stem cells for cardiovascular grafts tissue engineering.

PubMed

Wu, Kaihong; Liu, Ying Long; Cui, Bin; Han, Zhongchao

2006-06-01

Congenital and acquired heart diseases are leading causes of morbidity and mortality world-wide. Currently, the synthetic materials or bioprosthetic replacement devices for cardiovascular surgery are imperfect and subject patients to one or more ongoing risks including thrombosis, limited durability and need for reoperations due to lack of growth in children and young adults. Suitable replacement grafts should have appropriate characteristics, including resistance to infection, low immunogenicity, good biocompatability and thromboresistance, with appropriate mechanical and physiological properties. Tissue engineering is a new scientific field aiming at fabrication of living, autologous grafts having structure or function properties that can be used to restore, maintain or improve tissue function. The use of autologous stem cells in cardiovascular tissue engineering is quite promising due to their capacity of self-renewal, high proliferation, and differentiation into specialized progeny. Progress has been made in engineering the various components of the cardiovascular system, including myocardial constructs, heart valves, and vascular patches or conduits with autologous stem cells. This paper will review the current achievements in stem cell-based cardiovascular grafts tissue engineering, with an emphasis on its clinical or possible clinical use in cardiovascular surgery.

Regulation of stem cell therapies under attack in Europe: for whom the bell tolls

PubMed Central

Bianco, Paolo; Barker, Roger; Brüstle, Oliver; Cattaneo, Elena; Clevers, Hans; Daley, George Q; De Luca, Michele; Goldstein, Lawrence; Lindvall, Olle; Mummery, Christine; Robey, Pamela G; Sattler de Sousa e Brito, Clara; Smith, Austin

2013-01-01

At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context—social, financial, medical, legal—in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of ‘translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients. PMID:23644381

A GRFa2/Prop1/stem (GPS) cell niche in the pituitary.

PubMed

Garcia-Lavandeira, Montse; Quereda, Víctor; Flores, Ignacio; Saez, Carmen; Diaz-Rodriguez, Esther; Japon, Miguel A; Ryan, Aymee K; Blasco, Maria A; Dieguez, Carlos; Malumbres, Marcos; Alvarez, Clara V

2009-01-01

The adult endocrine pituitary is known to host several hormone-producing cells regulating major physiological processes during life. Some candidates to progenitor/stem cells have been proposed. However, not much is known about pituitary cell renewal throughout life and its homeostatic regulation during specific physiological changes, such as puberty or pregnancy, or in pathological conditions such as tumor development. We have identified in rodents and humans a niche of non-endocrine cells characterized by the expression of GFRa2, a Ret co-receptor for Neurturin. These cells also express b-Catenin and E-cadherin in an oriented manner suggesting a planar polarity organization for the niche. In addition, cells in the niche uniquely express the pituitary-specific transcription factor Prop1, as well as known progenitor/stem markers such as Sox2, Sox9 and Oct4. Half of these GPS (GFRa2/Prop1/Stem) cells express S-100 whereas surrounding elongated cells in contact with GPS cells express Vimentin. GFRa2+-cells form non-endocrine spheroids in culture. These spheroids can be differentiated to hormone-producing cells or neurons outlining the neuroectoderm potential of these progenitors. In vivo, GPSs cells display slow proliferation after birth, retain BrdU label and show long telomeres in its nuclei, indicating progenitor/stem cell properties in vivo. Our results suggest the presence in the adult pituitary of a specific niche of cells characterized by the expression of GFRa2, the pituitary-specific protein Prop1 and stem cell markers. These GPS cells are able to produce different hormone-producing and neuron-like cells and they may therefore contribute to postnatal pituitary homeostasis. Indeed, the relative abundance of GPS numbers is altered in Cdk4-deficient mice, a model of hypopituitarism induced by the lack of this cyclin-dependent kinase. Thus, GPS cells may display functional relevance in the physiological expansion of the pituitary gland throughout life as well as protection from pituitary disease.

A GRFa2/Prop1/Stem (GPS) Cell Niche in the Pituitary

PubMed Central

Garcia-Lavandeira, Montse; Quereda, Víctor; Flores, Ignacio; Saez, Carmen; Diaz-Rodriguez, Esther; Japon, Miguel A.; Ryan, Aymee K.; Blasco, Maria A.; Dieguez, Carlos; Malumbres, Marcos; Alvarez, Clara V.

2009-01-01

Background The adult endocrine pituitary is known to host several hormone-producing cells regulating major physiological processes during life. Some candidates to progenitor/stem cells have been proposed. However, not much is known about pituitary cell renewal throughout life and its homeostatic regulation during specific physiological changes, such as puberty or pregnancy, or in pathological conditions such as tumor development. Principal Findings We have identified in rodents and humans a niche of non-endocrine cells characterized by the expression of GFRa2, a Ret co-receptor for Neurturin. These cells also express b-Catenin and E-cadherin in an oriented manner suggesting a planar polarity organization for the niche. In addition, cells in the niche uniquely express the pituitary-specific transcription factor Prop1, as well as known progenitor/stem markers such as Sox2, Sox9 and Oct4. Half of these GPS (GFRa2/Prop1/Stem) cells express S-100 whereas surrounding elongated cells in contact with GPS cells express Vimentin. GFRa2+-cells form non-endocrine spheroids in culture. These spheroids can be differentiated to hormone-producing cells or neurons outlining the neuroectoderm potential of these progenitors. In vivo, GPSs cells display slow proliferation after birth, retain BrdU label and show long telomeres in its nuclei, indicating progenitor/stem cell properties in vivo. Significance Our results suggest the presence in the adult pituitary of a specific niche of cells characterized by the expression of GFRa2, the pituitary-specific protein Prop1 and stem cell markers. These GPS cells are able to produce different hormone-producing and neuron-like cells and they may therefore contribute to postnatal pituitary homeostasis. Indeed, the relative abundance of GPS numbers is altered in Cdk4-deficient mice, a model of hypopituitarism induced by the lack of this cyclin-dependent kinase. Thus, GPS cells may display functional relevance in the physiological expansion of the pituitary gland throughout life as well as protection from pituitary disease. PMID:19283075

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

PubMed

Jenny, Robert A; Hirst, Claire; Lim, Sue Mei; Goulburn, Adam L; Micallef, Suzanne J; Labonne, Tanya; Kicic, Anthony; Ling, Kak-Ming; Stick, Stephen M; Ng, Elizabeth S; Trounson, Alan; Giudice, Antonietta; Elefanty, Andrew G; Stanley, Edouard G

2015-06-01

Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold. ©AlphaMed Press.

Potential differentiation of islet-like cells from pregnant cow-derived placental stem cells.

PubMed

Peng, Shao-Yu; Chou, Chien-Wen; Kuo, Yu-Hsuan; Shen, Perng-Chih; Shaw, S W Steven

2017-06-01

Type 1 diabetes is an autoimmune disease that destroys islet cells and results in insufficient insulin secretion by pancreatic β-cells. Islet transplantation from donors is an approach used for treating patients with diabetes; however, this therapy is difficult to implement because of the lack of donors. Nevertheless, several stem cells have the potential to differentiate from islet-like cells and enable insulin secretion for treating diabetes in animal models. For example, placenta is considered a waste material and can be harvested noninvasively during delivery without ethical or moral concerns. To date, the differentiation of islet-like cells from cow-derived placental stem cells (CPSCs) has yet to be demonstrated. The investigation of potential differentiation of islet-like cells from CPSCs was conducted by supplementation with nicotinamide, exendin-4, glucose, and poly-d-lysine and was detected through reverse transcription polymerase chain reaction, dithizone staining, and immunocytochemical methods. Our results indicated that CPSCs are established and express mesenchymal stem cell surface antigen markers, such as CD73, CD166, β-integrin, and Oct-4, but not hematopoietic stem cell surface antigen markers, such as CD45. After induction, the CPSCs successfully differentiated into islet-like cells. The CPSC-derived islet-like cells expressed islet cell development-related genes, such as insulin, glucagon, pax-4, Nkx6.1, pax-6, and Fox. Moreover, CPSC-derived islet-like cells can be stained with zinc ions, which are widely distributed in the islet cells and enable insulin secretion. Altogether, islet-like cells have the potential to be differentiated from CPSCs without gene manipulation, and can be used in diabetic animal models in the future for preclinical and drug testing trial investigations. Copyright © 2017. Published by Elsevier B.V.

In vitro analysis of equine, bone marrow-derived mesenchymal stem cells demonstrates differences within age- and gender-matched horses.

PubMed

Carter-Arnold, J L; Neilsen, N L; Amelse, L L; Odoi, A; Dhar, M S

2014-09-01

Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. Descriptive study of stem cell characteristics. Equine BMMSCs from 6 horses were cultured in vitro and evaluated for viability, proliferation, osteogenesis, chondrogenesis, adipogenesis, cluster-of-differentiation markers and gene expression. Equine BMMSCs from all 6 donors demonstrated fibroblastic, cellular morphology, adherence to plastic and expression of cluster-of-differentiation markers. They varied in their rate of proliferation and trilineage differentiation. The equine BMMSCs of one of 6 donors demonstrated a higher rate of proliferation, enhanced ability for cell passaging and a more robust in vitro differentiation. Comparatively, equine BMMSCs from 2 donors demonstrated a lower rate of proliferation and lack of osteogenic and chondrogenic differentiation. The results of this study confirm that donor-to-donor variation in equine BMMSCs exists and this variation can be documented using in vitro assays. Subjective assessment suggests that the rate of proliferation tends to correlate with differentiation potential. © 2013 EVJ Ltd.

GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis.

PubMed

Dietrich, Philipp A; Yang, Chen; Leung, Halina H L; Lynch, Jennifer R; Gonzales, Estrella; Liu, Bing; Haber, Michelle; Norris, Murray D; Wang, Jianlong; Wang, Jenny Yingzi

2014-11-20

β-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of β-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active β-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and β-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant β-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/β-catenin signaling axis may represent a novel therapeutic strategy for AML. © 2014 by The American Society of Hematology.

Comparison of the glycosphingolipids of human-induced pluripotent stem cells and human embryonic stem cells.

PubMed

Säljö, Karin; Barone, Angela; Vizlin-Hodzic, Dzeneta; Johansson, Bengt R; Breimer, Michael E; Funa, Keiko; Teneberg, Susann

2017-04-01

High expectations are held for human-induced pluripotent stem cells (hiPSC) since they are established from autologous tissues thus overcoming the risk of allogeneic immune rejection when used in regenerative medicine. However, little is known regarding the cell-surface carbohydrate antigen profile of hiPSC compared with human embryonic stem cells (hESC). Here, glycosphingolipids were isolated from an adipocyte-derived hiPSC line, and hiPSC and hESC glycosphingolipids were compared by concurrent characterization by binding assays with carbohydrate-recognizing ligands and mass spectrometry. A high similarity between the nonacid glycosphingolipids of hiPSC and hESC was found. The nonacid glycosphingolipids P1 pentaosylceramide, x2 pentaosylceramide and H type 1 heptaosylceramide, not previously described in human pluripotent stem cells (hPSC), were characterized in both hiPSC and hESC. The composition of acid glycosphingolipids differed, with increased levels of GM3 ganglioside, and reduced levels of GD1a/GD1b in hiPSC when compared with hESC. In addition, the hESC glycosphingolipids sulf-globopentaosylceramide and sialyl-globotetraosylceramide were lacking in hiPSC. Neural stem cells differentiating from hiPSC had a reduced expression of sialyl-lactotetra, whereas expression of the GD1a ganglioside was significantly increased. Thus, while sialyl-lactotetra is a marker of undifferentiated hPSC, GD1a is a novel marker of neural differentiation. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

PubMed Central

Song, Min Jae; Dean, David; Knothe Tate, Melissa L.

2010-01-01

A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms. PMID:20862249

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

PubMed Central

Zhou, Jian; McDowell, William; Park, Jungeun; Haug, Jeff; Staehling, Karen; Tang, Hong; Xie, Ting

2011-01-01

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms. PMID:22216012

Novel MRI Contrast Agent from Magnetotactic Bacteria Enables In Vivo Tracking of iPSC-derived Cardiomyocytes.

PubMed

Mahmoudi, Morteza; Tachibana, Atsushi; Goldstone, Andrew B; Woo, Y Joseph; Chakraborty, Papia; Lee, Kayla R; Foote, Chandler S; Piecewicz, Stephanie; Barrozo, Joyce C; Wakeel, Abdul; Rice, Bradley W; Bell Iii, Caleb B; Yang, Phillip C

2016-06-06

Therapeutic delivery of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) represents a novel clinical approach to regenerate the injured myocardium. However, methods for robust and accurate in vivo monitoring of the iCMs are still lacking. Although superparamagnetic iron oxide nanoparticles (SPIOs) are recognized as a promising tool for in vivo tracking of stem cells using magnetic resonance imaging (MRI), their signal persists in the heart even weeks after the disappearance of the injected cells. This limitation highlights the inability of SPIOs to distinguish stem cell viability. In order to overcome this shortcoming, we demonstrate the use of a living contrast agent, magneto-endosymbionts (MEs) derived from magnetotactic bacteria for the labeling of iCMs. The ME-labeled iCMs were injected into the infarcted area of murine heart and probed by MRI and bioluminescence imaging (BLI). Our findings demonstrate that the MEs are robust and effective biological contrast agents to track iCMs in an in vivo murine model. We show that the MEs clear within one week of cell death whereas the SPIOs remain over 2 weeks after cell death. These findings will accelerate the clinical translation of in vivo MRI monitoring of transplanted stem cell at high spatial resolution and sensitivity.

Novel anticancer activity of phloroglucinol against breast cancer stem-like cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Rae-Kwon; Uddin, Nizam; Hyun, Jin-Won

Poor prognosis of breast cancer patients is closely associated with metastasis and relapse. There is substantial evidence supporting that cancer stem-like cells (CSCs) are primarily responsible for relapse in breast cancer after anticancer treatment. However, there is a lack of suitable drugs that target breast cancer stem-like cells (BCSCs). Here, we report that phloroglucinol (PG), a natural phlorotannin component of brown algae, suppresses sphere formation, anchorage-independent colony formation and in vivo tumorigenicity. In line with these observations, treatment with PG also decreased CD44{sup +} cancer cell population as well as expression of CSC regulators such as Sox2, CD44, Oct4, Notch2more » and β-catenin. Also, treatment with PG sensitized breast cancer cells to anticancer drugs such as cisplatin, etoposide, and taxol as well as to ionizing radiation. Importantly, PG inhibited KRAS and its downstream PI3K/AKT and RAF-1/ERK signaling pathways that regulate the maintenance of CSCs. Taken together, our findings implicate PG as a good candidate to target BCSCs and to prevent the disease relapse. - Highlights: • Phloroglucinol suppresses in vivo tumor formation. • Phloroglucinol sensitizes breast cancer cells to anticancer agents. • Phloroglucinol inhibits breast cancer stem-like cells. • Phloroglucinol inhibits PI3K/AKT and KRAS/RAF/ERK signaling pathways.« less

Isolation and biological characterization of tendon-derived stem cells from fetal bovine.

PubMed

Yang, Jinjuan; Zhao, Qianjun; Wang, Kunfu; Liu, Hao; Ma, Caiyun; Huang, Hongmei; Liu, Yingjie

2016-09-01

The lack of appropriate candidates of cell sources for cell transplantation has hampered efforts to develop therapies for tendon injuries, such as tendon rupture, tendonitis, and tendinopathy. Tendon-derived stem cells (TDSCs) are a type of stem cells which may be used in the treatment of tendon injuries. In this study, TDSCs were isolated from 5-mo-old Luxi Yellow fetal bovine and cultured in vitro and further analyzed for their biological characteristics using immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR) assays. It was found that primary TDSCs could be expanded for 42 passages in vitro maintaining proliferation. The expressions of stem cell marker nucleostemin and tenocyte-related markers, such as collagen I, collagen II, collagen III, and tenascin-C, were observed on different passage cells by immunofluorescence. The results from RT-PCR show that TDSCs were positive for collagen type I, CD44, tenascin-C, and collagen type III but negative for collagen type II. Meanwhile, TDSC passage 4 was successfully induced to differentiate into osteoblasts, adipocytes, and chondrocytes. Our results indicate that the fetal bovine TDSCs not only had strong self-renewal capacity but also possess the potential for multi-lineage differentiation. This study provides theoretical basis and experimental foundation for potential therapeutic application of the fetal bovine TDSCs in the treatment of tendon injuries.

The role of morphine on rat neural stem cells viability, neuro-angiogenesis and neuro-steroidgenesis properties.

PubMed

Abdyazdani, Nima; Nourazarian, Alireza; Nozad Charoudeh, Hojjatollah; Kazemi, Masoumeh; Feizy, Navid; Akbarzade, Maryam; Mehdizadeh, Amir; Rezaie, Jafar; Rahbarghazi, Reza

2017-01-01

A lack of comprehensive data exists on the effect of morphine on neural stem cell neuro-steroidogenesis and neuro-angiogenesis properties. We, herein, investigated the effects of morphine (100μM), naloxone (100μM) and their combination on rat neural stem cells viability, clonogenicity and Ki-67 expression over a period of 72h. Any alterations in the total fatty acids profile under treatment protocols were elucidated by direct transesterification method. We also monitored the expression of p53, aromatase and 5-alpha reductase by real-time PCR assay. To examine angiogenic capacity, in vitro tubulogenesis and the level of VE-cadherin transcript were investigated during neural to endothelial differentiation under the experimental procedure. Cells supplemented with morphine displayed reduced survival (p<0.01) and clonogenicity (p<0.001). Flow cytometric analysis showed a decrease in Ki-67 during 72h. Naloxone potentially blunted morphine-induced all effects. The normal levels of fatty acids, including saturated and unsaturated were altered by naloxone and morphine supplements. Following 48h, the up-regulation of p53, aromatase and 5-alpha reductase genes occurred in morphine-primed cells. Using three-dimensional culture models of angiogenesis and real time PCR assay, we showed morphine impaired the tubulogenesis properties of neural stem cells (p<0.001) by the inhibition of trans-differentiation into vascular cells and led to decrease of in VE-cadherin expression. Collectively, morphine strongly impaired the healthy status of neural stem cells by inducing p53 and concurrent elevation of aromatase and 5-alpha reductase activities especially during early 48h. Also, neural stem cells-being exposed to morphine lost their potency to elicit angiogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A cell-penetrating peptide based on the interaction between c-Src and connexin43 reverses glioma stem cell phenotype

PubMed Central

Gangoso, E; Thirant, C; Chneiweiss, H; Medina, J M; Tabernero, A

2014-01-01

Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is downregulated in malignant gliomas. These tumors are composed of a heterogeneous population of cells that include many with stem-cell-like properties, called glioma stem cells (GSCs), which are highly tumorigenic and lack Cx43 expression. Interestingly, restoring Cx43 reverses GSC phenotype and consequently reduces their tumorigenicity. In this study, we investigated the mechanism by which Cx43 exerts its antitumorigenic effects on GSCs. We have focused on the tyrosine kinase c-Src, which interacts with the intracellular carboxy tail of Cx43. We found that Cx43 regulates c-Src activity and proliferation in human GSCs expanded in adherent culture. Thus, restoring Cx43 in GSCs inhibited c-Src activity, which in turn promoted the downregulation of the inhibitor of differentiation Id1. Id1 sustains stem cell phenotype as it controls the expression of Sox2, responsible for stem cell self-renewal, and promotes cadherin switching, which has been associated to epithelial–mesenchymal transition. Our results show that both the ectopic expression of Cx43 and the inhibition of c-Src reduced Id1, Sox2 expression and promoted the switch from N- to E-cadherin, suggesting that Cx43, by inhibiting c-Src, downregulates Id1 with the subsequent changes in stem cell phenotype. On the basis of this mechanism, we found that a cell-penetrating peptide, containing the region of Cx43 that interacts with c-Src, mimics the effect of Cx43 on GSC phenotype, confirming the relevance of the interaction between Cx43 and c-Src in the regulation of the malignant phenotype and pinpointing this interaction as a promising therapeutic target. PMID:24457967

Culture on 3D Chitosan-Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells.

PubMed

Wang, Kui; Kievit, Forrest M; Erickson, Ariane E; Silber, John R; Ellenbogen, Richard G; Zhang, Miqin

2016-12-01

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

aPKCζ-dependent Repression of Yap is Necessary for Functional Restoration of Irradiated Salivary Glands with IGF-1.

PubMed

Chibly, Alejandro M; Wong, Wen Yu; Pier, Maricela; Cheng, Hongqiang; Mu, Yongxin; Chen, Ju; Ghosh, Sourav; Limesand, Kirsten H

2018-04-20

Xerostomia and salivary hypofunction often result as a consequence of radiation therapy for head and neck cancers, which are diagnosed in roughly 60,000 individuals every year in the U.S. Due to the lack of effective treatments for radiation-induced salivary hypofunction, stem cell-based therapies have been suggested to regenerate the irradiated salivary glands. Pharmacologically, restoration of salivary gland function has been accomplished in mice by administering IGF-1 shortly after radiation treatment, but it is not known if salivary stem and progenitor cells play a role. We show that radiation inactivates aPKCζ and promotes nuclear redistribution of Yap in a population of label-retaining cells in the acinar compartment of the parotid gland (PG)- which comprises a heterogeneous pool of salivary progenitors. Administration of IGF-1 post-radiation maintains activation of aPKCζ and partially rescues Yap's cellular localization in label retaining cells, while restoring salivary function. Finally, IGF-1 fails to restore saliva production in mice lacking aPKCζ, demonstrating the importance of the kinase as a potential therapeutic target.

Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

PubMed Central

Sansom, Stephen N.; Alsiö, Jessica M.; Kaneda, Masahiro; Smith, James; O'Carroll, Donal; Tarakhovsky, Alexander; Livesey, Frederick J.

2010-01-01

Background To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs. Principal Findings Dicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia. Conclusions The block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation. PMID:20976144

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9hi, SSEA-1− Cells in Embryonic Stem Cell-Derived Embryoid Bodies

PubMed Central

Lian, Qizhou; Yeo, KengSuan; Que, Jianwen; Tan, EileenKhiaWay; Yu, Fenggang; Yin, Yijun; Salto-Tellez, Manuel; Oakley, Reida Menshawe El; Lim, Sai-Kiang

2006-01-01

Background Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. Methodology/Principal Findings We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r2 = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9hi, SSEA-1− while ESCs are CD9lo, SSEA-1+. Isolation of CD9hi, SSEA-1− cells that constituted 1%–10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r2 = 0.95) and a propensity to differentiate into endothelial-like cells. Conclusions By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs. PMID:17183690

Nanoscale definition of substrate materials to direct human adult stem cells towards tissue specific populations.

PubMed

Curran, Judith M; Chen, Rui; Stokes, Robert; Irvine, Eleanor; Graham, Duncan; Gubbins, Earl; Delaney, Deany; Amro, Nabil; Sanedrin, Raymond; Jamil, Haris; Hunt, John A

2010-03-01

The development of homogenously nano-patterned chemically modified surfaces that can be used to initiate a cellular response, particularly stem cell differentiation, in a highly controlled manner without the need for exogenous biological factors has never been reported, due to that fact that precisely defined and reproducible systems have not been available that can be used to study cell/material interactions and unlock the potential of a material driven cell response. Until now material driven stem cell (furthermore any cell) responses have been variable due to the limitations in definition and reproducibility of the underlying substrate and the lack of true homogeneity of modifications that can dictate a cellular response at a sub-micron level that can effectively control initial cell interactions of all cells that contact the surface. Here we report the successful design and use of homogenously molecularly nanopatterned surfaces to control initial stem cell adhesion and hence function. The highly specified nano-patterned arrays were compared directly to silane modified bulk coated substrates that have previously been proven to initiate mesenchymal stem cell (MSC) differentiation in a heterogenous manner, the aim of this study was to prove the efficiency of these previously observed cell responses could be enhanced by the incorporation of nano-patterns. Nano-patterned surfaces were prepared by Dip Pen Nanolithography (DPN) to produce arrays of 70 nm sized dots separated by defined spacings of 140, 280 and 1000 nm with terminal functionalities of carboxyl, amino, methyl and hydroxyl and used to control cell growth. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and will change the capabilities for stem cell definition in vitro and then cell based medical therapies. In addition to highlighting the ability of the materials to control stem cell functionality on an unprecedented scale this research also introduces the successful scale-up of DPN and the novel chemistries and systems to facilitate the production of homogeneously patterned substrates (5 mm2) that are applicable for use in in vitro cell conditions over prolonged periods for complete control of material driven cell responses.

Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells.

PubMed

Nadadhur, Aishwarya G; Emperador Melero, Javier; Meijer, Marieke; Schut, Desiree; Jacobs, Gerbren; Li, Ka Wan; Hjorth, J J Johannes; Meredith, Rhiannon M; Toonen, Ruud F; Van Kesteren, Ronald E; Smit, August B; Verhage, Matthijs; Heine, Vivi M

2017-01-01

Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory-inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory-inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

PubMed Central

Danovi, Davide; Folarin, Amos; Gogolok, Sabine; Ender, Christine; Elbatsh, Ahmed M. O.; Engström, Pär G.; Stricker, Stefan H.; Gagrica, Sladjana; Georgian, Ana; Yu, Ding; U, Kin Pong; Harvey, Kevin J.; Ferretti, Patrizia; Paddison, Patrick J.; Preston, Jane E.; Abbott, N. Joan; Bertone, Paul; Smith, Austin; Pollard, Steven M.

2013-01-01

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF−/−, or p53−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value. PMID:24204733

Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

PubMed

Esain, Virginie; Cortes, Mauricio; North, Trista E

2016-01-01

Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

Do ray cells provide a pathway for radial water movement in the stems of conifer trees?

PubMed

Barnard, David M; Lachenbruch, Barbara; McCulloh, Katherine A; Kitin, Peter; Meinzer, Frederick C

2013-02-01

The pathway of radial water movement in tree stems presents an unknown with respect to whole-tree hydraulics. Radial profiles have shown substantial axial sap flow in deeper layers of sapwood (that may lack direct connection to transpiring leaves), which suggests the existence of a radial pathway for water movement. Rays in tree stems include ray tracheids and/or ray parenchyma cells and may offer such a pathway for radial water transport. This study investigated relationships between radial hydraulic conductivity (k(s-rad)) and ray anatomical and stem morphological characteristics in the stems of three conifer species whose distributions span a natural aridity gradient across the Cascade Mountain range in Oregon, United States. The k(s-rad) was measured with a high-pressure flow meter. Ray tracheid and ray parenchyma characteristics and water transport properties were visualized using autofluorescence or confocal microscopy. The k(s-rad) did not vary predictably with sapwood depth among species and populations. Dye tracer did not infiltrate ray tracheids, and infiltration into ray parenchyma was limited. Regression analyses revealed inconsistent relationships between k(s-rad) and selected anatomical or growth characteristics when ecotypes were analyzed individually and weak relationships between k(s-rad) and these characteristics when data were pooled by tree species. The lack of significant relationships between k(s-rad) and the ray and stem morphologies we studied, combined with the absence of dye tracer in ray tracheid and limited movement of dye into ray parenchyma suggests that rays may not facilitate radial water transport in the three conifer species studied.

Unproven stem cell-based interventions and achieving a compromise policy among the multiple stakeholders.

PubMed

Matthews, Kirstin R W; Iltis, Ana S

2015-11-04

In 2004, patient advocate groups were major players in helping pass and implement significant public policy and funding initiatives in stem cells and regenerative medicine. In the following years, advocates were also actively engaged in Washington DC, encouraging policy makers to broaden embryonic stem cell research funding, which was ultimately passed after President Barack Obama came into office. Many advocates did this because they were told stem cell research would lead to cures. After waiting more than 10 years, many of these same patients are now approaching clinics around the world offering experimental stem cell-based interventions instead of waiting for scientists in the US to complete clinical trials. How did the same groups who were once (and often still are) the strongest supporters of stem cell research become stem cell tourists? And how can scientists, clinicians, and regulators work to bring stem cell patients back home to the US and into the clinical trial process? In this paper, we argue that the continued marketing and use of experimental stem cell-based interventions is problematic and unsustainable. Central problems include the lack of patient protection, US liability standards, regulation of clinical sites, and clinician licensing. These interventions have insufficient evidence of safety and efficacy; patients may be wasting money and time, and they may be forgoing other opportunities for an intervention that has not been shown to be safe and effective. Current practices do not contribute to scientific progress because the data from the procedures are unsuitable for follow-up research to measure outcomes. In addition, there is no assurance for patients that they are receiving the interventions promised or of what dosage they are receiving. Furthermore, there is inconsistent or non-existent follow-up care. Public policy should be developed to correct the current situation. The current landscape of stem cell tourism should prompt a re-evaluation of current approaches to study cell-based interventions with respect to the design, initiation, and conduct of US clinical trials. Stakeholders, including scientists, clinicians, regulators and patient advocates, need to work together to find a compromise to keep patients in the US and within the clinical trial process. Using HIV/AIDS and breast cancer advocate cases as examples, we identify key priorities and goals for this policy effort.

Nociceptive tuning by stem cell factor/c-Kit signaling.

PubMed

Milenkovic, Nevena; Frahm, Christina; Gassmann, Max; Griffel, Carola; Erdmann, Bettina; Birchmeier, Carmen; Lewin, Gary R; Garratt, Alistair N

2007-12-06

The molecular mechanisms regulating the sensitivity of sensory circuits to environmental stimuli are poorly understood. We demonstrate here a central role for stem cell factor (SCF) and its receptor, c-Kit, in tuning the responsiveness of sensory neurons to natural stimuli. Mice lacking SCF/c-Kit signaling displayed profound thermal hypoalgesia, attributable to a marked elevation in the thermal threshold and reduction in spiking rate of heat-sensitive nociceptors. Acute activation of c-Kit by its ligand, SCF, resulted in a reduced thermal threshold and potentiation of heat-activated currents in isolated small-diameter neurons and thermal hyperalgesia in mice. SCF-induced thermal hyperalgesia required the TRP family cation channel TRPV1. Lack of c-Kit signaling during development resulted in hypersensitivity of discrete mechanoreceptive neuronal subtypes. Thus, c-Kit can now be grouped with a small family of receptor tyrosine kinases, including c-Ret and TrkA, that control the transduction properties of sensory neurons.

Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

PubMed

Sleeboom-Faulkner, Margaret

2010-01-01

This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

Electromechanical integration of cardiomyocytes derived from human embryonic stem cells.

PubMed

Kehat, Izhak; Khimovich, Leonid; Caspi, Oren; Gepstein, Amira; Shofti, Rona; Arbel, Gil; Huber, Irit; Satin, Jonathan; Itskovitz-Eldor, Joseph; Gepstein, Lior

2004-10-01

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

The relevance of human stem cell-derived organoid models for epithelial translational medicine

PubMed Central

Hynds, Robert E.; Giangreco, Adam

2014-01-01

Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. PMID:23203919

Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture.

PubMed

Dahlmann, Julia; Awad, George; Dolny, Carsten; Weinert, Sönke; Richter, Karin; Fischer, Klaus-Dieter; Munsch, Thomas; Leßmann, Volkmar; Volleth, Marianne; Zenker, Martin; Chen, Yaoyao; Merkl, Claudia; Schnieke, Angelika; Baraki, Hassina; Kutschka, Ingo; Kensah, George

2018-01-01

The possibility to generate cardiomyocytes from pluripotent stem cells in vitro has enormous significance for basic research, disease modeling, drug development and heart repair. The concept of heart muscle reconstruction has been studied and optimized in the rat model using rat primary cardiovascular cells or xenogeneic pluripotent stem cell derived-cardiomyocytes for years. However, the lack of rat pluripotent stem cells (rPSCs) and their cardiovascular derivatives prevented the establishment of an authentic clinically relevant syngeneic or allogeneic rat heart regeneration model. In this study, we comparatively explored the potential of recently available rat embryonic stem cells (rESCs) and induced pluripotent stem cells (riPSCs) as a source for cardiomyocytes (CMs). We developed feeder cell-free culture conditions facilitating the expansion of undifferentiated rPSCs and initiated cardiac differentiation by embryoid body (EB)-formation in agarose microwell arrays, which substituted the robust but labor-intensive hanging drop (HD) method. Ascorbic acid was identified as an efficient enhancer of cardiac differentiation in both rPSC types by significantly increasing the number of beating EBs (3.6 ± 1.6-fold for rESCs and 17.6 ± 3.2-fold for riPSCs). These optimizations resulted in a differentiation efficiency of up to 20% cTnTpos rPSC-derived CMs. CMs showed spontaneous contractions, expressed cardiac markers and had typical morphological features. Electrophysiology of riPSC-CMs revealed different cardiac subtypes and physiological responses to cardio-active drugs. In conclusion, we describe rPSCs as a robust source of CMs, which is a prerequisite for detailed preclinical studies of myocardial reconstruction in a physiologically and immunologically relevant small animal model.

Establishing a Quality Control System for Stem Cell-Based Medicinal Products in China

PubMed Central

2015-01-01

Stem cell-based medicinal products (SCMPs) are emerging as novel therapeutic products. The success of its development depends on the existence of an effective quality control system, which is constituted by quality control technologies, standards, reference materials, guidelines, and the associated management system in accordance with regulatory requirements along product lifespan. However, a worldwide, effective quality control system specific for SCMPs is still far from established partially due to the limited understanding of stem cell sciences and lack of quality control technologies for accurately assessing the safety and biological effectiveness of SCMPs before clinical use. Even though, based on the existing regulations and current stem cell sciences and technologies, initial actions toward the goal of establishing such a system have been taken as exemplified by recent development of new “interim guidelines” for governing quality control along development of SCMPs and new development of the associated quality control technologies in China. In this review, we first briefly introduced the major institutions involved in the regulation of cell substrates and therapeutic cell products in China and the existing regulatory documents and technical guidelines used as critical references for developing the new interim guidelines. With focus only on nonhematopoietic stem cells, we then discussed the principal quality attributes of SCMPs as well as our thinking of proper testing approaches to be established with relevant evaluation technologies to ensure all quality requirements of SCMPs along different manufacturing processes and development stages. At the end, some regulatory and technical challenges were also discussed with the conclusion that combined efforts should be taken to promote stem cell regulatory sciences to establish the effective quality control system for SCMPs. PMID:25471126

Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer.

PubMed

Siegfried, Jill M; Farooqui, Mariya; Rothenberger, Natalie J; Dacic, Sanja; Stabile, Laura P

2017-04-11

The estrogen receptor (ER) promotes non-small cell lung cancer (NSCLC) proliferation. Since fibroblast growth factors (FGFs) are known regulators of stem cell markers in ER positive breast cancer, we investigated whether a link between the ER, FGFs, and stem cell markers exists in NSCLC. In lung preneoplasias and adenomas of tobacco carcinogen exposed mice, the anti-estrogen fulvestrant and/or the aromatase inhibitor anastrozole blocked FGF2 and FGF9 secretion, and reduced expression of the stem cell markers SOX2 and nanog. Mice administered β-estradiol during carcinogen exposure showed increased FGF2, FGF9, SOX2, and Nanog expression in airway preneoplasias. In normal FGFR1 copy number NSCLC cell lines, multiple FGFR receptors were expressed and secreted several FGFs. β-estradiol caused enhanced FGF2 release, which was blocked by fulvestrant. Upon co-inhibition of ER and FGFRs using fulvestrant and the pan-FGFR inhibitor AZD4547, phosphorylation of FRS2, the FGFR docking protein, was maximally reduced, and enhanced anti-proliferative effects were observed. Combined AZD4547 and fulvestrant enhanced lung tumor xenograft growth inhibition and decreased Ki67 and stem cell marker expression. To verify a link between ERβ, the predominant ER in NSCLC, and FGFR signaling in patient tumors, mRNA analysis was performed comparing high versus low ERβ expressing tumors. The top differentially expressed genes in high ERβ tumors involved FGF signaling and human embryonic stem cell pluripotency. These results suggest interaction between the ER and FGFR pathways in NSCLC promotes a stem-like state. Combined FGFR and ER inhibition may increase the efficacy of FGFR inhibitors for NSCLC patients lacking FGFR genetic alterations.

Running rescues defective adult neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells.

PubMed

Farioli-Vecchioli, Stefano; Mattera, Andrea; Micheli, Laura; Ceccarelli, Manuela; Leonardi, Luca; Saraulli, Daniele; Costanzi, Marco; Cestari, Vincenzo; Rouault, Jean-Pierre; Tirone, Felice

2014-07-01

Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis. © 2014 AlphaMed Press.

[Therapeutic cloning: far from application at this stage].

PubMed

De Both, N J

2001-11-03

Therapeutic cloning has become possible since the discovery that nuclei from somatic cells of adult animal tissue can successfully be used for cloning and the fact that human embryonic stem cell lines have been established from preimplantation embryos. When nuclei from healthy tissue of a patient are transplanted into enucleated oocytes, these oocytes can be artificially activated so that embryos develop from which embryonic stem cells of the donor can be derived. These embryonic stem cells can be cultured as permanent lines in unlimited numbers and remain pluripotent, i.e. they can be induced to differentiate into the required cell type by adding one or more specific factors. These cells can then be transplanted back into the patient suffering from either a lack or dysfunction of these cells. This approach prevents the rejection of the transplanted cells by the patient's immunological system. As this type of cloning has a very low efficiency, a large number of unfertilized donor oocytes is required. It is questionable whether enough donors are or will be available for this purpose. The cultured cells must satisfy certain conditions before they can be used for transplantation. They must be checked for chromosomal abnormalities, and a complete differentiation of the embryonic stem cells into the cells types needed by the patient is necessary as after the transplantation, undifferentiated stem cells will form teratomas. Furthermore, it is difficult to ensure that the cells end up in the right place and to ensure that they fully integrate into the existing tissue to form functional connections. Due to this array of technical problems the question remains as to whether therapeutic cloning will become feasible in the near future.

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering.

PubMed

Neal, Christopher A; Nelson-Brantley, Jennifer G; Detamore, Michael S; Staecker, Hinrich; Mellott, Adam J

2018-01-01

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton's jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

Concise review: stem cell-based approaches to red blood cell production for transfusion.

PubMed

Shah, Siddharth; Huang, Xiaosong; Cheng, Linzhao

2014-03-01

Blood transfusion is a common procedure in modern medicine, and it is practiced throughout the world; however, many countries report a less than sufficient blood supply. Even in developed countries where the supply is currently adequate, projected demographics predict an insufficient supply as early as 2050. The blood supply is also strained during occasional widespread disasters and crises. Transfusion of blood components such as red blood cells (RBCs), platelets, or neutrophils is increasingly used from the same blood unit for multiple purposes and to reduce alloimmune responses. Even for RBCs and platelets lacking nuclei and many antigenic cell-surface molecules, alloimmunity could occur, especially in patients with chronic transfusion requirements. Once alloimmunization occurs, such patients require RBCs from donors with a different blood group antigen combination, making it a challenge to find donors after every successive episode of alloimmunization. Alternative blood substitutes such as synthetic oxygen carriers have so far proven unsuccessful. In this review, we focus on current research and technologies that permit RBC production ex vivo from hematopoietic stem cells, pluripotent stem cells, and immortalized erythroid precursors.

Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells.

PubMed

Lai, Ruenn Chai; Yeo, Ronne Wee Yeh; Padmanabhan, Jayanthi; Choo, Andre; de Kleijn, Dominique P V; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC.

The Use of Patient-Specific Induced Pluripotent Stem Cells (iPSCs) to Identify Osteoclast Defects in Rare Genetic Bone Disorders

PubMed Central

Chen, I-Ping

2014-01-01

More than 500 rare genetic bone disorders have been described, but for many of them only limited treatment options are available. Challenges for studying these bone diseases come from a lack of suitable animal models and unavailability of skeletal tissues for studies. Effectors for skeletal abnormalities of bone disorders may be abnormal bone formation directed by osteoblasts or anomalous bone resorption by osteoclasts, or both. Patient-specific induced pluripotent stem cells (iPSCs) can be generated from somatic cells of various tissue sources and in theory can be differentiated into any desired cell type. However, successful differentiation of hiPSCs into functional bone cells is still a challenge. Our group focuses on the use of human iPSCs (hiPSCs) to identify osteoclast defects in craniometaphyseal dysplasia. In this review, we describe the impact of stem cell technology on research for better treatment of such disorders, the generation of hiPSCs from patients with rare genetic bone disorders and current protocols for differentiating hiPSCs into osteoclasts. PMID:25621177

Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells.

PubMed

Taha, Masoumeh Fakhr; Hedayati, Vahideh

2010-08-01

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy. Copyright 2010 Elsevier Ltd. All rights reserved.

Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a clinical trial

PubMed Central

2012-01-01

The immune system plays an important role in the regulation of tissue homeostasis ("tissue immune physiology"). Function of distinct tissues during adulthood, including the ovary, requires (1) Renewal from stem cells, (2) Preservation of tissue-specific cells in a proper differentiated state, which differs among distinct tissues, and (3) Regulation of tissue quantity. Such morphostasis can be executed by the tissue control system, consisting of immune system-related components, vascular pericytes, and autonomic innervation. Morphostasis is established epigenetically, during morphogenetic (developmental) immune adaptation, i.e., during the critical developmental period. Subsequently, the tissues are maintained in a state of differentiation reached during the adaptation by a “stop effect” of resident and self renewing monocyte-derived cells. The later normal tissue is programmed to emerge (e.g., late emergence of ovarian granulosa cells), the earlier its function ceases. Alteration of certain tissue differentiation during the critical developmental period causes persistent alteration of that tissue function, including premature ovarian failure (POF) and primary amenorrhea. In fetal and adult human ovaries the ovarian surface epithelium cells called ovarian stem cells (OSC) are bipotent stem cells for the formation of ovarian germ and granulosa cells. Recently termed oogonial stem cells are, in reality, not stem but already germ cells which have the ability to divide. Immune system-related cells and molecules accompany asymmetric division of OSC resulting in the emergence of secondary germ cells, symmetric division, and migration of secondary germ cells, formation of new granulosa cells and fetal and adult primordial follicles (follicular renewal), and selection and growth of primary/preantral, and dominant follicles. The number of selected follicles during each ovarian cycle is determined by autonomic innervation. Morphostasis is altered with advancing age, due to degenerative changes of the immune system. This causes cessation of oocyte and follicular renewal at 38 +/-2 years of age due to the lack of formation of new granulosa cells. Oocytes in primordial follicles persisting after the end of the prime reproductive period accumulate genetic alterations resulting in an exponentially growing incidence of fetal trisomies and other genetic abnormalities with advanced maternal age. The secondary germ cells also develop in the OSC cultures derived from POF and aging ovaries. In vitro conditions are free of immune mechanisms, which prevent neo-oogenesis in vivo. Such germ cells are capable of differentiating in vitro into functional oocytes. This may provide fresh oocytes and genetically related children to women lacking the ability to produce their own follicular oocytes. Further study of "immune physiology" may help us to better understand ovarian physiology and pathology, including ovarian infertility caused by POF or by a lack of ovarian follicles with functional oocytes in aging ovaries. The observations indicating involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from OSC during the fetal and prime reproductive periods are reviewed as well as immune system and age-independent neo-oogenesis and oocyte maturation in OSC cultures, perimenopausal alteration of homeostasis causing disorders of many tissues, and the first OSC culture clinical trial. PMID:23176151

The Emerging Role of Insulin and Insulin-Like Growth Factor Signaling in Cancer Stem Cells

PubMed Central

Malaguarnera, Roberta; Belfiore, Antonino

2014-01-01

Cancer cells frequently exploit the IGF signaling, a fundamental pathway mediating development, cell growth, and survival. As a consequence, several components of the IGF signaling are deregulated in cancer and sustain cancer progression. However, specific targeting of IGF-IR in humans has resulted efficacious only in small subsets of cancers, making researches wondering whether IGF system targeting is still worth pursuing in the clinical setting. Although no definite answer is yet available, it has become increasingly clear that other components of the IGF signaling pathway, such as IR-A, may substitute for the lack of IGF-IR, and induce cancer resistance and/or clonal selection. Moreover, accumulating evidence now indicates that IGF signaling is a central player in the induction/maintenance of epithelial mesenchymal transition (EMT) and cell stemness, two strictly related programs, which play a key role in metastatic spread and resistance to cancer treatments. Here we review the evidences indicating that IGF signaling enhances the expression of transcription factors implicated in the EMT program and has extensive cross-talk with specific pathways involved in cell pluripotency and stemness maintenance. In turn, EMT and cell stemness activate positive feed-back mechanisms causing up-regulation of various IGF signaling components. These findings may have novel translational implications. PMID:24550888

Effect of gold nanoparticles on adipogenic differentiation of human mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Kohl, Yvonne; Gorjup, Erwin; Katsen-Globa, Alisa; Büchel, Claudia; von Briesen, Hagen; Thielecke, Hagen

2011-12-01

Gold nanoparticles are very attractive for biomedical products. However, there is a serious lack of information concerning the biological activity of nanosized gold in human tissue cells. An influence of nanoparticles on stem cells might lead to unforeseen consequences to organ and tissue functions as long as all cells arising from the initial stem cell might be subsequently damaged. Therefore the effect of negatively charged gold nanoparticles (9 and 95 nm), which are certified as reference material for preclinical biomedical research, on the adipogenic differentiation of human mesenchymal stem cells (hMSCs) is investigated here. Bone marrow hMSCs are chosen as differentiation model since bone marrow hMSCs are well characterized and their differentiation into the adipogenic lineage shows clear and easily detectable differentiation. In this study effects of gold nanoparticles on adipogenic differentiation are analyzed regarding fat storage and mitochondrial activity after different exposure times (4-21 days). Using time lapse microscopy the differentiation progress under chronically gold nanoparticle treatment is continuously investigated. In this preliminary study, chronically treatment of adipogenic differentiating hMSCs with gold nanoparticles resulted in a reduced number and size of lipid vacuoles and reduced mitochondrial activity depending on the applied concentration and the surface charge of the particles.

Concise Review: Aggressive Colorectal Cancer: Role of Epithelial Cell Adhesion Molecule in Cancer Stem Cells and Epithelial-to-Mesenchymal Transition.

PubMed

Boesch, Maximilian; Spizzo, Gilbert; Seeber, Andreas

2018-06-01

Colorectal cancer (CRC) is one of the most common malignancies worldwide. In spite of various attempts to ameliorate outcome by escalating treatment, significant improvement is lacking particularly in the adjuvant setting. It has been proposed that cancer stem cells (CSCs) and the epithelial-to-mesenchymal transition (EMT) are at least partially responsible for therapy resistance in CRC. The epithelial cell adhesion molecule (EpCAM) was one of the first CSC antigens to be described. Furthermore, an EpCAM-specific antibody (edrecolomab) has the merit of having launched the era of monoclonal antibody treatment in oncology in the 1990s. However, despite great initial enthusiasm, monoclonal antibody treatment has not proven successful in the adjuvant treatment of CRC patients. In the meantime, new insights into the function of EpCAM in CRC have emerged and new drugs targeting various epitopes have been developed. In this review article, we provide an update on the role of EpCAM in CSCs and EMT, and emphasize the potential predictive selection criteria for novel treatment strategies and refined clinical trial design. Stem Cells Translational Medicine 2018;7:495-501. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Perivascular Mesenchymal Stem Cells From the Adult Human Brain Harbor No Instrinsic Neuroectodermal but High Mesodermal Differentiation Potential.

PubMed

Lojewski, Xenia; Srimasorn, Sumitra; Rauh, Juliane; Francke, Silvan; Wobus, Manja; Taylor, Verdon; Araúzo-Bravo, Marcos J; Hallmeyer-Elgner, Susanne; Kirsch, Matthias; Schwarz, Sigrid; Schwarz, Johannes; Storch, Alexander; Hermann, Andreas

2015-10-01

Brain perivascular cells have recently been identified as a novel mesodermal cell type in the human brain. These cells reside in the perivascular niche and were shown to have mesodermal and, to a lesser extent, tissue-specific differentiation potential. Mesenchymal stem cells (MSCs) are widely proposed for use in cell therapy in many neurological disorders; therefore, it is of importance to better understand the "intrinsic" MSC population of the human brain. We systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation and compared these cells with fetal and adult human brain-derived neural stem cells (NSCs) and adult human bone marrow-derived MSCs. We found that adult human brain pericytes, which can be isolated from the hippocampus and from subcortical white matter, are-in contrast to adult human NSCs-easily expandable in monolayer cultures and show many similarities to human bone marrow-derived MSCs both regarding both surface marker expression and after whole transcriptome profile. Human brain pericytes showed a negligible propensity for neuroectodermal differentiation under various differentiation conditions but efficiently generated mesodermal progeny. Consequently, human brain pericytes resemble bone marrow-derived MSCs and might be very interesting for possible autologous and endogenous stem cell-based treatment strategies and cell therapeutic approaches for treating neurological diseases. Perivascular mesenchymal stem cells (MSCs) recently gained significant interest because of their appearance in many tissues including the human brain. MSCs were often reported as being beneficial after transplantation in the central nervous system in different neurological diseases; therefore, adult brain perivascular cells derived from human neural tissue were systematically characterized concerning neural stem cell and MSC marker expression, transcriptomics, and mesodermal and inherent neuroectodermal differentiation potential in vitro and in vivo after in utero transplantation. This study showed the lack of an innate neuronal but high mesodermal differentiation potential. Because of their relationship to mesenchymal stem cells, these adult brain perivascular mesodermal cells are of great interest for possible autologous therapeutic use. ©AlphaMed Press.

Integrin-binding elastin-like polypeptide as an in situ gelling delivery matrix enhances the therapeutic efficacy of adipose stem cells in healing full-thickness cutaneous wounds.

PubMed

Choi, Seong-Kyoon; Park, Jin-Kyu; Kim, Jung-Hee; Lee, Kyeong-Min; Kim, Enjoo; Jeong, Kyu-Shik; Jeon, Won Bae

2016-09-10

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair. Copyright © 2016 Elsevier B.V. All rights reserved.

Ethics and synthetic gametes.

PubMed

Testa, Giuseppe; Harris, John

2005-04-01

The recent in vitro derivation of gamete-like cells from mouse embryonic stem (mES) cells is a major breakthrough and lays down several challenges, both for the further scientific investigation and for the bioethical and biolegal discourse. We refer here to these cells as gamete-like (sperm-like or oocyte-like, respectively), because at present there is still no evidence that these cells behave fully like bona fide sperm or oocytes, lacking the fundamental proof, i.e. combination with a normally derived gamete of the opposite sex to yield a normal individual. However, the results published so far do show that these cells share some defining features of gametes. We discuss these results in the light of the bioethical and legal questions that are likely to arise would the same process become possible with human embryonic stem (hES) cells.

Satellite Cells in Muscular Dystrophy - Lost in Polarity

PubMed Central

Chang, Natasha C.; Chevalier, Fabien P.; Rudnicki, Michael A.

2016-01-01

Recent findings employing the mdx mouse model for Duchenne muscular dystrophy (DMD) have revealed that muscle satellite stem cells play a direct role in contributing to disease etiology and progression of DMD, the most common and severe form of muscular dystrophy. Lack of dystrophin expression in DMD has critical consequences in satellite cells including an inability to establish cell polarity, abrogation of asymmetric satellite stem cell divisions and failure to enter the myogenic program. Thus, muscle wasting in dystrophic mice is not only caused by myofiber fragility, but is exacerbated by intrinsic satellite cell dysfunction leading to impaired regeneration. Despite intense research and clinical efforts, there is still no effective cure for DMD. In this review, we highlight recent research advances in DMD and discuss the current state of treatment and importantly, how we can incorporate satellite cell-targeted therapeutic strategies to correct satellite cell dysfunction in DMD. PMID:27161598

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

PubMed

Dalley, Andrew J; AbdulMajeed, Ahmad A; Upton, Zee; Farah, Camile S

2013-01-01

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. © 2012 John Wiley & Sons A/S.

Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

PubMed

Kim, Yoon Young; Ku, Seung-Yup; Huh, Yul; Liu, Hung-Ching; Kim, Seok Hyun; Choi, Young Min; Moon, Shin Yong

2013-10-01

Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 μM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

Stem cells as anticancer drug carrier to reduce the chemotherapy side effect

NASA Astrophysics Data System (ADS)

Salehi, Hamideh; Al-Arag, Siham; Middendorp, Elodie; Gergley, Csilla; Cuisinier, Frederic

2017-02-01

Chemotherapy used for cancer treatment, due to the lack of specificity of drugs, is associated to various damaging side effects that have severe impact on patients' quality of life. Over the past 30 years, increasing efforts have been placed on optimizing chemotherapy dosing with the main goal of increasing antitumor efficacy while reducing drug-associated toxicity. A novel research shows that stem cells may act as a reservoir for the anticancer agent, which will subsequently release some of the drug's metabolites, or even the drug in its original form, in vicinity of the cancer cells. These cells may play a dual role in controlling drug toxicity depending on their capacity to uptake and release the chemotherapeutic drug. In our study, we show that Dental Pulp Stem Cells DPSCs are able to rapidly uptake Paclitaxel PTX, and to release it in the culture medium in a time-dependent manner. This resulting conditioned culture medium is to be transferred to breast cancer cells, the MCF-7. By applying Confocal Raman Microscopy, the anticancer drug uptake by the MCF-7 was measured. Surprisingly, the cancer cells -without any direct contact with PTX- showed a drug uptake. This proves that the stem cells carried and delivered the anticancer drug without its modification. It could be a revolution in chemotherapy to avoid the drug's side effects and increase its efficacy.

Zinc oxide nanoparticles as selective killers of proliferating cells

PubMed Central

Taccola, Liuba; Raffa, Vittoria; Riggio, Cristina; Vittorio, Orazio; Iorio, Maria Carla; Vanacore, Renato; Pietrabissa, Andrea; Cuschieri, Alfred

2011-01-01

Background: It has recently been demonstrated that zinc oxide nanoparticles (ZnO NPs) induce death of cancerous cells whilst having no cytotoxic effect on normal cells. However, there are several issues which need to be resolved before translation of zinc oxide nanoparticles into medical use, including lack of suitable biocompatible dispersion protocols and a better understanding being needed of the mechanism of their selective cytotoxic action. Methods: Nanoparticle dose affecting cell viability was evaluated in a model of proliferating cells both experimentally and mathematically. The key issue of selective toxicity of ZnO NPs toward proliferating cells was addressed by experiments using a biological model of noncancerous cells, ie, mesenchymal stem cells before and after cell differentiation to the osteogenic lineage. Results: In this paper, we report a biocompatible protocol for preparation of stable aqueous solutions of monodispersed zinc oxide nanoparticles. We found that the threshold of intracellular ZnO NP concentration required to induce cell death in proliferating cells is 0.4 ± 0.02 mM. Finally, flow cytometry analysis revealed that the threshold dose of zinc oxide nanoparticles was lethal to proliferating pluripotent mesenchymal stem cells but exhibited negligible cytotoxic effects to osteogenically differentiated mesenchymal stem cells. Conclusion: Results confirm the ZnO NP selective cytotoxic action on rapidly proliferating cells, whether benign or malignant. PMID:21698081

Zinc oxide nanoparticles as selective killers of proliferating cells.

PubMed

Taccola, Liuba; Raffa, Vittoria; Riggio, Cristina; Vittorio, Orazio; Iorio, Maria Carla; Vanacore, Renato; Pietrabissa, Andrea; Cuschieri, Alfred

2011-01-01

It has recently been demonstrated that zinc oxide nanoparticles (ZnO NPs) induce death of cancerous cells whilst having no cytotoxic effect on normal cells. However, there are several issues which need to be resolved before translation of zinc oxide nanoparticles into medical use, including lack of suitable biocompatible dispersion protocols and a better understanding being needed of the mechanism of their selective cytotoxic action. Nanoparticle dose affecting cell viability was evaluated in a model of proliferating cells both experimentally and mathematically. The key issue of selective toxicity of ZnO NPs toward proliferating cells was addressed by experiments using a biological model of noncancerous cells, ie, mesenchymal stem cells before and after cell differentiation to the osteogenic lineage. In this paper, we report a biocompatible protocol for preparation of stable aqueous solutions of monodispersed zinc oxide nanoparticles. We found that the threshold of intracellular ZnO NP concentration required to induce cell death in proliferating cells is 0.4 ± 0.02 mM. Finally, flow cytometry analysis revealed that the threshold dose of zinc oxide nanoparticles was lethal to proliferating pluripotent mesenchymal stem cells but exhibited negligible cytotoxic effects to osteogenically differentiated mesenchymal stem cells. Results confirm the ZnO NP selective cytotoxic action on rapidly proliferating cells, whether benign or malignant.

Breast Cancer Subtypes: Two decades of Journey from Cell Culture to Patients

PubMed Central

Zhao, Xiangshan; Gurumurthy, Channabasavaiah Basavaraju; Malhotra, Gautam; Mirza, Sameer; Mohibi, Shakur; Bele, Aditya; Quinn, Meghan G; Band, Hamid; Band, Vimla

2014-01-01

Breast cancer remains the second leading cause of cancer-related deaths among women. Clinically breast cancer patients present with distinct diseases with vastly different outcomes. Recent molecular profiling has identified five major subtypes of breast cancers. Importantly, survival analyses have shown significantly different outcomes for patients belonging to various subgroups. These studies strongly support the idea that breast tumor subtypes may represent malignancies of biologically distinct cell types producing distinct disease entities that may also require different treatment strategies. Alternatively, different types of breast cancers may arise from a common precursor based on oncogene-driven reprogramming. Experimental systems that clearly define cancer cell heterogeneity and link this process to cancer stem/progenitor cells have not been developed. It is also unclear if oncogenic transformation of committed progenitors drives them along their committed pathway, and hence the cell of origin determines the histological features of breast cancer, or if different oncogenic pathways can transform the same precursor along distinct phenotypes. One major hurdle to addressing these fundamental questions about the origin and heterogeneity of human breast cancer is the lack of immortal human stem/progenitor cells that could be interrogated with breast cancer-relevant oncogenesis protocols. We have now identified, isolated and immortalized (using hTERT) such mammary stem/progenitor cells that are immortal and still maintain their progenitor/stem cell properties (self-renewal and differentiation into myoepithelial and luminal cells). Our research using these progenitor/stem cells that are highly susceptible to oncogenesis and various models of mammary cell immortalization has allowed us to define several novel cellular pathways and demonstration of their involvement in oncogenesis and breast cancer progression. Given the emerging evidence that stem/progenitor cells are precursors of cancers and distinct subtypes of breast cancer have different survival outcome, these studies are timely and carry the potential of developing novel therapeutics in the future as well as provide potentially novel markers for diagnostic/prognostic use in breast cancer. PMID:21901624

Imatinib-loaded polyelectrolyte microcapsules for sustained targeting of BCR-ABL+ leukemia stem cells.

PubMed

Palamà, Ilaria E; Leporatti, Stefano; de Luca, Emanuela; Di Renzo, Nicola; Maffia, Michele; Gambacorti-Passerini, Carlo; Rinaldi, Ross; Gigli, Giuseppe; Cingolani, Roberto; Coluccia, Addolorata M L

2010-04-01

The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts. Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested. Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro. This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

Biodistribution of Liver-Derived Mesenchymal Stem Cells After Peripheral Injection in a Hemophilia A Patient.

PubMed

Sokal, Etienne M; Lombard, Catherine Anne; Roelants, Véronique; Najimi, Mustapha; Varma, Sharat; Sargiacomo, Camillo; Ravau, Joachim; Mazza, Giuseppe; Jamar, François; Versavau, Julia; Jacobs, Vanessa; Jacquemin, Marc; Eeckhoudt, Stéphane; Lambert, Catherine; Stéphenne, Xavier; Smets, Françoise; Hermans, Cédric

2017-08-01

With the exception of liver transplantation, there is no cure for hemophilia, which is currently managed by preemptive replacement therapy. Liver-derived stem cells are in clinical development for inborn and acquired liver diseases and could represent a curative treatment for hemophilia A. The liver is a major factor VIII (FVIII) synthesis site, and mesenchymal stem cells have been shown to control joint bleeding in animal models of hemophilia. Adult-derived human liver stem cells (ADHLSCs) have mesenchymal characteristics and have been shown able to engraft in and repopulate both animal and human livers. Thus, the objectives were to evaluate the potency of ADHLSCs to control bleeding in a hemophilia A patient and assess the biodistribution of the cells after intravenous injection. A patient suffering from hemophilia A was injected with repeated doses of ADHLSCs via a peripheral vein (35 million In-oxine-labeled cells, followed by 125 million cells the next day, and 3 infusions of 250 million cells every 2 weeks thereafter; total infusion period, 50 days). After cell therapy, we found a temporary (15 weeks) decrease in the patient's FVIII requirements and severe bleeding complications, despite a lack of increase in circulating FVIII. The cells were safely administered to the patient via a peripheral vein. Biodistribution analysis revealed an initial temporary entrapment of the cells in the lungs, followed by homing to the liver and to a joint afflicted with hemarthrosis. These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.

Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

PubMed

Neuenhahn, M; Albrecht, J; Odendahl, M; Schlott, F; Dössinger, G; Schiemann, M; Lakshmipathi, S; Martin, K; Bunjes, D; Harsdorf, S; Weissinger, E M; Menzel, H; Verbeek, M; Uharek, L; Kröger, N; Wagner, E; Kobbe, G; Schroeder, T; Schmitt, M; Held, G; Herr, W; Germeroth, L; Bonig, H; Tonn, T; Einsele, H; Busch, D H; Grigoleit, G U

2017-10-01

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D + repl; n=28) or T-cell-depleted (D + depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D + depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D - ) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D + depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D - patients.

Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells

PubMed Central

Bennett, John A.; Singh, Kameshwar P.; Unnisa, Zeenath; Welle, Stephen L.; Gasiewicz, Thomas A.

2015-01-01

Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice. PMID:26208102

Efficient, Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly-Defined and Fully-Defined Conditions

PubMed Central

van Wilgenburg, Bonnie; Browne, Cathy; Vowles, Jane; Cowley, Sally A.

2013-01-01

Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively, up to ∼3×107 monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14+, CD16low, CD163+). Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology. PMID:23951090

When Is an Alveolar Type 2 Cell an Alveolar Type 2 Cell? A Conundrum for Lung Stem Cell Biology and Regenerative Medicine.

PubMed

Beers, Michael F; Moodley, Yuben

2017-07-01

Generating mature, differentiated, adult lung cells from pluripotent cells, such as induced pluripotent stem cells and embryonic stem cells, offers the hope of both generating disease-specific in vitro models and creating definitive and personalized therapies for a host of debilitating lung parenchymal and airway diseases. With the goal of advancing lung-regenerative medicine, several groups have developed and reported on protocols using defined media, coculture with mesenchymal components, or sequential treatments mimicking lung development, to obtain distal lung epithelial cells from stem cell precursors. However, there remains significant controversy about the degree of differentiation of these cells compared with their primary counterparts, coupled with a lack of consistency or uniformity in assessing the resultant phenotypes. Given the inevitable, exponential expansion of these approaches and the probable, but yet-to-emerge second and higher generation techniques to create such assets, we were prompted to pose the question, what makes a lung epithelial cell a lung epithelial cell? More specifically for this Perspective, we also posed the question, what are the minimum features that constitute an alveolar type (AT) 2 epithelial cell? In addressing this, we summarize a body of work spanning nearly five decades, amassed by a series of "lung epithelial cell biology pioneers," which carefully describes well characterized molecular, functional, and morphological features critical for discriminately assessing an AT2 phenotype. Armed with this, we propose a series of core criteria to assist the field in confirming that cells obtained following a differentiation protocol are indeed mature and functional AT2 epithelial cells.

The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca(2+) as a secondary cytosolic messenger.

PubMed

Chou, Hsuan; Zhu, Yingfang; Ma, Yi; Berkowitz, Gerald A

2016-02-01

CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non-cell-autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca(2+) elevations, cyclic nucleotide (cGMP)-activated Ca(2+) channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca(2+) elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca(2+) and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP-activated Ca(2+) channel. In wild-type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca(2+) channel blocker or a guanylyl cyclase inhibitor. When CLV3-dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca(2+) channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca(2+), and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

The synergistic effect of beta-boswellic acid and Nurr1 overexpression on dopaminergic programming of antioxidant glutathione peroxidase-1-expressing murine embryonic stem cells.

PubMed

Abasi, M; Massumi, M; Riazi, G; Amini, H

2012-10-11

Parkinson's disease (PD) is a neurodegenerative disorder in which the nigro-striatal dopaminergic (DAergic) neurons have been selectively lost. Due to side effects of levodopa, a dopamine precursor drug, recently cell replacement therapy for PD has been considered. Lack of sufficient amounts of, embryos and ethical problems regarding the use of dopamine-rich embryonic neural cells have limited the application of these cells for PD cell therapy. Therefore, many investigators have focused on using the pluripotent stem cells to generate DAergic neurons. This study is aimed first to establish a mouse embryonic stem (mES) cell line that can stably co-express Nurr1 (Nuclear receptor subfamily 4, group A, member 2) transcription factor in order to efficiently generate DAergic neurons, and glutathione peroxidase-1 (GPX-1) to protect the differentiated DAergic-like cells against oxidative stress. In addition to genetic engineering of ES cells, the effect of Beta-boswellic acid (BBA) on DAergic differentiation course of mES cells was sought in the present study. To that end, the feeder-independent CGR8 mouse embryonic stem cells were transduced by Nurr1- and GPX-1-harboring Lentiviruses and the generated Nurr1/GPX-1-expresssing ES clones were characterized and verified. Gene expression analyses demonstrated that BBA treatment and overexpression of Nurr1 has a synergistic effect on derivation of DAergic neurons from Nurr1/GPX-1-expressing ES cells. The differentiated cells could exclusively synthesize and secrete dopamine in response to stimuli. Overexpression of GPX-1 in genetically engineered Nurr1/GPX-1-ES cells increased the viability of these cells during their differentiation into CNS stem cells. In conclusion, the results demonstrated that Nurr1-overexpressing feeder-independent ES cells like the feeder-dependent ES cells, can be efficiently programmed into functional DAergic neurons and additional treatment of cells by BBA can even augment this efficiency. GPX-1 overexpression in Nurr1/GPX-1-ES cells increases the viability of differentiated CNS stem-like cells. The result of this study may have impact on future stem cell therapy of PD. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Development of Mouse Embryonic Stem (ES) Cells: IV Differentiation to Mature T and B Lymphocytes after Implantation of Embryoid Bodies Into Nude Mice

PubMed Central

Mok, Hoyan

1995-01-01

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed. PMID:9700357

Characterization of immortalized dairy goat male germline stem cells (mGSCs).

PubMed

Zhu, Haijing; Ma, Jing; Du, Rui; Zheng, Liming; Wu, Jiang; Song, Wencong; Niu, Zhiwei; He, Xin; Du, Enqi; Zhao, Shanting; Hua, Jinlian

2014-09-01

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs. © 2014 Wiley Periodicals, Inc.

Gelatinized Copper–Capillary Alginate Gel Functions as an Injectable Tissue Scaffolding System for Stem Cell Transplants

PubMed Central

Willenberg, Bradley Jay; Zheng, Tong; Meng, Fan-Wei; Meneses, Juan Carlos; Rossignol, Candace; Batich, Christopher D.; Terada, Naohiro; Steindler, Dennis A.; Weiss, Michael D.

2013-01-01

In severe hypoxic–ischemic brain injury, cellular components such as neurons and astrocytes are injured or destroyed along with the supporting extracellular matrix. This presents a challenge to the field of regenerative medicine since the lack of extracellular matrix and supporting structures makes the transplant milieu inhospitable to the transplanted cells. A potential solution to this problem is the use of a biomaterial to provide the extracellular components needed to keep cells localized in cystic brain regions, allowing the cells to form connections and repair lost brain tissue. Ideally, this biomaterial would be combined with stem cells, which have been proven to have therapeutic potentials, and could be delivered via an injection. To study this approach, we derived a hydrogel biomaterial tissue scaffold from oligomeric gelatin and copper–capillary alginate gel (GCCAG). We then demonstrated that our multipotent astrocytic stem cells (MASCs) could be maintained in GCCAG scaffolds for up to 2 weeks in vitro and that the cells retained their multipotency. We next performed a pilot transplant study in which GCCAG was mixed with MASCs and injected into the brain of a neonatal rat pup. After a week in vivo, our results showed that: the GCCAG biomaterial did not cause a significant reactive gliosis; viable cells were retained within the injected scaffolds; and some delivered cells migrated into the surrounding brain tissue. Therefore, GCCAG tissue scaffolds are a promising, novel injectable system for transplantation of stem cells to the brain. PMID:20699061

Adipose-derived stem cells seeded in Pluronic F-127 hydrogel promotes diabetic wound healing.

PubMed

Kaisang, Lin; Siyu, Wang; Lijun, Fan; Daoyan, Pan; Xian, Cory J; Jie, Shen

2017-09-01

Chronic nonhealing wound is a multifactorial complication of diabetes that results specifically as a consequence of impaired angiogenesis and currently lacks in effective treatments. Although a stem cell-based therapy may provide a novel treatment to augment diabetic wound healing, inferior cell survival at the diabetic skin wound is one of the key causes that are responsible for the low efficacy of the stem cell therapy. In this work, we used an injectable, biocompatible, and thermosensitive hydrogel Pluronic F-127 to encapsulate allogeneic nondiabetic adipose-derived stem cells (ADSCs) and topically applied the cells to a full-thickness cutaneous wound in the streptozotocin-induced diabetic model in rats. The cells seeded in the hydrogel enhanced angiogenesis (CD31 marker) and promoted the cell proliferation (Ki67 marker) at the wound site and significantly accelerated wound closure, which was accompanied by facilitated regeneration of granulation tissue. Consistently, levels of the messenger RNA expression of key angiogenesis growth factor, vascular endothelial growth factor, and key wound healing growth factor, transforming growth factor beta 1, were also upregulated in the cell-treated wounds when compared with untreated wounds. The results indicated that the transplantation of allogeneic ADSCs via the hydrogel improves the efficiency of cell delivery and optimizes the performance of ADSCs for augmenting diabetic wound healing. In conclusion, this ADSC-based therapy may provide a novel therapeutic strategy for the treatment of nonhealing diabetic foot ulcers. Copyright © 2017 Elsevier Inc. All rights reserved.

Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds

PubMed Central

Skardal, Aleksander; Mack, David; Kapetanovic, Edi; Atala, Anthony; Jackson, John D.; Yoo, James

2012-01-01

Stem cells obtained from amniotic fluid show high proliferative capacity in culture and multilineage differentiation potential. Because of the lack of significant immunogenicity and the ability of the amniotic fluid-derived stem (AFS) cells to modulate the inflammatory response, we investigated whether they could augment wound healing in a mouse model of skin regeneration. We used bioprinting technology to treat full-thickness skin wounds in nu/nu mice. AFS cells and bone marrow-derived mesenchymal stem cells (MSCs) were resuspended in fibrin-collagen gel and “printed” over the wound site. At days 0, 7, and 14, AFS cell- and MSC-driven wound closure and re-epithelialization were significantly greater than closure and re-epithelialization in wounds treated by fibrin-collagen gel only. Histological examination showed increased microvessel density and capillary diameters in the AFS cell-treated wounds compared with the MSC-treated wounds, whereas the skin treated only with gel showed the lowest amount of microvessels. However, tracking of fluorescently labeled AFS cells and MSCs revealed that the cells remained transiently and did not permanently integrate in the tissue. These observations suggest that the increased wound closure rates and angiogenesis may be due to delivery of secreted trophic factors, rather than direct cell-cell interactions. Accordingly, we performed proteomic analysis, which showed that AFS cells secreted a number of growth factors at concentrations higher than those of MSCs. In parallel, we showed that AFS cell-conditioned media induced endothelial cell migration in vitro. Taken together, our results indicate that bioprinting AFS cells could be an effective treatment for large-scale wounds and burns. PMID:23197691

Macrophages control vascular stem/progenitor cell plasticity through tumor necrosis factor-α-mediated nuclear factor-κB activation.

PubMed

Wong, Mei Mei; Chen, Yikuan; Margariti, Andriani; Winkler, Bernhard; Campagnolo, Paola; Potter, Claire; Hu, Yanhua; Xu, Qingbo

2014-03-01

Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process. We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α-mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation. Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α-mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.

Fos Promotes Early Stage Teno-Lineage Differentiation of Tendon Stem/Progenitor Cells in Tendon.

PubMed

Chen, Jialin; Zhang, Erchen; Zhang, Wei; Liu, Zeyu; Lu, Ping; Zhu, Ting; Yin, Zi; Backman, Ludvig J; Liu, Huanhuan; Chen, Xiao; Ouyang, Hongwei

2017-11-01

Stem cells have been widely used in tendon tissue engineering. The lack of refined and controlled differentiation strategy hampers the tendon repair and regeneration. This study aimed to find new effective differentiation factors for stepwise tenogenic differentiation. By microarray screening, the transcript factor Fos was found to be expressed in significantly higher amounts in postnatal Achilles tendon tissue derived from 1 day as compared with 7-days-old rats. It was further confirmed that expression of Fos decreased with time in postnatal rat Achilles tendon, which was accompanied with the decreased expression of multiply tendon markers. The expression of Fos also declined during regular in vitro cell culture, which corresponded to the loss of tendon phenotype. In a cell-sheet and a three-dimensional cell culture model, the expression of Fos was upregulated as compared with in regular cell culture, together with the recovery of tendon phenotype. In addition, significant higher expression of tendon markers was found in Fos-overexpressed tendon stem/progenitor cells (TSPCs), and Fos knock-down gave opposite results. In situ rat tendon repair experiments found more normal tendon-like tissue formed and higher tendon markers expression at 4 weeks postimplantation of Fos-overexpressed TSPCs derived nonscaffold engineering tendon (cell-sheet), as compared with the control group. This study identifies Fos as a new marker and functional driver in the early stage teno-lineage differentiation of tendon, which paves the way for effective stepwise tendon differentiation and future tendon regeneration. Stem Cells Translational Medicine 2017;6:2009-2019. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1.

PubMed

Ocaña, Oscar H; Córcoles, Rebeca; Fabra, Angels; Moreno-Bueno, Gema; Acloque, Hervé; Vega, Sonia; Barrallo-Gimeno, Alejandro; Cano, Amparo; Nieto, M Angela

2012-12-11

The epithelial-mesenchymal transition (EMT) is required in the embryo for the formation of tissues for which cells originate far from their final destination. Carcinoma cells hijack this program for tumor dissemination. The relevance of the EMT in cancer is still debated because it is unclear how these migratory cells colonize distant tissues to form macrometastases. We show that the homeobox factor Prrx1 is an EMT inducer conferring migratory and invasive properties. The loss of Prrx1 is required for cancer cells to metastasize in vivo, which revert to the epithelial phenotype concomitant with the acquisition of stem cell properties. Thus, unlike the classical EMT transcription factors, Prrx1 uncouples EMT and stemness, and is a biomarker associated with patient survival and lack of metastasis. Copyright © 2012 Elsevier Inc. All rights reserved.

Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs.

PubMed

Fu, Lina; Xu, Xiuling; Ren, Ruotong; Wu, Jun; Zhang, Weiqi; Yang, Jiping; Ren, Xiaoqing; Wang, Si; Zhao, Yang; Sun, Liang; Yu, Yang; Wang, Zhaoxia; Yang, Ze; Yuan, Yun; Qiao, Jie; Izpisua Belmonte, Juan Carlos; Qu, Jing; Liu, Guang-Hui

2016-03-01

Xeroderma pigmentosum (XP) is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patient-specific induced pluripotent stem cells (iPSCs) harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells (NSCs) or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clarify the molecular mechanisms of neurological abnormalities in the XP patients.

Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells

PubMed Central

Kumar, Nathan; Richter, Jenna; Cutts, Josh; Bush, Kevin T; Trujillo, Cleber; Nigam, Sanjay K; Gaasterland, Terry; Brafman, David; Willert, Karl

2015-01-01

The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate. DOI: http://dx.doi.org/10.7554/eLife.08413.001 PMID:26554899

Characterization and isolation of highly purified porcine satellite cells

PubMed Central

Ding, Shijie; Wang, Fei; Liu, Yan; Li, Sheng; Zhou, Guanghong; Hu, Ping

2017-01-01

Pig is an important food source and an excellent system to model human diseases. Careful characterization of the swine skeletal muscle stem cells (satellite cells) will shed lights on generation of swine skeletal muscle disease model and efficient production of porcine meat for the food industry. Paired box protein 7 (Pax7) is a highly conserved transcription factor shared by satellite cells from various species. However, the sequence of Pax7 has not been characterized in pig. The lack of method to isolate highly purified satellite cells hinders the thorough characterization of the swine satellite cells. Here we found molecular markers for swine satellite cells and revealed that the porcine satellite cells were heterogeneous in various pieces of skeletal muscle. We further developed a method to isolate highly purified satellite cells directly from porcine muscles using fluorescence-activated cell sorting. We next characterized the proliferation and differentiation abilities of isolated satellite cells in vitro; and found that long-term culturing of satellite cells in vitro led to stemness loss. PMID:28417015

Human Natural Killer Cells Exhibit Direct Activity Against Aspergillus fumigatus Hyphae, But Not Against Resting Conidia

PubMed Central

Schmidt, Stanislaw; Tramsen, Lars; Hanisch, Mitra; Latgé, Jean-Paul; Huenecke, Sabine; Koehl, Ulrike

2011-01-01

Because natural killer (NK) cells kill tumor cells and combat infections, there is growing interest in adoptively transferring NK cells to hematopoietic stem cell recipients. Unfortunately, in humans, the activity of NK cells against Aspergillus species, the major cause of invasive fungal infection in stem cell recipients, are poorly characterized. Our results show that unstimulated and interleukin-2 prestimulated human NK cells kill Aspergillus fumigatus hyphae but do not affect resting conidia. Killing is also induced by the supernatant of prestimulated NK cells and human perforin. The high levels of interferon-γ and granulocyte macrophage colony-stimulating factor produced by prestimulated NK cells are significantly reduced by Aspergillus, indicating an immunosuppressive effect of the fungus. Whereas Aspergillus hyphae activate NK cells, resting, and germinating, conidia and conidia of ΔrodA mutants lacking the hydrophobic surface layer do not. Our results suggest that adoptively transferred human NK cells may be a potential antifungal tool in the transplantation context. PMID:21208932

Assessing stemness and proliferation properties of the newly established colon cancer 'stem' cell line, CSC480 and novel approaches to identify dormant cancer cells.

PubMed

Alowaidi, Faisal; Hashimi, Saeed Mujahid; Alqurashi, Naif; Alhulais, Reem; Ivanovski, Saso; Bellette, Bernadette; Meedenyia, Adrian; Lam, Alfred; Wood, Stephen

2018-06-01

To date two questions that remain unanswered regarding cancer are the following: i) how is it initiated, and ii) what is the role that cancer stem cells (CSCs) play in the disease process? Understanding the biology of CSCs and how they are generated is pivotal for the development of successful treatment regimens. To date, the lack of a representative cell model has prevented the successful identification and eradication of CSCs in vivo. The current methods of CSC identification are dependent on the protocol used to generate these cells, which has introduced variation and made the identification process more complicated. Furthermore, the list of possible markers is increasing in complexity. This is further confounded by the fact that there is insufficient information to determine whether the cells these markers detect are truly self‑renewing stem cells or, instead, progenitor cells. In the present study, we investigated a novel cell line model, CSC480, which can be employed to assess CSC markers and for testing novel therapeutic regimens. CSC480 cells have been revealed to express markers of CSCs such as CD44, ALDH1 and Sox2, that have lower expression in the SW480 cell line. CSC480 cells also expressed higher levels of the cancer resistance marker, ABCG2 and had higher proliferative and growth capacity than SW480 cells. In the present study, we also evaluated a novel approach to identify different cell types present in heterogeneous cancer cell populations according to their proliferative ability using the proliferation marker 5‑ethynyl‑2'‑deoxyuridine (EdU). Furthermore, using EdU, we identified dormant cells with a modified label‑retaining cell (LRC) protocol. Through this novel LRC method, we assessed newly discovered markers of stemness to ascertain their capability to identify quiescent from dividing CSCs. In conclusion, the CSC480 cell line was an important model to be used in unravelling the underlying mechanisms that control fast‑dividing and partially self‑renewing stem cells (SCs) that may give rise to cancer.

[Clinical effect of stem cell transplantation via hepatic artery in the treatment of type II hyperammonemia: a report on 6 cases].

PubMed

DU, Kan; Luan, Zuo; Qu, Su-Qing; Yang, Hui; Yang, Yin-Xiang; Wang, Zhao-Yan; Jin, Hui-Yu; Liu, Wei-Peng

2013-11-01

This study aimed to investigate the clinical effect of transplantation of CD133⁺ peripheral blood stem cells or umbilical cord mesenchymal stem cells via the hepatic artery in children with type II hyperammonemia and its possible action mechanism. Umbilical cord mesenchymal stem cells were obtained by collecting cord blood (100-150 mL) from healthy fetuses and separating stem cell suspension (5 mL) from the cord blood by hydroxyethyl starch sedimentation. CD133⁺ peripheral blood stem cells were obtained by mobilizing peripheral blood from the fathers of sick children using recombinant human granulocyte colony-stimulating factor for 5 days, collecting mononuclear cells (120 mL), and separating out CD133⁺ cells by sorting. With catheterization and percutaneous puncture, the obtained stem cells were slowly injected into the liver of sick children via the hepatic artery. The changes in clinical symptoms and laboratory indices such as blood ammonia, liver function, and arginine and citrulline concentrations were observed. After stem cell transplantation via the hepatic artery, the 6 children showed significantly decreased blood ammonia levels, and their blood ammonia levels slowly increased 1 to 2 weeks later, but remained below 100 μmol/L, and changes in glutamic-pyruvic transaminase levels were similar to blood ammonia. Plasma citrulline and arginine concentrations increased significantly after transplantation and the increase in citrulline level exceeded the increase in arginine level. An 8 months follow-up visit for one typical patient showed that the weight and height increased after transplantation and sleep was improved without night crying. The child could actively gaze at interesting objects instead of responding indifferently and started to say simple words. With regard to fine motor skills, the child could pinch things with the thumb and middle finger instead of displaying a lack of hand-eye coordination and progress was also made in gross motor skills. Gesell test showed that the child made progress for an average of 3.82 months in all areas. It was concluded that after stem cell transplantation, children with type II hyperammonemia have decreased blood ammonia levels, stable and improved liver function and steadily increased plasma citrulline and arginine concentrations. They display a progressive trend in such aspects as movement, language and environmental adaptability. It is hypothesized that stem cell transplantation via the hepatic artery partially or totally activates, or provides supplementary ornithine carbamoyl transferase, so that plasma citrulline and arginine concentrations increase and urea cycle disorder can be corrected to some extent.

Stem cell transplantation (cord blood transplants).

PubMed

Chao, Nelson J; Emerson, Stephen G; Weinberg, Kenneth I

2004-01-01

Allogeneic stem cell transplantation is an accepted treatment modality for selected malignant and non-malignant diseases. However, the ability to identify suitably matched related or unrelated donors can be difficult in some patients. Alternative sources of stem cells such as cord blood provide a readily available graft for such patients. Data accumulated over the past several years have demonstrated that the use of cord blood is an accepted source of stem cells for pediatric patients. Since the cell numbers of hematopoietic progenitors in cord blood is limited and the collection can occur only in a single occasion, its use in adult patients can be more problematic. Here, new developments in the use of cord blood for adults and studies aimed at expansion of cord blood cells and immune reconstitution are described. In Section I, Dr. Nelson Chao describes the early data in cord blood transplantation in adult patients. The patient outcomes are reviewed and analyzed for various factors such as cell dose, HLA typing, and patient selection that could have contributed to the final outcome of these adult patients. Myeloablative as well as nonmyeloablative approaches are presented. Discussion of the various benefits and risks are presented. More recent data from multiple single institutions as well as larger registry data comparisons are also provided. Analyses of these studies suggest methods to improve on the outcome. These newer data should lead to a logical progression in the use of cord blood cells in adult patients. In Section II, Dr. Stephen Emerson describes the historical efforts associated with expansion of hematopoietic stem cells, specifically with cord blood cells. These efforts to expand cord blood cells continue with novel methods. Moreover, a better understanding of stem cell biology and signaling is critical if we are to be able to effectively expand these cells for clinical use. An alternative, more direct, approach to expanding stem cells could be achieved by specific genetic pathways known or believed to support primitive HSC proliferation such as Notch-1 receptor activation, Wnt/LEF-1 pathway induction, telomerase or the Homeobox (Hox) gene products. The clinical experience with the use of expanded cord blood cells is also discussed. In Section III, Dr. Kenneth Weinberg describes immune reconstitution or lack thereof following cord blood transplantation. One of the hallmarks of successful hematopoietic stem cell transplantation is the ability to fully reconstitute the immune system of the recipient. Thus, the relationship between stem cell source and the development of T lymphocyte functions required for protection of the recipient from infection will be described, and cord blood recipients will be compared with those receiving other sources of stem cells. T cell development is described in detail, tracking from prethymic to postthymic lymphocytes with specific attention to umbilical cord blood as the source of stem cells. Moreover, a discussion of the placenta as a special microenvironment for umbilical cord blood is presented. Strategies to overcome the immunological defects are presented to improve the outcome of these recipients.

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

PubMed

Thoma, Eva C; Heckel, Tobias; Keller, David; Giroud, Nicolas; Leonard, Brian; Christensen, Klaus; Roth, Adrian; Bertinetti-Lapatki, Cristina; Graf, Martin; Patsch, Christoph

2016-10-25

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Quantitative Raman spectral changes of the differentiation of mesenchymal stem cells into islet-like cells by biochemical component analysis and multiple peak fitting

NASA Astrophysics Data System (ADS)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; He, Yingtian; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-12-01

Mesenchymal stem cells (MSCs) differentiate into islet-like cells, providing a possible solution for type I diabetes treatment. To search for the precise molecular mechanism of the directional differentiation of MSC-derived islet-like cells, biomolecular composition, and structural conformation information during MSC differentiation, is required. Because islet-like cells lack specific surface markers, the commonly employed immunostaining technique is not suitable for their identification, physical separation, and enrichment. Combining Raman spectroscopic data, a fitting accuracy-improved biochemical component analysis, and multiple peaks fitting approach, we identified the quantitative biochemical and intensity change of Raman peaks that show the differentiation of MSCs into islet-like cells. Along with increases in protein and glycogen content, and decreases in deoxyribonucleic acid and ribonucleic acid content, in islet-like cells relative to MSCs, it was found that a characteristic peak of insulin (665 cm-1) has twice the intensity in islet-like cells relative to MSCs, indicating differentiation of MSCs into islet-like cells was successful. Importantly, these Raman signatures provide useful information on the structural and pathological states during MSC differentiation and help to develop noninvasive and label-free Raman sorting methods for stem cells and their lineages.

[Unresolved issues in the evaluation of research projects involving induced pluripotent stem cells (iPS)].

PubMed

Casado, María; de Lecuona, Itziar

2013-01-01

This paper identifies problems and analyzes those conflicts posed by the evaluation of research projects involving the collection and use of human induced pluripotent stem cells (iPS) in Spain. Current legislation is causing problems of interpretation, circular and unnecessary referrals, legal uncertainty and undue delays. Actually, this situation may cause a lack of control and monitoring, and even some paralysis in regenerative medicine and cell therapy research, that is a priority nowadays. The analysis of the current legislation and its bioethical implications, led us to conclude that the review of iPS research projects cannot be assimilated to the evaluation of research projects that involve human embryonic stem cell (hESC). In this context, our proposal is based on the review by the Research Ethics Committees and the checkout by the Spanish Comission of Guarantees for Donation and Use of Human Cells and Tissues (CGDUCTH) of human iPS cells research projects. Moreover, this article claims for a more transparent research system, by effectively articulating the Registry on Research Projects. Finally, a model of verification protocol (checklist) for checking out biomedical research projects involving human iPS cells is suggested.

Concise Review: Fat and Furious: Harnessing the Full Potential of Adipose‐Derived Stromal Vascular Fraction

PubMed Central

Dykstra, Jordan A.; Facile, Tiffany; Patrick, Ryan J.; Francis, Kevin R.; Milanovich, Samuel; Weimer, Jill M.

2017-01-01

Abstract Due to their capacity to self‐renew, proliferate and generate multi‐lineage cells, adult‐derived stem cells offer great potential for use in regenerative therapies to stop and/or reverse degenerative diseases such as diabetes, heart failure, Alzheimer's disease and others. However, these subsets of cells can be isolated from different niches, each with differing potential for therapeutic applications. The stromal vascular fraction (SVF), a stem cell enriched and adipose‐derived cell population, has garnered interest as a therapeutic in regenerative medicine due to its ability to secrete paracrine factors that accelerate endogenous repair, ease of accessibility and lack of identified major adverse effects. Thus, one can easily understand the rush to employ adipose‐derived SVF to treat human disease. Perhaps faster than any other cell preparation, SVF is making its way to clinics worldwide, while critical preclinical research needed to establish SVF safety, efficacy and optimal, standardized clinical procedures are underway. Here, we will provide an overview of the current knowledge driving this phenomenon, its regulatory issues and existing studies, and propose potential unmapped applications. Stem Cells Translational Medicine 2017;6:1096–1108 PMID:28186685

Hydra, the everlasting embryo, confronts aging.

PubMed

Martínez, Daniel E; Bridge, Diane

2012-01-01

Existing data imply that the cnidarian Hydra vulgaris does not undergo senescence. In contrast, the related species Hydra oligactis shows increased mortality and physiological deterioration following sexual reproduction. Hydra thus offers the chance to study a striking difference in lifespan in members of the same genus. Adult Hydra possess three well-characterized stem cell populations, one of which gives rise to both somatic cells and gametes. The lack of senescence in Hydra vulgaris raises the question of how these stem cell populations are maintained over long periods of time. Investigation of the roles in Hydra of proteins involved in cellular stress responses in other organisms should provide insight into this issue. Proteins of particular interest include the Hsp70 family proteins and the transcription factor FoxO.

Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells

PubMed Central

Niu, N; Mercado-Uribe, I; Liu, J

2017-01-01

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse. PMID:28436947

Enabling Interactive Measurements from Large Coverage Microscopy

PubMed Central

Bajcsy, Peter; Vandecreme, Antoine; Amelot, Julien; Chalfoun, Joe; Majurski, Michael; Brady, Mary

2017-01-01

Microscopy could be an important tool for characterizing stem cell products if quantitative measurements could be collected over multiple spatial and temporal scales. With the cells changing states over time and being several orders of magnitude smaller than cell products, modern microscopes are already capable of imaging large spatial areas, repeat imaging over time, and acquiring images over several spectra. However, characterizing stem cell products from such large image collections is challenging because of data size, required computations, and lack of interactive quantitative measurements needed to determine release criteria. We present a measurement web system consisting of available algorithms, extensions to a client-server framework using Deep Zoom, and the configuration know-how to provide the information needed for inspecting the quality of a cell product. The cell and other data sets are accessible via the prototype web-based system at http://isg.nist.gov/deepzoomweb. PMID:28663600

Role of T cells in sex differences in syngeneic bone marrow transfers.

PubMed

Raveche, E S; Santoro, T; Brecher, G; Tjio, J H

1985-11-01

Transferred marrow cells will proliferate in normal mice not exposed to irradiation or any other type of stem cell depletion when five consecutive transfers of 40 million cells are given. Approximately 25% of the mitotic cells are of male donor origin observed cytogenetically in all of the female recipient spleens and marrow analyzed from two weeks to one and one-half years after transfusions. Male donor stem cells are accepted and form a stable component of the self-renewing stem cell pool. In contrast, only 5% female cells are found in male recipients. This sex difference in engraftment is not hormonal since castration of recipients does not alter the percentage of donor cells. Rigorous T depletion of female donor bone marrow, however, increases the percentage of donor engraftment to the level observed when male marrow, either whole or T depleted, is transferred to female recipients. The success of T-depleted female stem cells to seed male recipients is observed in both C57BL/6, a responder strain in which females readily respond to the H-Y antigen as manifest by skin graft rejection, and CBA/J, a strain in which females do not readily respond to H-Y. In addition, recipient nude BALB/c males, which lack a thymus, fail to accept whole bone marrow from BALB/c females. However, male bone marrow cells seed BALB/c nude females. These studies demonstrate that the poor engraftment of female cells in transfused male recipients is abrogated by the removal of T cells from the donor female marrow.

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katano, Takahito; Ootani, Akifumi; Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501

2013-03-22

Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system withinmore » the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.« less

Therapeutic cloning in individual parkinsonian mice

PubMed Central

Tabar, Viviane; Tomishima, Mark; Panagiotakos, Georgia; Wakayama, Sayaka; Menon, Jayanthi; Chan, Bill; Mizutani, Eiji; Al-Shamy, George; Ohta, Hiroshi; Wakayama, Teruhiko; Studer, Lorenz

2009-01-01

Cell transplantation with embryonic stem (ES) cell progeny requires immunological compatibility with host tissue. ‘Therapeutic cloning’ is a strategy to overcome this limitation by generating nuclear transfer (nt)ES cells that are genetically matched to an individual. Here we establish the feasibility of treating individual mice via therapeutic cloning. Derivation of 187 ntES cell lines from 24 parkinsonian mice, dopaminergic differentiation, and transplantation into individually matched host mice showed therapeutic efficacy and lack of immunological response. PMID:18376409

From bench to FDA to bedside: US regulatory trends for new stem cell therapies.

PubMed

Knoepfler, Paul S

2015-03-01

The phrase "bench-to-bedside" is commonly used to describe the translation of basic discoveries such as those on stem cells to the clinic for therapeutic use in human patients. However, there is a key intermediate step in between the bench and the bedside involving governmental regulatory oversight such as by the Food and Drug Administration (FDA) in the United States (US). Thus, it might be more accurate in most cases to describe the stem cell biological drug development process in this way: from bench to FDA to bedside. The intermediate development and regulatory stage for stem cell-based biological drugs is a multifactorial, continually evolving part of the process of developing a biological drug such as a stem cell-based regenerative medicine product. In some situations, stem cell-related products may not be classified as biological drugs in which case the FDA plays a relatively minor role. However, this middle stage is generally a major element of the process and is often colloquially referred to in an ominous way as "The Valley of Death". This moniker seems appropriate because it is at this point, and in particular in the work that ensues after Phase 1, clinical trials that most drug product development is terminated, often due to lack of funding, diseases being refractory to treatment, or regulatory issues. Not surprisingly, workarounds to deal with or entirely avoid this difficult stage of the process are evolving both inside and outside the domains of official regulatory authorities. In some cases these efforts involve the FDA invoking new mechanisms of accelerating the bench to beside process, but in other cases these new pathways bypass the FDA in part or entirely. Together these rapidly changing stem cell product development and regulatory pathways raise many scientific, ethical, and medical questions. These emerging trends and their potential consequences are reviewed here. Copyright © 2014 Elsevier B.V. All rights reserved.

Beyond the Mouse Monopoly: Studying the Male Germ Line in Domestic Animal Models

PubMed Central

González, Raquel; Dobrinski, Ina

2015-01-01

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals. PMID:25991701

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation.

PubMed

Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe; Bustin, Michael

2017-04-07

An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation

PubMed Central

Deng, Tao; Postnikov, Yuri; Zhang, Shaofei; Garrett, Lillian; Becker, Lore; Rácz, Ildikó; Hölter, Sabine M.; Wurst, Wolfgang; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe

2017-01-01

Abstract An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior. PMID:27923998

Just-in-time rescue plerixafor in combination with chemotherapy and granulocyte-colony stimulating factor for peripheral blood progenitor cell mobilization

PubMed Central

Smith, Veronica R.; Popat, Uday; Ciurea, Stefan; Nieto, Yago; Anderlini, Paolo; Rondon, Gabriela; Alousi, Amin; Qazilbash, Muzaffar; Kebriaei, Partow; Khouri, Issa; de Lima, Marcos; Champlin, Richard; Hosing, Chitra

2014-01-01

Plerixafor, a recently approved peripheral blood progenitor cell mobilizing agent, is often added to granulocyte-colony stimulating factor (G-CSF) to mobilize peripheral blood progenitor cells in patients with lymphoma or myeloma who cannot mobilize enough CD34+ cells with G-CSF alone to undergo autologous stem cell transplantation. However, data are lacking regarding the feasibility and efficacy of just-in-time plerixafor in combination with chemotherapy and G-CSF. We reviewed the peripheral blood stem cell collection data of 38 consecutive patients with lymphoma (Hodgkin’s and non-Hodgkin’s) and multiple myeloma who underwent chemomobilization and high-dose G-CSF and just-in-time plerixafor to evaluate the efficacy of this treatment combination. All patients with multiple myeloma and all but 1 patient with lymphoma collected the minimum required number of CD34+ cells to proceed with autologous stem cell transplantation (>2 × 106/kilogram of body weight). The median CD34+ cell dose collected in patients with non-Hodgkin lymphoma was 4.93 × 106/kilogram of body weight. The median CD34+ cell dose collected for patients with multiple myeloma was 8.81 × 106/kilogram of body weight. Plerixafor was well tolerated; no grade 2 or higher non- hematologic toxic effects were observed. PMID:23749720

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

PubMed Central

Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

2015-01-01

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

Cryopreservation in Closed Bag Systems as an Alternative to Clean Rooms for Preparations of Peripheral Blood Stem Cells.

PubMed

Spoerl, Silvia; Peter, Robert; Krackhardt, Angela M

2016-01-01

Autologous and allogeneic stem cell transplantation (SCT) represents a therapeutic option widely used for hematopoietic malignancies. One important milestone in the development of this treatment strategy was the development of effective cryopreservation technologies resulting in a high quality with respect to cell viability as well as lack of contamination of the graft.Stem cell preparations have been initially performed within standard laboratories as it is routinely still the case in many countries. With the emergence of cleanrooms, manufacturing of stem cell preparations within these facilities has become a new standard mandatory in Europe. However, due to high costs and laborious procedures, novel developments recently emerged using closed bag systems as reliable alternatives to conventional cleanrooms. Several hurdles needed to be overcome including the addition of the cryoprotectant dimethylsulfoxide (DMSO) as a relevant manipulation. As a result of the development, closed bag systems proved to be comparable in terms of product quality and patient outcome to cleanroom products. They also comply with the strict regulations of good manufacturing practice.With closed systems being available, costs and efforts of a cleanroom facility may be substantially reduced in the future. The process can be easily extended for other cell preparations requiring minor modifications as donor lymphocyte preparations. Moreover, novel developments may provide solutions for the production of advanced-therapy medicinal products in closed systems.

Effects of Intravenous Administration of Human Umbilical Cord Blood Stem Cells in 3-Acetylpyridine-Lesioned Rats

PubMed Central

Calatrava-Ferreras, Lucía; Gonzalo-Gobernado, Rafael; Herranz, Antonio S.; Reimers, Diana; Montero Vega, Teresa; Jiménez-Escrig, Adriano; Richart López, Luis Alberto; Bazán, Eulalia

2012-01-01

Cerebellar ataxias include a heterogeneous group of infrequent diseases characterized by lack of motor coordination caused by disturbances in the cerebellum and its associated circuits. Current therapies are based on the use of drugs that correct some of the molecular processes involved in their pathogenesis. Although these treatments yielded promising results, there is not yet an effective therapy for these diseases. Cell replacement strategies using human umbilical cord blood mononuclear cells (HuUCBMCs) have emerged as a promising approach for restoration of function in neurodegenerative diseases. The aim of this work was to investigate the potential therapeutic activity of HuUCBMCs in the 3-acetylpyridine (3-AP) rat model of cerebellar ataxia. Intravenous administered HuUCBMCs reached the cerebellum and brain stem of 3-AP ataxic rats. Grafted cells reduced 3-AP-induced neuronal loss promoted the activation of microglia in the brain stem, and prevented the overexpression of GFAP elicited by 3-AP in the cerebellum. In addition, HuUCBMCs upregulated the expression of proteins that are critical for cell survival, such as phospho-Akt and Bcl-2, in the cerebellum and brain stem of 3-AP ataxic rats. As all these effects were accompanied by a temporal but significant improvement in motor coordination, HuUCBMCs grafts can be considered as an effective cell replacement therapy for cerebellar disorders. PMID:23150735

Cure for thalassemia major – from allogeneic hematopoietic stem cell transplantation to gene therapy

PubMed Central

Srivastava, Alok; Shaji, Ramachandran V.

2017-01-01

Allogeneic hematopoietic stem cell transplantation has been well established for several decades as gene replacement therapy for patients with thalassemia major, and now offers very high rates of cure for patients who have access to this therapy. Outcomes have improved tremendously over the last decade, even in high-risk patients. The limited data available suggests that the long-term outcome is also excellent, with a >90% survival rate, but for the best results, hematopoietic stem cell transplantation should be offered early, before any end organ damage occurs. However, access to this therapy is limited in more than half the patients by the lack of suitable donors. Inadequate hematopoietic stem cell transplantation services and the high cost of therapy are other reasons for this limited access, particularly in those parts of the world which have a high prevalence of this condition. As a result, fewer than 10% of eligible patients are actually able to avail of this therapy. Other options for curative therapies are therefore needed. Recently, gene correction of autologous hematopoietic stem cells has been successfully established using lentiviral vectors, and several clinical trials have been initiated. A gene editing approach to correct the β-globin mutation or disrupt the BCL11A gene to increase fetal hemoglobin production has also been reported, and is expected to be introduced in clinical trials soon. Curative possibilities for the major hemoglobin disorders are expanding. Providing access to these therapies around the world will remain a challenge. PMID:27909215

Assessment of human MAPCs for stem cell transplantation and cardiac regeneration after myocardial infarction in SCID mice.

PubMed

Dimomeletis, Ilias; Deindl, Elisabeth; Zaruba, Marc; Groebner, Michael; Zahler, Stefan; Laslo, Saskia M; David, Robert; Kostin, Sawa; Deutsch, Markus A; Assmann, Gerd; Mueller-Hoecker, Josef; Feuring-Buske, Michaela; Franz, Wolfgang M

2010-11-01

Clinical studies suggest that transplantation of total bone marrow (BM) after myocardial infarction (MI) is feasible and potentially effective. However, focusing on a defined BM-derived stem cell type may enable a more specific and optimized treatment. Multilineage differentiation potential makes BM-derived multipotent adult progenitor cells (MAPCs) a promising stem cell pool for regenerative purposes. We analyzed the cardioregenerative potential of human MAPCs in a murine model of myocardial infarction. Human MAPCs were selected by negative depletion of CD45(+)/glycophorin(+) BM cells and plated on fibronectin-coated dishes. In vitro, stem cells were analyzed by reverse transcription polymerase chain reaction. In vivo, we transplanted human MAPCs (5 × 10(5)) by intramyocardial injection after MI in severe combined immunodeficient (SCID) beige mice. Six and 30 days after the surgical procedure, pressure-volume relationships were investigated in vivo. Heart tissues were analyzed immunohistochemically. Reverse transcription polymerase chain reaction experiments on early human MAPC passages evidenced an expression of Oct-4, a stem cell marker indicating pluripotency. In later passages, cardiac markers (Nkx2.5, GATA4, MLC-2v, MLC-2a, ANP, cTnT, cTnI,) and smooth muscle cell markers (SMA, SM22α) were expressed. Transplantation of human MAPCs into the ischemic border zone after MI resulted in an improved cardiac function at day 6 (ejection fraction, 26% vs 20%) and day 30 (ejection fraction, 30% vs 23%). Confirmation of human MAPC marker vimentin in immunohistochemistry demonstrated that human MAPC integrated in the peri-infarct region. The proliferation marker Ki67 was absent in immunohistochemistry and teratoma formation was not found, indicating no tumorous potential of transplanted human MAPCs in the tumor-sensitive SCID model. Transplantation of human MAPCs after MI ameliorates myocardial function, which may be explained by trophic effects of human MAPCs. Lack of evidence of tumorous potential in the tumor-sensitive SCID model indicates that human MAPCs may deliver an effective and safe stem cell pool for potential treatment of ischemic heart disease. Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

PubMed Central

Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

2013-01-01

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

Radiosensitivity of cancer-initiating cells and normal stem cells (or what the Heisenberg uncertainly principle has to do with biology).

PubMed

Woodward, Wendy Ann; Bristow, Robert Glen

2009-04-01

Mounting evidence suggests that parallels between normal stem cell biology and cancer biology may provide new targets for cancer therapy. Prospective identification and isolation of cancer-initiating cells from solid tumors has promoted the descriptive and functional identification of these cells allowing for characterization of their response to contemporary cancer therapies, including chemotherapy and radiation. In clinical radiation therapy, the failure to clinically eradicate all tumor cells (eg, a lack of response, partial response, or nonpermanent complete response by imaging) is considered a treatment failure. As such, biologists have explored the characteristics of the small population of clonogenic cancer cells that can survive and are capable of repopulating the tumor after subcurative therapy. Herein, we discuss the convergence of these clonogenic studies with contemporary radiosensitivity studies that use cell surface markers to identify cancer-initiating cells. Implications for and uncertainties regarding incorporation of these concepts into the practice of modern radiation oncology are discussed.

FOXN1 Transcription Factor in Epithelial Growth and Wound Healing.

PubMed

Grabowska, Anna I; Wilanowski, Tomasz

2017-09-01

FOXN1 is a prodifferentiation transcription factor in the skin epithelium. Recently, it has also emerged as an important player in controlling the skin wound healing process, as it actively participates in reepithelialization and is thought to be responsible for scar formation. FOXN1 positivity is also a feature of pigmented keratinocytes, including nevi, and FOXN1 is an attribute of benign epithelial tumors. The lack of FOXN1 favors the skin regeneration process displayed by nude mice, pointing to FOXN1 as a switch between regeneration and reparative processes. The stem cell niche provides a functional source of cells after the loss of tissue following wounding. The involvement of prodifferentiation factors in the regulation of this pool of stem cells is suggested. However, the exact mechanism is still under question, and we speculate that the FOXN1 transcription factor is involved in this process. This review analyzes the pleiotropic effects of FOXN1 in the skin, its function in the tumorigenesis process, and its potential role in depletion of the stem cell niche after injury, as well as its suggested mechanistic role, acting in a cell-autonomous and a non-cell-autonomous manner during skin self-renewal. Copyright © 2017 American Society for Microbiology.

Stem cells, blood vessels, and angiogenesis as major determinants for musculoskeletal tissue repair.

PubMed

Huard, Johnny

2018-05-22

This manuscript summarizes 20 years of research from my laboratories at the University of Pittsburgh and more recently, at the University of Texas Health Science Center at Houston and the Steadman Philippon Research Institute in Vail, Colorado. The discovery of muscle-derived stem cells (MDSCs) did not arise from a deliberate search to find a novel population of muscle cells with high regenerative potential, but instead was conceived in response to setbacks encountered while working in muscle cell transplantation for Duchenne muscular dystrophy (DMD). DMD is a devastating inherited X-linked muscle disease characterized by progressive muscle weakness due to lack of dystrophin expression in muscle fiber sarcolemma. Although the transplantation of normal myoblasts into dystrophin-deficient muscle can restore dystrophin, this approach has been hindered by limited survival (less than 1%) of the injected cells. The fact that 99% of the cells were not surviving implantation was seen as a major weakness with this technology by most. My research team decided to investigate which cells represent the 1% of the cells surviving post-implantation. We have subsequently confirmed that the few cells which exhibit high survival post-implantation also display stem cell characteristics, and were termed "muscle-derived stem cells" or MDSCs. Herein, I will describe the origin of these MDSCs, the mechanisms of MDSC action during tissue repair, and finally the development of therapeutic strategies to improve regeneration and repair of musculoskeletal tissues. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-9, 2018. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silva Meirelles, Lindolfo da, E-mail: lindolfomeirelles@gmail.com; Laboratory for Stem Cells and Tissue Engineering, PPGBioSaúde, Lutheran University of Brazil, Av. Farroupilha 8001, 92425-900 Canoas, RS; Deus Wagatsuma, Virgínia Mara de

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with anmore » AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.« less

Fulminant infectious mononucleosis and recurrent Epstein-Barr virus reactivation in an adolescent.

PubMed

Nourse, Jamie P; Jones, Kimberley; Dua, Ujjwal; Runnegar, Naomi; Looke, David; Schmidt, Chris; Tey, Siok-Keen; Kennedy, Glen; Gandhi, Maher K

2010-03-15

We describe a unique case of fulminant infectious mononucleosis and recurrent Epstein-Barr virus reactivation presenting in an adolescent. Detailed assays of Epstein-Barr virus-specific T cell immunity revealed defects in the patient's T cell receptor signalling pathway characterized by a lack of interleukin-2 and CD25 expression, which may have contributed to her clinical course. Allogeneic stem cell transplantation reversed the clinical and laboratory phenotype.

Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.

PubMed

Tilgner, Katarzyna; Atkinson, Stuart P; Yung, Sun; Golebiewska, Anna; Stojkovic, Miodrag; Moreno, Ruben; Lako, Majlinda; Armstrong, Lyle

2010-01-01

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.

Gli3 is a negative regulator of Tas1r3-expressing taste cells

PubMed Central

Jyotaki, Masafumi; Redding, Kevin; Jiang, Peihua

2018-01-01

Mouse taste receptor cells survive from 3–24 days, necessitating their regeneration throughout adulthood. In anterior tongue, sonic hedgehog (SHH), released by a subpopulation of basal taste cells, regulates transcription factors Gli2 and Gli3 in stem cells to control taste cell regeneration. Using single-cell RNA-Seq we found that Gli3 is highly expressed in Tas1r3-expressing taste receptor cells and Lgr5+ taste stem cells in posterior tongue. By PCR and immunohistochemistry we found that Gli3 was expressed in taste buds in all taste fields. Conditional knockout mice lacking Gli3 in the posterior tongue (Gli3CKO) had larger taste buds containing more taste cells than did control wild-type (Gli3WT) mice. In comparison to wild-type mice, Gli3CKO mice had more Lgr5+ and Tas1r3+ cells, but fewer type III cells. Similar changes were observed ex vivo in Gli3CKO taste organoids cultured from Lgr5+ taste stem cells. Further, the expression of several taste marker and Gli3 target genes was altered in Gli3CKO mice and/or organoids. Mirroring these changes, Gli3CKO mice had increased lick responses to sweet and umami stimuli, decreased lick responses to bitter and sour taste stimuli, and increased glossopharyngeal taste nerve responses to sweet and bitter compounds. Our results indicate that Gli3 is a suppressor of stem cell proliferation that affects the number and function of mature taste cells, especially Tas1r3+ cells, in adult posterior tongue. Our findings shed light on the role of the Shh pathway in adult taste cell regeneration and may help devise strategies for treating taste distortions from chemotherapy and aging. PMID:29415007

Enhanced periodontal regeneration using collagen, stem cells or growth factors.

PubMed

Basan, Tanja; Welly, Daniel; Kriebel, Katja; Scholz, Malte; Brosemann, Anne; Liese, Jan; Vollmar, Brigitte; Frerich, Bernhard; Lang, Hermann

2017-01-01

The regeneration of periodontal tissues still remains a challenge in periodontology. The aim of the present study was to examine the regenerative potential of a) different collagen support versus blank, b) different collagen support +/- a growth factor cocktail (GF) and c) a collagen powder versus collagen powder + periodontal ligament stem cells (PDLSCs) comparatively in a large animal model. The stem cells (SC) were isolated from extracted teeth of 15 adult miniature pigs. A total of 60 class II furcation defects were treated with the materials named above. Concluding, a histological evaluation followed. A significant increase in regeneration was observed in all treatment groups. The new attachment formation reached a maximum of 77 percent. In the control group a new attachment formation of 13 percent was observed. The study shows that all implanted materials improved periodontal regeneration, though there were no significant differences between the experimental groups. Within the limitations of this study, it can be assumed that the lack of significant differences is due to the complexity of the clinical setting.

Stem cell treatment for chronic lung diseases.

PubMed

Tzouvelekis, Argyris; Ntolios, Paschalis; Bouros, Demosthenes

2013-01-01

Chronic lung diseases such as idiopathic pulmonary fibrosis and cystic fibrosis or chronic obstructive pulmonary disease and asthma are leading causes of morbidity and mortality worldwide with a considerable human, societal and financial burden. In view of the current disappointing status of available pharmaceutical agents, there is an urgent need for alternative more effective therapeutic approaches that will not only help to relieve patient symptoms but will also affect the natural course of the respective disease. Regenerative medicine represents a promising option with several fruitful therapeutic applications in patients suffering from chronic lung diseases. Nevertheless, despite relative enthusiasm arising from experimental data, application of stem cell therapy in the clinical setting has been severely hampered by several safety concerns arising from the major lack of knowledge on the fate of exogenously administered stem cells within chronically injured lung as well as the mechanisms regulating the activation of resident progenitor cells. On the other hand, salient data arising from few 'brave' pilot investigations of the safety of stem cell treatment in chronic lung diseases seem promising. The main scope of this review article is to summarize the current state of knowledge regarding the application status of stem cell treatment in chronic lung diseases, address important safety and efficacy issues and present future challenges and perspectives. In this review, we argue in favor of large multicenter clinical trials setting realistic goals to assess treatment efficacy. We propose the use of biomarkers that reflect clinically inconspicuous alterations of the disease molecular phenotype before rigid conclusions can be safely drawn. Copyright © 2013 S. Karger AG, Basel.

Male gametogenesis without centrioles.

PubMed

Riparbelli, Maria Giovanna; Callaini, Giuliano

2011-01-15

The orientation of the mitotic spindle plays a central role in specifying stem cell-renewal by enabling interaction of the daughter cells with external cues: the daughter cell closest to the hub region is instructed to self-renew, whereas the distal one starts to differentiate. Here, we have analyzed male gametogenesis in DSas-4 Drosophila mutants and we have reported that spindle alignment and asymmetric divisions are properly executed in male germline stem cells that lack centrioles. Spermatogonial divisions also correctly proceed in the absence of centrioles, giving rise to cysts of 16 primary spermatocytes. By contrast, abnormal meiotic spindles assemble in primary spermatocytes. These results point to different requirements for centrioles during male gametogenesis of Drosophila. Spindle formation during germ cell mitosis may be successfully supported by an acentrosomal pathway that is inadequate to warrant the proper execution of meiosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Overcoming the Roadblocks to Cardiac Cell Therapy Using Tissue Engineering.

PubMed

Yanamandala, Mounica; Zhu, Wuqiang; Garry, Daniel J; Kamp, Timothy J; Hare, Joshua M; Jun, Ho-Wook; Yoon, Young-Sup; Bursac, Nenad; Prabhu, Sumanth D; Dorn, Gerald W; Bolli, Roberto; Kitsis, Richard N; Zhang, Jianyi

2017-08-08

Transplantations of various stem cells or their progeny have repeatedly improved cardiac performance in animal models of myocardial injury; however, the benefits observed in clinical trials have been generally less consistent. Some of the recognized challenges are poor engraftment of implanted cells and, in the case of human cardiomyocytes, functional immaturity and lack of electrical integration, leading to limited contribution to the heart's contractile activity and increased arrhythmogenic risks. Advances in tissue and genetic engineering techniques are expected to improve the survival and integration of transplanted cells, and to support structural, functional, and bioenergetic recovery of the recipient hearts. Specifically, application of a prefabricated cardiac tissue patch to prevent dilation and to improve pumping efficiency of the infarcted heart offers a promising strategy for making stem cell therapy a clinical reality. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

First step in developing SWNT nano-sensor for C17.2 neural stem cells

NASA Astrophysics Data System (ADS)

Ignatova, Tetyana; Pirbhai, Massooma; Chandrasekar, Swetha; Rotkin, Slava V.; Jedlicka, Sabrina

Nanomaterials are widely used for biomedical applications and diagnostics, including as drug and gene delivery agents, imaging objects, and biosensors. As single-wall carbon nanotubes (SWNTs) possess a size similar to intracellular components, including fibrillar proteins and some organelles, the potential for use in a wide variety of intracellular applications is significant. However, implementation of an SWNT based nano-sensor is difficult due to lack of understanding of SWNT-cell interaction on both the cellular and molecular level. In this study, C17.2 neural stem cells have been tested after uptake of SWNTs wrapped with ssDNA over a wide variety of time periods, allowing for broad localization of SWNTs inside of the cells over long time periods. The localization data is being used to develop a predictive model of how, upon uptake of SWNT, the cytoskeleton and other cellular structures of the adherent cells is perturbed.

Programming and Isolation of Highly Pure Physiologically and Pharmacologically Functional Sinus-Nodal Bodies from Pluripotent Stem Cells

PubMed Central

Jung, Julia Jeannine; Husse, Britta; Rimmbach, Christian; Krebs, Stefan; Stieber, Juliane; Steinhoff, Gustav; Dendorfer, Andreas; Franz, Wolfgang-Michael; David, Robert

2014-01-01

Summary Therapeutic approaches for “sick sinus syndrome” rely on electrical pacemakers, which lack hormone responsiveness and bear hazards such as infection and battery failure. These issues may be overcome via “biological pacemakers” derived from pluripotent stem cells (PSCs). Here, we show that forward programming of PSCs with the nodal cell inducer TBX3 plus an additional Myh6-promoter-based antibiotic selection leads to cardiomyocyte aggregates consisting of >80% physiologically and pharmacologically functional pacemaker cells. These induced sinoatrial bodies (iSABs) exhibited highly increased beating rates (300–400 bpm), coming close to those found in mouse hearts, and were able to robustly pace myocardium ex vivo. Our study introduces iSABs as highly pure, functional nodal tissue that is derived from PSCs and may be important for future cell therapies and drug testing in vitro. PMID:24936448

Genetic separation of phototropism and blue light inhibition of stem elongation

NASA Technical Reports Server (NTRS)

Liscum, E.; Young, J. C.; Poff, K. L.; Hangarter, R. P.

1992-01-01

Blue light-induced regulation of cell elongation is a component of the signal response pathway for both phototropic curvature and inhibition of stem elongation in higher plants. To determine if blue light regulates cell elongation in these responses through shared or discrete pathways, phototropism and hypocotyl elongation were investigated in several blue light response mutants in Arabidopsis thaliana. Specifically, the blu mutants that lack blue light-dependent inhibition of hypocotyl elongation were found to exhibit a normal phototropic response. In contrast, a phototropic null mutant (JK218) and a mutant that has a 20- to 30-fold shift in the fluence dependence for first positive phototropism (JK224) showed normal inhibition of hypocotyl elongation in blue light. F1 progeny of crosses between the blu mutants and JK218 showed normal phototropism and inhibition of hypocotyl elongation, and approximately 1 in 16 F2 progeny were double mutants lacking both responses. Thus, blue light-dependent inhibition of hypocotyl elongation and phototropism operate through at least some genetically distinct components.

Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS− cell populations

PubMed Central

El-Sayed, Karim M Fawzy; Paris, Sebastian; Graetz, Christian; Kassem, Neemat; Mekhemar, Mohamed; Ungefroren, Hendrick; Fändrich, Fred; Dörfer, Christof

2015-01-01

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells. PMID:25257881

Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

PubMed

Sharrow, Allison C; Perkins, Brandy; Collector, Michael I; Yu, Wayne; Simons, Brian W; Jones, Richard J

2016-08-01

The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes. Copyright © 2016 Elsevier Inc. All rights reserved.

Genetic modification of embryonic stem cells with VEGF enhances cell survival and improves cardiac function.

PubMed

Xie, Xiaoyan; Cao, Feng; Sheikh, Ahmad Y; Li, Zongjin; Connolly, Andrew J; Pei, Xuetao; Li, Ren-Ke; Robbins, Robert C; Wu, Joseph C

2007-01-01

Cardiac stem cell therapy remains hampered by acute donor cell death posttransplantation and the lack of reliable methods for tracking cell survival in vivo. We hypothesize that cells transfected with inducible vascular endothelial growth factor 165 (VEGF(165)) can improve their survival as monitored by novel molecular imaging techniques. Mouse embryonic stem (ES) cells were transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF(165) and renilla luciferase (Rluc). Addition of doxycycline induced Bi-Tet expression of VEGF(165) and Rluc significantly compared to baseline (p<0.05). Expression of VEGF(165) enhanced ES cell proliferation and inhibited apoptosis as determined by Annexin-V staining. For noninvasive imaging, ES cells were transduced with a double fusion (DF) reporter gene consisting of firefly luciferase and enhanced green fluorescence protein (Fluc-eGFP). There was a robust correlation between cell number and Fluc activity (R(2)=0.99). Analysis by immunostaining, histology, and RT-PCR confirmed that expression of Bi-Tet and DF systems did not affect ES cell self-renewal or pluripotency. ES cells were differentiated into beating embryoid bodies expressing cardiac markers such as troponin, Nkx2.5, and beta-MHC. Afterward, 5 x 10(5) cells obtained from these beating embryoid bodies or saline were injected into the myocardium of SV129 mice (n=36) following ligation of the left anterior descending (LAD) artery. Bioluminescence imaging (BLI) and echocardiography showed that VEGF(165) induction led to significant improvements in both transplanted cell survival and cardiac function (p<0.05). This is the first study to demonstrate imaging of embryonic stem cell-mediated gene therapy targeting cardiovascular disease. With further validation, this platform may have broad applications for current basic research and further clinical studies.

Current and Future Perspectives on Alginate Encapsulated Pancreatic Islet.

PubMed

Strand, Berit L; Coron, Abba E; Skjak-Braek, Gudmund

2017-04-01

Transplantation of pancreatic islets in immune protective capsules holds the promise as a functional cure for type 1 diabetes, also about 40 years after the first proof of principal study. The concept is simple in using semipermeable capsules that allow the ingress of oxygen and nutrients, but limit the access of the immune system. Encapsulated human islets have been evaluated in four small clinical trials where the procedure has been evaluated as safe, but lacking long-term efficacy. Host reactions toward the biomaterials used in the capsules may be one parameter limiting the long-term function of the graft in humans. The present article briefly discusses important capsule properties such as stability, permeability and biocompatibility, as well as possible strategies to overcome current challenges. Also, recent progress in capsule development as well as the production of insulin-producing cells from human stem cells that gives promising perspectives for the transplantation of encapsulated insulin-producing tissue is briefly discussed. Stem Cells Translational Medicine 2017;6:1053-1058. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

The effect of mesenchymal stem cells delivered via hydrogel-based tissue engineered periosteum on bone allograft healing

PubMed Central

Hoffman, Michael D.; Xie, Chao; Zhang, Xinping; Benoit, Danielle S.W.

2013-01-01

Allografts remain the clinical “gold standard” for treatment of critical sized bone defects despite minimal engraftment and ~60% long-term failure rates. Therefore, the development of strategies to improve allograft healing and integration are necessary. The periosteum and its associated stem cell population, which are lacking in allografts, coordinate autograft healing. Herein we utilized hydrolytically degradable hydrogels to transplant and localize mesenchymal stem cells (MSCs) to allograft surfaces, creating a periosteum mimetic, termed a ‘tissue engineered periosteum’. Our results demonstrated that this tissue engineering approach resulted in increased graft vascularization (~2.4-fold), endochondral bone formation (~2.8-fold), and biomechanical strength (1.8-fold), as compared to untreated allografts, over 16 weeks of healing. Despite this enhancement in healing, the process of endochondral ossification was delayed compared to autografts, requiring further modifications for this approach to be clinically acceptable. However, this bottom-up biomaterials approach, the engineered periosteum, can be augmented with alternative cell types, matrix cues, growth factors, and/or other small molecule drugs to expedite the process of ossification. PMID:23958029

Tissue- and cell-type–specific manifestations of heteroplasmic mtDNA 3243A>G mutation in human induced pluripotent stem cell-derived disease model

PubMed Central

Hämäläinen, Riikka H.; Manninen, Tuula; Koivumäki, Hanna; Kislin, Mikhail; Otonkoski, Timo; Suomalainen, Anu

2013-01-01

Mitochondrial DNA (mtDNA) mutations manifest with vast clinical heterogeneity. The molecular basis of this variability is mostly unknown because the lack of model systems has hampered mechanistic studies. We generated induced pluripotent stem cells from patients carrying the most common human disease mutation in mtDNA, m.3243A>G, underlying mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. During reprogramming, heteroplasmic mtDNA showed bimodal segregation toward homoplasmy, with concomitant changes in mtDNA organization, mimicking mtDNA bottleneck during epiblast specification. Induced pluripotent stem cell–derived neurons and various tissues derived from teratomas manifested cell-type specific respiratory chain (RC) deficiency patterns. Similar to MELAS patient tissues, complex I defect predominated. Upon neuronal differentiation, complex I specifically was sequestered in perinuclear PTEN-induced putative kinase 1 (PINK1) and Parkin-positive autophagosomes, suggesting active degradation through mitophagy. Other RC enzymes showed normal mitochondrial network distribution. Our data show that cellular context actively modifies RC deficiency manifestation in MELAS and that autophagy is a significant component of neuronal MELAS pathogenesis. PMID:24003133

EMP2 is a novel therapeutic target for endometrial cancer stem cells

PubMed Central

Kiyohara, Meagan H.; Dillard, Christen; Tsui, Jessica; Kim, Sara Ruth; Lu, Jianyi; Sachdev, Divya; Goodglick, Lee; Tong, Maomeng; Torous, Vanda Farahmand; Aryasomayajula, Chinmayi; Wang, Wei; Najafzadeh, Parisa; Gordon, Lynn K.; Braun, Jonathan; McDermott, Sean; Wicha, Max S.; Wadehra, Madhuri

2017-01-01

Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1). ALDH expression significantly promotes tumor initiation and correlates with the levels of EMP2 expression in both patient samples and tumor cell lines. As therapy against CSCs in endometrial cancer is lacking, the ability of anti-EMP2 IgG1 therapy to reduce primary and secondary tumor formation using xenograft HEC1A models was determined. Anti-EMP2 IgG1 reduced the expression and activity of ALDH and correspondingly reduced both primary and secondary tumor load. Our results collectively suggest that anti-EMP2 therapy may be a novel method of reducing endometrial cancer stem cells. PMID:28604744

A Novel Differentiation Therapy Approach to Reduce the Metastatic Potential of Basal, Highly Metastatic, Triple-Negative Breast Cancers

DTIC Science & Technology

2012-05-01

subset enriched in epithelial-to- mesenchymal transition and stem cell characteristics. Cancer Res 69: 4116–4124. Hoenerhoff MJ, Chu I, Barkan D, Liu ZY...expression of epithelial markers and loss of mesenchymal markers in MB- 231 cells (Task1a) Our analyses of m icroarray data com paring 231-Empty cells ...is considered a hallmark of EMT (Yang and Weinberg 2008). MB-231 cells lack E-cadherin expression and exhibit a more mesenchymal phenotype

Physical confinement signals regulate the organization of stem cells in three dimensions

PubMed Central

Sean, David; Ignacio, Maxime; Godin, Michel; Slater, Gary W.; Pelling, Andrew E.

2016-01-01

During embryogenesis, the spherical inner cell mass (ICM) proliferates in the confined environment of a blastocyst. Embryonic stem cells (ESCs) are derived from the ICM, and mimicking embryogenesis in vitro, mouse ESCs (mESCs) are often cultured in hanging droplets. This promotes the formation of a spheroid as the cells sediment and aggregate owing to increased physical confinement and cell–cell interactions. In contrast, mESCs form two-dimensional monolayers on flat substrates and it remains unclear if the difference in organization is owing to a lack of physical confinement or increased cell–substrate versus cell–cell interactions. Employing microfabricated substrates, we demonstrate that a single geometric degree of physical confinement on a surface can also initiate spherogenesis. Experiment and computation reveal that a balance between cell–cell and cell–substrate interactions finely controls the morphology and organization of mESC aggregates. Physical confinement is thus an important regulatory cue in the three-dimensional organization and morphogenesis of developing cells. PMID:27798278

PTEN expression and function in adult cancer stem cells and prospects for therapeutic targeting.

PubMed

Ciuffreda, Ludovica; Falcone, Italia; Incani, Ursula Cesta; Del Curatolo, Anais; Conciatori, Fabiana; Matteoni, Silvia; Vari, Sabrina; Vaccaro, Vanja; Cognetti, Francesco; Milella, Michele

2014-09-01

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a non-redundant lipid phosphatase that restrains and fine tunes the phosphatidylinositol-3-kinase (PI3K) signaling pathway. PTEN is involved in inherited syndromes, which predispose to different types of cancers and is among the most frequently inactivated tumor suppressor genes in sporadic cancers. Indeed, loss of PTEN function occurs in a wide spectrum of human cancers through a variety of mechanisms, including mutations, deletions, transcriptional silencing, or protein instability. PTEN prevents tumorigenesis through multiple mechanisms and regulates a plethora of cellular processes, including survival, proliferation, energy metabolism and cellular architecture. Moreover, recent studies have demonstrated that PTEN is able to exit, exist, and function outside the cell, allowing for inhibition of the PI3K pathway in neighboring cells in a paracrine fashion. Most recently, studies have shown that PTEN is also critical for stem cell maintenance and that PTEN loss can lead to the emergence and proliferation of cancer stem cell (CSC) clones. Depending on the cellular and tissue context of origin, PTEN deletion may result in increased self-renewal capacity or normal stem cell exhaustion and PTEN-defìcient stem and progenitor cells have been reported in prostate, lung, intestinal, and pancreatic tissues before tumor formation; moreover, reversible or irreversible PTEN loss is frequently observed in CSC from a variety of solid and hematologic malignancies, where it may contribute to the functional phenotype of CSC. In this review, we will focus on the role of PTEN expression and function and downstream pathway activation in cancer stem cell biology and regulation of the tumorigenic potential; the emerging role of PTEN in mediating the crosstalk between the PI3K and MAPK pathways will also be discussed, together with prospects for the therapeutic targeting of tumors lacking PTEN expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dynamic Network-Based Relevance Score Reveals Essential Proteins and Functional Modules in Directed Differentiation

PubMed Central

Wu, Chia-Chou; Lin, Che

2015-01-01

The induction of stem cells toward a desired differentiation direction is required for the advancement of stem cell-based therapies. Despite successful demonstrations of the control of differentiation direction, the effective use of stem cell-based therapies suffers from a lack of systematic knowledge regarding the mechanisms underlying directed differentiation. Using dynamic modeling and the temporal microarray data of three differentiation stages, three dynamic protein-protein interaction networks were constructed. The interaction difference networks derived from the constructed networks systematically delineated the evolution of interaction variations and the underlying mechanisms. A proposed relevance score identified the essential components in the directed differentiation. Inspection of well-known proteins and functional modules in the directed differentiation showed the plausibility of the proposed relevance score, with the higher scores of several proteins and function modules indicating their essential roles in the directed differentiation. During the differentiation process, the proteins and functional modules with higher relevance scores also became more specific to the neuronal identity. Ultimately, the essential components revealed by the relevance scores may play a role in controlling the direction of differentiation. In addition, these components may serve as a starting point for understanding the systematic mechanisms of directed differentiation and for increasing the efficiency of stem cell-based therapies. PMID:25977693

Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells

PubMed Central

Gruenloh, William; Kambal, Amal; Sondergaard, Claus; McGee, Jeannine; Nacey, Catherine; Kalomoiris, Stefanos; Pepper, Karen; Olson, Scott; Fierro, Fernando

2011-01-01

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48 h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties. PMID:21275830

Notch2 blockade enhances hematopoietic stem cell mobilization and homing.

PubMed

Wang, Weihuan; Yu, Shuiliang; Myers, Jay; Wang, Yiwei; Xin, William W; Albakri, Marwah; Xin, Alison W; Li, Ming; Huang, Alex Y; Xin, Wei; Siebel, Christian W; Lazarus, Hillard M; Zhou, Lan

2017-10-01

Despite use of newer approaches, some patients being considered for autologous hematopoietic cell transplantation (HCT) may only mobilize limited numbers of hematopoietic progenitor cells (HPCs) into blood, precluding use of the procedure, or being placed at increased risk of complications due to slow hematopoietic reconstitution. Developing more efficacious HPC mobilization regimens and strategies may enhance the mobilization process and improve patient outcome. Although Notch signaling is not essential for homeostasis of adult hematopoietic stem cells (HSCs), Notch-ligand adhesive interaction maintains HSC quiescence and niche retention. Using Notch receptor blocking antibodies, we report that Notch2 blockade, but not Notch1 blockade, sensitizes hematopoietic stem cells and progenitors (HSPCs) to mobilization stimuli and leads to enhanced egress from marrow to the periphery. Notch2 blockade leads to transient myeloid progenitor expansion without affecting HSC homeostasis and self-renewal. We show that transient Notch2 blockade or Notch2-loss in mice lacking Notch2 receptor lead to decreased CXCR4 expression by HSC but increased cell cycling with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. In addition, we found that Notch2-blocked or Notch2-deficient marrow HSPCs show an increased homing to the marrow, while mobilized Notch2-blocked, but not Notch2-deficient stem cells and progenitors, displayed a competitive repopulating advantage and enhanced hematopoietic reconstitution. These findings suggest that blocking Notch2 combined with the current clinical regimen may further enhance HPC mobilization and improve engraftment during HCT. Copyright© 2017 Ferrata Storti Foundation.

Electrical and Mechanical Strategies to Enable Cardiac Repair and Regeneration

PubMed Central

Cao, Hung; Kang, Bong Jin; Lee, Chia-An; Shung, K. Kirk; Hsiai, Tzung K.

2015-01-01

Inadequate replacement of lost ventricular myocardium from myocardial infarction leads to heart failure. Investigating the regenerative capacity of mammalian hearts represents an emerging direction for tissue engineering and cell-based therapy. Recent advances in stem cells hold promise to restore cardiac functions. However, embryonic or induced pluripotent stem cell-derived cardiomyocytes lack functional phenotypes of the native myocardium, and transplanted tissues are not fully integrated for synchronized electrical and mechanical coupling with the host. In this context, this review highlights the mechanical and electrical strategies to promote cardiomyocyte maturation and integration, and to assess the functional phenotypes of regenerating myocardium. Simultaneous micro-electrocardiogram and high-frequency ultrasound techniques will also be introduced to assess electrical and mechanical coupling for small animal models of heart regeneration. PMID:25974948

Stem Cell Technology for (Epi)genetic Brain Disorders.

PubMed

Riemens, Renzo J M; Soares, Edilene S; Esteller, Manel; Delgado-Morales, Raul

2017-01-01

Despite the enormous efforts of the scientific community over the years, effective therapeutics for many (epi)genetic brain disorders remain unidentified. The common and persistent failures to translate preclinical findings into clinical success are partially attributed to the limited efficiency of current disease models. Although animal and cellular models have substantially improved our knowledge of the pathological processes involved in these disorders, human brain research has generally been hampered by a lack of satisfactory humanized model systems. This, together with our incomplete knowledge of the multifactorial causes in the majority of these disorders, as well as a thorough understanding of associated (epi)genetic alterations, has been impeding progress in gaining more mechanistic insights from translational studies. Over the last years, however, stem cell technology has been offering an alternative approach to study and treat human brain disorders. Owing to this technology, we are now able to obtain a theoretically inexhaustible source of human neural cells and precursors in vitro that offer a platform for disease modeling and the establishment of therapeutic interventions. In addition to the potential to increase our general understanding of how (epi)genetic alterations contribute to the pathology of brain disorders, stem cells and derivatives allow for high-throughput drugs and toxicity testing, and provide a cell source for transplant therapies in regenerative medicine. In the current chapter, we will demonstrate the validity of human stem cell-based models and address the utility of other stem cell-based applications for several human brain disorders with multifactorial and (epi)genetic bases, including Parkinson's disease (PD), Alzheimer's disease (AD), fragile X syndrome (FXS), Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Rett syndrome (RTT).

FoxG1 interacts with Bmi1 to regulate self-renewal and tumorigenicity of medulloblastoma stem cells.

PubMed

Manoranjan, Branavan; Wang, Xin; Hallett, Robin M; Venugopal, Chitra; Mack, Stephen C; McFarlane, Nicole; Nolte, Sara M; Scheinemann, Katrin; Gunnarsson, Thorsteinn; Hassell, John A; Taylor, Michael D; Lee, Cathy; Triscott, Joanna; Foster, Colleen M; Dunham, Christopher; Hawkins, Cynthia; Dunn, Sandra E; Singh, Sheila K

2013-07-01

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. Recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Groups 1 and 2 are characterized by relatively good clinical outcomes and activation of the Wnt and Shh pathways, respectively. In contrast, groups 3 and 4 ("non-Shh/Wnt MBs") are distinguished by metastatic disease, poor patient outcome, and lack a molecular pathway phenotype. Current gene expression platforms have not detected brain tumor-initiating cell (BTIC) self-renewal genes in groups 3 and 4 MBs as BTICs typically comprise a minority of tumor cells and may therefore go undetected on bulk tumor analyses. Since increasing BTIC frequency has been associated with increasing tumor aggressiveness and poor patient outcome, we investigated the subgroup-specific gene expression profile of candidate stem cell genes within 251 primary human MBs from four nonoverlapping MB transcriptional databases (Amsterdam, Memphis, Toronto, Boston) and 74 NanoString-subgrouped MBs (Vancouver). We assessed the functional relevance of two genes, FoxG1 and Bmi1, which were significantly enriched in non-Shh/Wnt MBs and showed these genes to mediate MB stem cell self-renewal and tumor initiation in mice. We also identified their transcriptional regulation through reciprocal promoter occupancy in CD15+ MB stem cells. Our work demonstrates the application of stem cell data gathered from genomic platforms to guide functional BTIC assays, which may then be used to develop novel BTIC self-renewal mechanisms amenable to therapeutic targeting. Copyright © 2013 AlphaMed Press.

[Preliminary establishment of transplanted human chronic myeloid leukemia model in nude mice].

PubMed

Li, Xian-Min; Ding, Xin; Zhang, Long-Zhen; Cen, Jian-Nong; Chen, Zi-Xing

2011-12-01

Chronic myeloid leukemia (CML) is a malignant clonal disease derived from hematopoietic stem cells. CML stem cells were thought to be the root which could lead disease development and ultimately rapid change. However, a stable animal model for studying the characteristics of CML stem cells is currently lacking. This study was aimed to establish a transplanted human CML nude-mice model to further explore the biological behavior of CML stem cells in vivo, and to enrich CML stem cells in nude mice by series transplantation. The 4 - 6 weeks old BALB/c nude mice pretreated by splenectomy (S), cytoxan intraperitoneal injection (C) and sublethal irradiation (I) were transplanted intravenously with (5 - 7) × 10(7) of bone marrow mononuclear cells from CML patients in chronic phase. Alternatively, 4 - 6 weeks old BALB/c nude mice pretreated by lethal irradiation were transplanted intravenously with 5 × 10(6) homologous bone marrow cells of BALB/c nude mice together with (5 - 7) × 10(7) of bone marrow mononuclear cells from CML patients in chronic phase simultaneously. The leukemic cells engrafted and infiltrated in organs and bone marrow of the mice were tracked by reverse transcription-polymerase chain reaction (RT-PCR), plastic-embedded biopsy and flow cytometry. The results of these two methods were compared. The results showed that human CML cells engrafted and infiltrating into the bone marrow of two nude mice pretreated with SCI could be detected. In spite of the low successful rate, results suggested the feasibility of this method by using BALB/c nude mice as a human CML animal model. In contrast, in nude mice pretreated by the lethal dose irradiation, CML cells in the bone marrow could not be found. It is concluded that human bone marrow CML cells can results in leukemia in nude mice pretreated by SCI. Thus this study provides a new strategy for establishment of CML animal models which deserves further elaboration.

Raman spectroscopy for discrimination of neural progenitor cells and their lineages (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Keren; Ong, William; Chew, Sing Yian; Liu, Quan

2017-02-01

Neurological diseases are one of the leading causes of adult disability and they are estimated to cause more deaths than cancer in the elderly population by 2040. Stem cell therapy has shown great potential in treating neurological diseases. However, before cell therapy can be widely adopted in the long term, a number of challenges need to be addressed, including the fundamental research about cellular development of neural progenitor cells. To facilitate the fundamental research of neural progenitor cells, many methods have been developed to identify neural progenitor cells. Although great progress has been made, there is still lack of an effective method to achieve fast, label-free and noninvasive differentiation of neural progenitor cells and their lineages. As a fast, label-free and noninvasive technique, spontaneous Raman spectroscopy has been conducted to characterize many types of stem cells including neural stem cells. However, to our best knowledge, it has not been studied for the discrimination of neural progenitor cells from specific lineages. Here we report the differentiation of neural progenitor cell from their lineages including astrocytes, oligodendrocytes and neurons using spontaneous Raman spectroscopy. Moreover, we also evaluate the influence of system parameters during spectral acquisition on the quality of measured Raman spectra and the accuracy of classification using the spectra, which yield a set of optimal system parameters facilitating future studies.

The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium.

PubMed

Franzetti, Eleonora; Romanelli, Davide; Caccia, Silvia; Cappellozza, Silvia; Congiu, Terenzio; Rajagopalan, Muthukumaran; Grimaldi, Annalisa; de Eguileor, Magda; Casartelli, Morena; Tettamanti, Gianluca

2015-08-01

The midgut represents the middle part of the alimentary canal and is responsible for nutrient digestion and absorption in insect larva. Despite the growing interest in this organ for different purposes, such as studies on morphogenesis and differentiation, stem cell biology, cell death processes and transport mechanisms, basic information on midgut development is still lacking for a large proportion of insect species. Undoubtedly, this lack of data could hinder the full exploitation of practical applications that involve midgut as their primary target. This may represent in particular a significant problem for Lepidoptera, an insect order that includes some of the most important species of high economic importance. With the aim of overcoming this fragmentation of knowledge, we performed a detailed morphofunctional analysis of the midgut of the silkworm, Bombyx mori, a representative model among Lepidoptera, during its development from the larval up to the adult stage, focusing attention on stem cells. Our data demonstrate stem cell proliferation and differentiation, not only in the larval midgut but also in the pupal and adult midgut epithelium. Moreover, we present evidence for a complex trophic relationship between the dying larval epithelium and the new adult one, which is established during metamorphosis. This study, besides representing the first morphological and functional characterization of the changes that occur in the midgut of a lepidopteron during the transition from the larva to the moth, provides a detailed analysis of the midgut of the adult insect, a stage that has been neglected up to now.

Airway delivery of mesenchymal stem cells prevents arrested alveolar growth in neonatal lung injury in rats.

PubMed

van Haaften, Timothy; Byrne, Roisin; Bonnet, Sebastien; Rochefort, Gael Y; Akabutu, John; Bouchentouf, Manaf; Rey-Parra, Gloria J; Galipeau, Jacques; Haromy, Alois; Eaton, Farah; Chen, Ming; Hashimoto, Kyoko; Abley, Doris; Korbutt, Greg; Archer, Stephen L; Thébaud, Bernard

2009-12-01

Bronchopulmonary dysplasia (BPD) and emphysema are characterized by arrested alveolar development or loss of alveoli; both are significant global health problems and currently lack effective therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown. We hypothesized that intratracheal delivery of BMSCs would prevent alveolar destruction in experimental BPD. In vitro, BMSC differentiation and migration were assessed using co-culture assays and a modified Boyden chamber. In vivo, the therapeutic potential of BMSCs was assessed in a chronic hyperoxia-induced model of BPD in newborn rats. In vitro, BMSCs developed immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2) (surfactant protein C expression and lamellar bodies) when co-cultured with lung tissue, but not with culture medium alone or liver. Migration assays revealed preferential attraction of BMSCs toward oxygen-damaged lung versus normal lung. In vivo, chronic hyperoxia in newborn rats led to air space enlargement and loss of lung capillaries, and this was associated with a decrease in circulating and resident lung BMSCs. Intratracheal delivery of BMSCs on Postnatal Day 4 improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and pulmonary hypertension. Engrafted BMSCs coexpressed the AEC2-specific marker surfactant protein C. However, engraftment was disproportionately low for cell replacement to account for the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived conditioned medium prevented O(2)-induced AEC2 apoptosis, accelerated AEC2 wound healing, and enhanced endothelial cord formation. BMSCs prevent arrested alveolar and vascular growth in part through paracrine activity. Stem cell-based therapies may offer new therapeutic avenues for lung diseases that currently lack efficient treatments.

Attenuated Innate Immunity in Embryonic Stem Cells and Its Implications in Developmental Biology and Regenerative Medicine.

PubMed

Guo, Yan-Lin; Carmichael, Gordon G; Wang, Ruoxing; Hong, Xiaoxiao; Acharya, Dhiraj; Huang, Faqing; Bai, Fengwei

2015-11-01

Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past 2 decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology, and in regenerative medicine. © 2015 AlphaMed Press.

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

PubMed

Thompson, Heather L; van Rooijen, Nico; McLelland, Bryce T; Manilay, Jennifer O

2016-01-01

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin - BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo . Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80 + macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80 + macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro . Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo .

Resveratrol Exerts Dosage and Duration Dependent Effect on Human Mesenchymal Stem Cell Development

PubMed Central

Peltz, Lindsay; Gomez, Jessica; Marquez, Maribel; Alencastro, Frances; Atashpanjeh, Negar; Quang, Tara; Bach, Thuy; Zhao, Yuanxiang

2012-01-01

Studies in the past have illuminated the potential benefit of resveratrol as an anticancer (pro-apoptosis) and life-extending (pro-survival) compound. However, these two different effects were observed at different concentration ranges. Studies of resveratrol in a wide range of concentrations on the same cell type are lacking, which is necessary to comprehend its diverse and sometimes contradictory cellular effects. In this study, we examined the effects of resveratrol on cell self-renewal and differentiation of human mesenchymal stem cells (hMSCs), a type of adult stem cells that reside in a number of tissues, at concentrations ranging from 0.1 to 10 µM after both short- and long-term exposure. Our results reveal that at 0.1 µM, resveratrol promotes cell self-renewal by inhibiting cellular senescence, whereas at 5 µM or above, resveratrol inhibits cell self-renewal by increasing senescence rate, cell doubling time and S-phase cell cycle arrest. At 1 µM, its effect on cell self-renewal is minimal but after long-term exposure it exerts an inhibitory effect, accompanied with increased senescence rate. At all concentrations, resveratrol promotes osteogenic differentiation in a dosage dependent manner, which is offset by its inhibitory effect on cell self-renewal at high concentrations. On the contrary, resveratrol suppresses adipogenic differentiation during short-term exposure but promotes this process after long-term exposure. Our study implicates that resveratrol is the most beneficial to stem cell development at 0.1 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its dosage dependent effect on hMSCs. PMID:22615926

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

PubMed

Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

2016-04-01

The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hiPSCs), followed by the differentiation of these cells toward a retinal lineage, including photoreceptors, retinal ganglion cells, and retinal pigment epithelium, has been demonstrated. The use of mRNA reprogramming to yield pluripotency represents a unique ability to derive pluripotent stem cells without the use of DNA vectors, ensuring the lack of genomic integration and constitutive expression. The studies reported in the present article serve to establish a more reproducible system with which to derive retinal cell types from hiPSCs through the prevention of genomic integration of delivered genes and should also eliminate the risk of constitutive expression of these genes. Such ability has important implications for the study of, and development of potential treatments for, retinal degenerative disorders and the development of novel therapeutic approaches to the treatment of these diseases. ©AlphaMed Press.

In vitro model of cerebral ischemia by using brain microvascular endothelial cells derived from human induced pluripotent stem cells.

PubMed

Kokubu, Yasuhiro; Yamaguchi, Tomoko; Kawabata, Kenji

2017-04-29

Brain-derived microvascular endothelial cells (BMECs), which play a central role in blood brain barrier (BBB), can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported, they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy, drug screening, and pathological study. In the recent study, an induction method of BMECs from PSCs has been established, making it possible to more precisely study the in vitro human BBB function. Here, using induced pluripotent stem (iPS) cell-derived BMECs, we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function, and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly, TNF-α, which is known to be secreted from astrocytes and microglia in the cerebral ischemia, prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus, we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs. Copyright © 2017 Elsevier Inc. All rights reserved.

Omcg1 is critically required for mitosis in rapidly dividing mouse intestinal progenitors and embryonic stem cells.

PubMed

Léguillier, Teddy; Vandormael-Pournin, Sandrine; Artus, Jérôme; Houlard, Martin; Picard, Christel; Bernex, Florence; Robine, Sylvie; Cohen-Tannoudji, Michel

2012-07-15

Recent studies have shown that factors involved in transcription-coupled mRNA processing are important for the maintenance of genome integrity. How these processes are linked and regulated in vivo remains largely unknown. In this study, we addressed in the mouse model the function of Omcg1, which has been shown to participate in co-transcriptional processes, including splicing and transcription-coupled repair. Using inducible mouse models, we found that Omcg1 is most critically required in intestinal progenitors. In absence of OMCG1, proliferating intestinal epithelial cells underwent abnormal mitosis followed by apoptotic cell death. As a consequence, the crypt proliferative compartment of the small intestine was quickly and totally abrogated leading to the rapid death of the mice. Lack of OMCG1 in embryonic stem cells led to a similar cellular phenotype, with multiple mitotic defects and rapid cell death. We showed that mutant intestinal progenitors and embryonic stem cells exhibited a reduced cell cycle arrest following irradiation, suggesting that mitotic defects may be consecutive to M phase entry with unrepaired DNA damages. These findings unravel a crucial role for pre-mRNA processing in the homeostasis of the small intestine and point to a major role of OMCG1 in the maintenance of genome integrity.

Generation and Long-term Maintenance of Nerve-free Hydra.

PubMed

Tran, Cassidy M; Fu, Sharon; Rowe, Trevor; Collins, Eva-Maria S

2017-07-07

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Just-in-time rescue plerixafor in combination with chemotherapy and granulocyte-colony stimulating factor for peripheral blood progenitor cell mobilization.

PubMed

Smith, Veronica R; Popat, Uday; Ciurea, Stefan; Nieto, Yago; Anderlini, Paolo; Rondon, Gabriela; Alousi, Amin; Qazilbash, Muzaffar; Kebriaei, Partow; Khouri, Issa; de Lima, Marcos; Champlin, Richard; Hosing, Chitra

2013-09-01

Plerixafor, a recently approved peripheral blood progenitor cell mobilizing agent, is often added to granulocyte-colony stimulating factor (G-CSF) to mobilize peripheral blood progenitor cells in patients with lymphoma or myeloma who cannot mobilize enough CD34+ cells with G-CSF alone to undergo autologous stem cell transplantation. However, data are lacking regarding the feasibility and efficacy of just-in-time plerixafor in combination with chemotherapy and G-CSF. We reviewed the peripheral blood stem cell collection data of 38 consecutive patients with lymphoma (Hodgkin's and non-Hodgkin's) and multiple myeloma who underwent chemomobilization and high-dose G-CSF and just-in-time plerixafor to evaluate the efficacy of this treatment combination. All patients with multiple myeloma and all but one patient with lymphoma collected the minimum required number of CD34+ cells to proceed with autologous stem cell transplantation (>2 × 10(6) /kg of body weight). The median CD34+ cell dose collected in patients with non-Hodgkin lymphoma was 4.93 × 10(6) /kg of body weight. The median CD34+ cell dose collected for patients with multiple myeloma was 8.81 × 10(6) /kg of body weight. Plerixafor was well tolerated; no grade 2 or higher non-hematologic toxic effects were observed. Copyright © 2013 Wiley Periodicals, Inc.

UTX regulates mesoderm differentiation of embryonic stem cells independent of H3K27 demethylase activity.

PubMed

Wang, Chaochen; Lee, Ji-Eun; Cho, Young-Wook; Xiao, Ying; Jin, Qihuang; Liu, Chengyu; Ge, Kai

2012-09-18

To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/β-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.

A myogenic precursor cell that could contribute to regeneration in zebrafish and its similarity to the satellite cell.

PubMed

Siegel, Ashley L; Gurevich, David B; Currie, Peter D

2013-09-01

The cellular basis for mammalian muscle regeneration has been an area of intense investigation over recent decades. The consensus is that a specialized self-renewing stem cell, termed the satellite cell, plays a major role during the process of regeneration in amniotes. How broadly this mechanism is deployed within the vertebrate phylogeny remains an open question. A lack of information on the role of cells analogous to the satellite cell in other vertebrate systems is even more unexpected given the fact that satellite cells were first designated in frogs. An intriguing aspect of this debate is that a number of amphibia and many fish species exhibit epimorphic regenerative processes in specific tissues, whereby regeneration occurs by the dedifferentiation of the damaged tissue, without deploying specialized stem cell populations analogous to satellite cells. Hence, it is feasible that a cellular process completely distinct from that deployed during mammalian muscle regeneration could operate in species capable of epimorphic regeneration. In this minireview, we examine the evidence for the broad phylogenetic distribution of satellite cells. We conclude that, in the vertebrates examined so far, epimorphosis does not appear to be deployed during muscle regeneration, and that analogous cells expressing similar marker genes to satellite cells appear to be deployed during the regenerative process. However, the functional definition of these cells as self-renewing muscle stem cells remains a final hurdle to the definition of the satellite cell as a generic vertebrate cell type. © 2013 FEBS.

Articular cartilage tissue engineering with plasma-rich in growth factors and stem cells with nano scaffolds

NASA Astrophysics Data System (ADS)

Montaser, Laila M.; Abbassy, Hadeer A.; Fawzy, Sherin M.

2016-09-01

The ability to heal soft tissue injuries and regenerate cartilage is the Holy Grail of musculoskeletal medicine. Articular cartilage repair and regeneration is considered to be largely intractable due to the poor regenerative properties of this tissue. Due to their low self-repair ability, cartilage defects that result from joint injury, aging, or osteoarthritis, are the most often irreversible and are a major cause of joint pain and chronic disability. However, current methods do not perfectly restore hyaline cartilage and may lead to the apparition of fibro- or continue hypertrophic cartilage. The lack of efficient modalities of treatment has prompted research into tissue engineering combining stem cells, scaffold materials and environmental factors. The field of articular cartilage tissue engineering, which aims to repair, regenerate, and/or improve injured or diseased cartilage functionality, has evoked intense interest and holds great potential for improving cartilage therapy. Plasma-rich in growth factors (PRGF) and/or stem cells may be effective for tissue repair as well as cartilage regenerative processes. There is a great promise to advance current cartilage therapies toward achieving a consistently successful approach for addressing cartilage afflictions. Tissue engineering may be the best way to reach this objective via the use of stem cells, novel biologically inspired scaffolds and, emerging nanotechnology. In this paper, current and emergent approach in the field of cartilage tissue engineering is presented for specific application. In the next years, the development of new strategies using stem cells, in scaffolds, with supplementation of culture medium could improve the quality of new formed cartilage.

Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet.

PubMed

Li, Dan; Zhu, Lian; Liu, Yu; Yin, Zongqi; Liu, Yi; Liu, Fangjun; He, Aijuan; Feng, Shaoqing; Zhang, Yixin; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

2017-05-01

In vivo niche plays an important role in regulating differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. This study explored the feasibility that chondrocyte sheet created chondrogenic niche retained chondrogenic phenotype of BMSC engineered cartilage (BEC) in subcutaneous environments. Porcine BMSCs were seeded into biodegradable scaffolds followed by 4weeks of chondrogenic induction in vitro to form BEC, which were wrapped with chondrocyte sheets (Sheet group), acellular small intestinal submucosa (SIS, SIS group), or nothing (Blank group) respectively and then implanted subcutaneously into nude mice to trace the maintenance of chondrogenic phenotype. The results showed that all the constructs in Sheet group displayed typical cartilaginous features with abundant lacunae and cartilage specific matrices deposition. These samples became more mature with prolonged in vivo implantation, and few signs of ossification were observed at all time points except for one sample that had not been wrapped completely. Cell labeling results in Sheet group further revealed that the implanted BEC directly participated in cartilage formation. Samples in both SIS and Blank groups mainly showed ossified tissue at all time points with partial fibrogenesis in a few samples. These results suggested that chondrocyte sheet could create a chondrogenic niche for retaining chondrogenic phenotype of BEC in subcutaneous environment and thus provide a novel research model for stable ectopic cartilage regeneration based on stem cells. In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by high-density culture of chondrocytes in vitro could cearte a chondrogenic niche in subcutaneous environment and efficiently retain the chondrogenic phenotype of in vitro BMSC engineered cartilage (vitro-BEC). Furthermore, cell tracing results revealed that the regenerated cartilage mainly derived from the implanted vitro-BEC. The current study not only proposes a novel research model for microenvironment simulation but also provides a useful strategy for stable ectopic cartilage regeneration of stem cells. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Scalloped and Yorkie are required for cell cycle re-entry of quiescent cells after tissue damage.

PubMed

Meserve, Joy H; Duronio, Robert J

2015-08-15

Regeneration of damaged tissues typically requires a population of active stem cells. How damaged tissue is regenerated in quiescent tissues lacking a stem cell population is less well understood. We used a genetic screen in the developing Drosophila melanogaster eye to investigate the mechanisms that trigger quiescent cells to re-enter the cell cycle and proliferate in response to tissue damage. We discovered that Hippo signaling regulates compensatory proliferation after extensive cell death in the developing eye. Scalloped and Yorkie, transcriptional effectors of the Hippo pathway, drive Cyclin E expression to induce cell cycle re-entry in cells that normally remain quiescent in the absence of damage. Ajuba, an upstream regulator of Hippo signaling that functions as a sensor of epithelial integrity, is also required for cell cycle re-entry. Thus, in addition to its well-established role in modulating proliferation during periods of tissue growth, Hippo signaling maintains homeostasis by regulating quiescent cell populations affected by tissue damage. © 2015. Published by The Company of Biologists Ltd.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

PubMed

Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

2017-10-10

Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

PubMed

Antonucci, Ivana; Di Pietro, Roberta; Alfonsi, Melissa; Centurione, Maria Antonietta; Centurione, Lucia; Sancilio, Silvia; Pelagatti, Francesca; D'Amico, Maria Angela; Di Baldassarre, Angela; Piattelli, Adriano; Tetè, Stefano; Palka, Giandomenico; Borlongan, Cesar V; Stuppia, Liborio

2014-01-01

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

Recording axonal conduction to evaluate the integration of pluripotent cell-derived neurons into a neuronal network.

PubMed

Shimba, Kenta; Sakai, Koji; Takayama, Yuzo; Kotani, Kiyoshi; Jimbo, Yasuhiko

2015-10-01

Stem cell transplantation is a promising therapy to treat neurodegenerative disorders, and a number of in vitro models have been developed for studying interactions between grafted neurons and the host neuronal network to promote drug discovery. However, methods capable of evaluating the process by which stem cells integrate into the host neuronal network are lacking. In this study, we applied an axonal conduction-based analysis to a co-culture study of primary and differentiated neurons. Mouse cortical neurons and neuronal cells differentiated from P19 embryonal carcinoma cells, a model for early neural differentiation of pluripotent stem cells, were co-cultured in a microfabricated device. The somata of these cells were separated by the co-culture device, but their axons were able to elongate through microtunnels and then form synaptic contacts. Propagating action potentials were recorded from these axons by microelectrodes embedded at the bottom of the microtunnels and sorted into clusters representing individual axons. While the number of axons of cortical neurons increased until 14 days in vitro and then decreased, those of P19 neurons increased throughout the culture period. Network burst analysis showed that P19 neurons participated in approximately 80% of the bursting activity after 14 days in vitro. Interestingly, the axonal conduction delay of P19 neurons was significantly greater than that of cortical neurons, suggesting that there are some physiological differences in their axons. These results suggest that our method is feasible to evaluate the process by which stem cell-derived neurons integrate into a host neuronal network.

Bone Tissue Engineering: Recent Advances and Challenges

PubMed Central

Amini, Ami R.; Laurencin, Cato T.; Nukavarapu, Syam P.

2013-01-01

The worldwide incidence of bone disorders and conditions has trended steeply upward and is expected to double by 2020, especially in populations where aging is coupled with increased obesity and poor physical activity. Engineered bone tissue has been viewed as a potential alternative to the conventional use of bone grafts, due to their limitless supply and no disease transmission. However, bone tissue engineering practices have not proceeded to clinical practice due to several limitations or challenges. Bone tissue engineering aims to induce new functional bone regeneration via the synergistic combination of biomaterials, cells, and factor therapy. In this review, we discuss the fundamentals of bone tissue engineering, highlighting the current state of this field. Further, we review the recent advances of biomaterial and cell-based research, as well as approaches used to enhance bone regeneration. Specifically, we discuss widely investigated biomaterial scaffolds, micro- and nano-structural properties of these scaffolds, and the incorporation of biomimetic properties and/or growth factors. In addition, we examine various cellular approaches, including the use of mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), and platelet-rich plasma (PRP), and their clinical application strengths and limitations. We conclude by overviewing the challenges that face the bone tissue engineering field, such as the lack of sufficient vascularization at the defect site, and the research aimed at functional bone tissue engineering. These challenges will drive future research in the field. PMID:23339648

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud

PubMed Central

Norrie, Jacqueline L.; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C.; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T.; Galli, Antonella; Ji, Hongkai

2016-01-01

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4. Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. PMID:27827819

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

PubMed

Norrie, Jacqueline L; Li, Qiang; Co, Swanie; Huang, Bau-Lin; Ding, Ding; Uy, Jann C; Ji, Zhicheng; Mackem, Susan; Bedford, Mark T; Galli, Antonella; Ji, Hongkai; Vokes, Steven A

2016-12-15

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis. © 2016. Published by The Company of Biologists Ltd.

Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth

NASA Astrophysics Data System (ADS)

Yao, Shenglian; Liu, Xi; Yu, Shukui; Wang, Xiumei; Zhang, Shuming; Wu, Qiong; Sun, Xiaodan; Mao, Haiquan

2016-05-01

The development of novel biomaterials that deliver precise regulatory signals to direct stem cell fate for nerve regeneration is the focus of current intensive research efforts. In this study, a hierarchically aligned fibrillar fibrin hydrogel (AFG) that was fabricated through electrospinning and the concurrent molecular self-assembly process mimics both the soft and oriented features of nerve tissue, thus providing hybrid biophysical cues to instruct cell behavior in vitro and in vivo. The electrospun hydrogels were examined by scanning electron microscopy (SEM), polarized light microscopy, small angle X-ray scattering assay and atomic force microscopy (AFM), showing a hierarchically linear-ordered structure from the nanoscale to the macroscale with a soft elastic character (elasticity ~1 kPa). We found that this low elasticity and aligned topography of AFG exhibit co-effects on promoting the neurogenic differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) in comparison to random fibrin hydrogel (RFG) and tissue culture plate (TCP) control after two week cell culture in growth medium lacking supplementation with soluble neurogenic induction factors. In addition, AFG also induces dorsal root ganglion (DRG) neurons to rapidly project numerous long neurite outgrowths longitudinally along the AFG fibers for a total neurite extension distance of 1.96 mm in three days in the absence of neurotrophic factor supplementation. Moreover, the AFG implanted in a rat T9 dorsal hemisection spinal cord injury model was found to promote endogenous neural cell fast migration and axonal invasion along AFG fibers, resulting in aligned tissue cables in vivo. Our results suggest that matrix stiffness and aligned topography may instruct stem cell neurogenic differentiation and rapid neurite outgrowth, providing great promise for biomaterial design for applications in nerve regeneration.The development of novel biomaterials that deliver precise regulatory signals to direct stem cell fate for nerve regeneration is the focus of current intensive research efforts. In this study, a hierarchically aligned fibrillar fibrin hydrogel (AFG) that was fabricated through electrospinning and the concurrent molecular self-assembly process mimics both the soft and oriented features of nerve tissue, thus providing hybrid biophysical cues to instruct cell behavior in vitro and in vivo. The electrospun hydrogels were examined by scanning electron microscopy (SEM), polarized light microscopy, small angle X-ray scattering assay and atomic force microscopy (AFM), showing a hierarchically linear-ordered structure from the nanoscale to the macroscale with a soft elastic character (elasticity ~1 kPa). We found that this low elasticity and aligned topography of AFG exhibit co-effects on promoting the neurogenic differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) in comparison to random fibrin hydrogel (RFG) and tissue culture plate (TCP) control after two week cell culture in growth medium lacking supplementation with soluble neurogenic induction factors. In addition, AFG also induces dorsal root ganglion (DRG) neurons to rapidly project numerous long neurite outgrowths longitudinally along the AFG fibers for a total neurite extension distance of 1.96 mm in three days in the absence of neurotrophic factor supplementation. Moreover, the AFG implanted in a rat T9 dorsal hemisection spinal cord injury model was found to promote endogenous neural cell fast migration and axonal invasion along AFG fibers, resulting in aligned tissue cables in vivo. Our results suggest that matrix stiffness and aligned topography may instruct stem cell neurogenic differentiation and rapid neurite outgrowth, providing great promise for biomaterial design for applications in nerve regeneration. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01169a

RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

PubMed

Ge, Jianfeng; Burnier, Laurent; Adamopoulou, Maria; Kwa, Mei Qi; Schaks, Matthias; Rottner, Klemens; Brakebusch, Cord

2018-06-15

Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFβ-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFβ-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 ( col1a1 ). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFβ-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFβ-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFβ signaling and have implications for our understanding of MSC function in fibrosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The specific linker phosphorylation of Smad2/3 indicates epithelial stem cells in stomach; particularly increasing in mucosae of Helicobacter-associated gastritis.

PubMed

Fukui, Toshiro; Kishimoto, Masanobu; Nakajima, Atsushi; Yamashina, Masao; Nakayama, Shinji; Kusuda, Takeo; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2011-04-01

The gastric corpus and antrum are believed to contain epithelial stem cells in the isthmus. However, the lack of useful markers has hindered studies of their origin. We explored whether Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), could serve as a marker for stem cells. Stomachs, small intestines, and colons from Helicobacter felis-infected and noninfected C57BL/6 mice were examined. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, or doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) was performed, and pSmad2/3L-Thr immunostaining-positive cells were counted. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. In infected mice, pSmad2/3L-Thr immunostaining-positive cells were significantly increased in the corpus and antrum compared with those of noninfected mice (p < 0.0001). The number of Ki67 immunostaining-positive cells in the corpus and antrum of infected mice was also much greater than in the noninfected mice. Although pSmad2/3L-Thr immunostaining-positive cells were detected among the Ki67 cells, immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr immunostaining-positive cells showed immunohistochemical co-localization with cytokeratin 8, but some of them showed co-localization or adjacent localization with DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features and were confirmed in the isthmus. In small intestines and colons, pSmad2/3L-Thr immunostaining-positive cells were detected in specific epithelial cells around crypt bases, where the respective putative stem cells are thought to exist. We have identified the significant expression of pSmad2/3L-Thr in specific epithelial cells of the murine stomach and have suggested these cells to be epithelial stem cells.

MicroRNA-137 is downregulated in glioblastoma and inhibits the stemness of glioma stem cells by targeting RTVP-1

PubMed Central

Bier, Ariel; Giladi, Nis; Kronfeld, Noam; Lee, Hae Kyung; Cazacu, Simona; Finniss, Susan; Xiang, Cunli; Poisson, Laila; deCarvalho, Ana C.; Slavin, Shimon; Jacoby, Elad; Yalon, Michal; Toren, Amos; Mikkelsen, Tom; Brodie, Chaya

2013-01-01

Glioblastomas (GBM), the most common and aggressive malignant astrocytic tumors, contain a small subpopulation of cancer stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Here, we study the expression and function of miR-137, a putative suppressor miRNA, in GBM and GSCs. We found that the expression of miR-137 was significantly lower in GBM and GSCs compared to normal brains and neural stem cells (NSCs) and that the miR-137 promoter was hypermethylated in the GBM specimens. The expression of miR-137 was increased in differentiated NSCs and GSCs and overexpression of miR-137 promoted the neural differentiation of both cell types. Moreover, pre-miR-137 significantly decreased the self-renewal of GSCs and the stem cell markers Oct4, Nanog, Sox2 and Shh. We identified RTVP-1 as a novel target of miR-137 in GSCs; transfection of the cells with miR-137 decreased the expression of RTVP-1 and the luciferase activity of RTVP-1 3'-UTR reporter plasmid. Furthermore, overexpression of RTVP-1 plasmid lacking its 3'-UTR abrogated the inhibitory effect of miR-137 on the self-renewal of GSCs. Silencing of RTVP-1 decreased the self-renewal of GSCs and the expression of CXCR4 and overexpression of CXCR4 abrogated the inhibitory effect of RTVP-1 silencing on GSC self-renewal. These results demonstrate that miR-137 is downregulated in GBM probably due to promoter hypermethylation. miR-137 inhibits GSC self-renewal and promotes their differentiation by targeting RTVP-1 which downregulates CXCR4. Thus, miR-137 and RTVP-1 are attractive therapeutic targets for the eradication of GSCs and for the treatment of GBM. PMID:23714687

In vitro Culture of Naïve Human Bone Marrow Mesenchymal Stem Cells: A Stemness Based Approach

PubMed Central

Pal, Bidisha; Das, Bikul

2017-01-01

Human bone marrow derived mesenchymal stem cells (BM-MSCs) resides in their niches in close proximity to hematopoietic stem cells (HSCs). These naïve MSCs have tremendous potential in regenerative therapeutics, and may also be exploited by cancer and infectious disease agents. Hence, it is important to study the physiological and pathological roles of naïve MSC. However, our knowledge of naïve MSCs is limited by lack of appropriate isolation and in vitro culture methods. Established culture methods use serum rich media, and serial passaging for retrospective isolation of MSCs. These primed MSCs may not reflect the true physiological and pathological roles of naive MSCs (Figure 1). Therefore, there is a strong need for direct isolation and in vitro culture of naïve MSCs to study their stemness (self-renewal and undifferentiated state) and developmental ontogeny. We have taken a niche-based approach on stemness to better maintain naïve MSCs in vitro. In this approach, stemness is broadly divided as niche dependent (extrinsic), niche independent (intrinsic) and niche modulatory (altruistic or competitive). Using this approach, we were able to maintain naïve CD271+/CD133+ BM-MSCs for 2 weeks. Furthermore, this in vitro culture system helped us to identify naïve MSCs as a protective niche site for Mycobacterium tuberculosis, the causative organism of pulmonary tuberculosis. In this review, we discuss the in vitro culture of primed vs. naïve human BM derived MSCs with a special focus on how a stemness based approach could facilitate the study of naïve BM-MSCs. PMID:28884113

PITX2 Enhances the Regenerative Potential of Dystrophic Skeletal Muscle Stem Cells.

PubMed

Vallejo, Daniel; Hernández-Torres, Francisco; Lozano-Velasco, Estefanía; Rodriguez-Outeiriño, Lara; Carvajal, Alejandra; Creus, Carlota; Franco, Diego; Aránega, Amelia Eva

2018-04-10

Duchenne muscular dystrophy (DMD), one of the most lethal genetic disorders, involves progressive muscle degeneration resulting from the absence of DYSTROPHIN. Lack of DYSTROPHIN expression in DMD has critical consequences in muscle satellite stem cells including a reduced capacity to generate myogenic precursors. Here, we demonstrate that the c-isoform of PITX2 transcription factor modifies the myogenic potential of dystrophic-deficient satellite cells. We further show that PITX2c enhances the regenerative capability of mouse DYSTROPHIN-deficient satellite cells by increasing cell proliferation and the number of myogenic committed cells, but importantly also increasing dystrophin-positive (revertant) myofibers by regulating miR-31. These PITX2-mediated effects finally lead to improved muscle function in dystrophic (DMD/mdx) mice. Our studies reveal a critical role for PITX2 in skeletal muscle repair and may help to develop therapeutic strategies for muscular disorders. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Lymphomatoid hypersensitivity reaction to levofloxacin during autologous stem cell transplantation: a potential diagnostic pitfall in patients treated for lymphoma or leukemia.

PubMed

Esparza, Edward M; Takeshita, Junko; George, Evan

2011-01-01

Drug-associated cutaneous lymphomatoid hypersensitivity reactions are rare eruptions that can clinically and microscopically mimic a bona fide lymphomatous process. Clinically, the appearance ranges from papulosquamous to purpuric. Histopathologically, these reactions simulate a wide variety of lymphoma subtypes; the most frequently reported examples resemble mycosis fungoides. We report a 61-year-old female who developed a purpuric eruption prior to engraftment of an autologous hematopoietic stem cell transplant for stage IV mantle cell lymphoma. Skin biopsies showed a superficial perivascular and interstitial infiltrate of large, immature-appearing mononuclear cells associated with spongiosis, papillary dermal edema and erythrocyte extravasation. The cells were immunoreactive for T-cell markers and lacked B-cell marker expression, excluding recurrence of the underlying mantle cell lymphoma as a diagnostic possibility. The cutaneous eruption was temporally linked to levofloxacin administration and resolved after discontinuation of this medication. This is the first report of a lymphomatoid hypersensitivity reaction associated with fluoroquinolone use. The histopathologic features presented in this paper underscore the potential for misdiagnosis of such lesions as lymphoma or acute myeloid leukemia, particularly in the setting of hematopoietic stem cell transplantation for underlying lymphoma or leukemia. Clinical correlation, morphologic comparison to the original malignancy and immunohistochemical studies aid the dermatopathologist in rendering the correct diagnosis. Copyright © 2010 John Wiley & Sons A/S.

Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration.

PubMed

Hackelberg, Sandra; Tuck, Samuel J; He, Long; Rastogi, Arjun; White, Christina; Liu, Liqian; Prieskorn, Diane M; Miller, Ryan J; Chan, Che; Loomis, Benjamin R; Corey, Joseph M; Miller, Josef M; Duncan, R Keith

2017-01-01

Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution.

PubMed

Afrimzon, E; Botchkina, G; Zurgil, N; Shafran, Y; Sobolev, M; Moshkov, S; Ravid-Hermesh, O; Ojima, I; Deutsch, M

2016-03-21

Specific phenotypic subpopulations of cancer stem cells (CSCs) are responsible for tumor development, production of heterogeneous differentiated tumor mass, metastasis, and resistance to therapies. The development of therapeutic approaches based on targeting rare CSCs has been limited partially due to the lack of appropriate experimental models and measurement approaches. The current study presents new tools and methodologies based on a hydrogel microstructure array (HMA) for identification and multiplex analyses of CSCs. Low-melt agarose integrated with type I collagen, a major component of the extracellular matrix (ECM), was used to form a solid hydrogel array with natural non-adhesive characteristics and high optical quality. The array contained thousands of individual pyramidal shaped, nanoliter-volume micro-chambers (MCs), allowing concomitant generation and measurement of large populations of free-floating CSC spheroids from single cells, each in an individual micro-chamber (MC). The optical live cell platform, based on an imaging plate patterned with HMA, was validated using CSC-enriched prostate and colon cancer cell lines. The HMA methodology and quantitative image analysis at single-element resolution clearly demonstrates several levels of tumor cell heterogeneity, including morphological and phenotypic variability, differences in proliferation capacity and in drug response. Moreover, the system facilitates real-time examination of single stem cell (SC) fate, as well as drug-induced alteration in expression of stemness markers. The technology may be applicable in personalized cancer treatment, including multiplex ex vivo analysis of heterogeneous patient-derived tumor specimens, precise detection and characterization of potentially dangerous cell phenotypes, and for representative evaluation of drug sensitivity of CSCs and other types of tumor cells.

Three-dimensional polymer scaffolds for enhanced differentiation of human mesenchymal stem cells to hepatocyte-like cells: a comparative study.

PubMed

Chitrangi, Swati; Nair, Prabha; Khanna, Aparna

2017-08-01

Stem cell-based tissue engineering has emerged as a promising avenue for the treatment of liver diseases and as drug metabolism and toxicity models in drug discovery and development. The in vitro simulation of a micro-environmental niche for hepatic differentiation remains elusive, due to lack of information about crucial factors for the stem cell niche. For generation of functional hepatocytes, an in vivo three-dimensional (3D) micro-environment and architecture should be reproduced. Towards this, we fabricated three scaffolds as dextran-gelatin (DG1), chitosan-hyaluronic acid (CH1) and gelatin-vinyl acetate (GEVAC). Hepatic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) was induced by culturing hUC-MSCs on these scaffolds. The scaffolds support hepatic differentiation by mimicking the native extracellular matrix (ECM) micro-environment and architecture to facilitate 3D cell-cell and cell-matrix interactions. The expression of hepatic markers, glycogen storage, urea production, albumin secretion and cytochrome P450 (CYP450) activity indicated the hepatic differentiation of hUC-MSCs. The differentiated hUC-MSCs on the 3D scaffolds formed hepatospheroids (3D hepatocyte aggregates), as illustrated by scanning electron microscopy (SEM), confocal microscopy and cytoskeleton organization. It was observed that the 3D scaffolds supported improved cell morphology, expression of hepatic markers and metabolic activities, as compared to Matrigel-coated plates. To the best of our knowledge, this is the first report demonstrating the use of a well-characterized scaffold (GEVAC) for enhanced differentiation of hUC-MSCs to hepatocyte-like cells (HLCs). Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

PubMed

Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

2016-01-01

A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

Encapsulation of mesenchymal stem cells in chitosan/β-glycerophosphate hydrogel for seeding on a novel calcium phosphate cement scaffold.

PubMed

Liu, Tao; Li, Jian; Shao, Zengwu; Ma, Kaige; Zhang, Zhicai; Wang, Baichuan; Zhang, Yannan

2018-06-01

Due to its moldability, biocompatibility, osteoconductivity and resorbability, calcium phosphate cement (CPC) is a highly promising scaffold material for orthopedic applications. However, pH changes and ionic activity during the CPC setting reaction may adversely affect cells seeded directly on CPC. Moreover, a lack of macropores in CPC limits ingrowth of new bone. The objectives of this study were to prepare macroporous CPC scaffolds via porogen leaching, using mannitol crystals as the porogen and to evaluate the in vitro proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) encapsulated in chitosan/β-glycerophosphate (C/GP) hydrogel prior to exposure to the novel CPC scaffold. MSCs were found to be adhered to the surfaces of CPC macropores via scanning electron microscopy. The viability and osteogenic differentiation of MSCs in C/GP hydrogel with or without exposure to CPC constructs containing mannitol crystals indicated that coating with C/GP hydrogel protected the cells during cement mixing and setting. In conclusion, novel, macroporous CPC scaffolds were prepared, and our data indicate that a hydrogel encapsulation-based strategy can be used to protect cells during scaffold formation. Thus, the MSC-laden CPC scaffolds show promise for the delivery of stem cells to promote bone regeneration. Copyright © 2018 IPEM. Published by Elsevier Ltd. All rights reserved.

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

PubMed

Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-08-01

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Aberrant Gene Expression Profiles in Pluripotent Stem Cells Induced from Fibroblasts of a Klinefelter Syndrome Patient*

PubMed Central

Ma, Yu; Li, Chunliang; Gu, Junjie; Tang, Fan; Li, Chun; Li, Peng; Ping, Ping; Yang, Shi; Li, Zheng; Jin, Ying

2012-01-01

Klinefelter syndrome (KS) is the most common male chromosome aneuploidy. Its pathophysiology is largely unexplained due to the lack of adequate models. Here, we report the derivation of induced pluripotent stem cell (iPSCs) lines from a KS patient with a karyotype of 47, XXY. Derived KS-iPSCs meet all criteria of normal iPSCs with the potential for germ cell differentiation. Although X chromosome inactivation occurs in all KS-iPSCs, genome-wide transcriptome analysis identifies aberrantly expressed genes associated with the clinical features of KS. Our KS-iPSCs can serve as a cellular model for KS research. Identified genes may become biomarkers for early diagnosis or potential therapeutic targets for KS and significantly accelerate the understanding, diagnosis, and treatment of Klinefelter syndrome. PMID:23019320

Adult hematopoietic stem cells lacking Hif-1α self-renew normally.

PubMed

Vukovic, Milica; Sepulveda, Catarina; Subramani, Chithra; Guitart, Amélie V; Mohr, Jasmine; Allen, Lewis; Panagopoulou, Theano I; Paris, Jasmin; Lawson, Hannah; Villacreces, Arnaud; Armesilla-Diaz, Alejandro; Gezer, Deniz; Holyoake, Tessa L; Ratcliffe, Peter J; Kranc, Kamil R

2016-06-09

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance. © 2016 by The American Society of Hematology.

Adult hematopoietic stem cells lacking Hif-1α self-renew normally

PubMed Central

Vukovic, Milica; Sepulveda, Catarina; Subramani, Chithra; Guitart, Amélie V.; Mohr, Jasmine; Allen, Lewis; Panagopoulou, Theano I.; Paris, Jasmin; Lawson, Hannah; Villacreces, Arnaud; Armesilla-Diaz, Alejandro; Gezer, Deniz; Holyoake, Tessa L.; Ratcliffe, Peter J.

2016-01-01

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α–deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α–deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance. PMID:27060169

β-Arrestin2 mediates progression of murine primary myelofibrosis.

PubMed

Rein, Lindsay Am; Wisler, James W; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Minyong; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia Kl; Premont, Richard T; Lefkowitz, Robert J

2017-12-21

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2-/- cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

A novel autologous stem cell procedure for the treatment of aplastic anaemia using reprogrammed mature adult cells: a pilot study

PubMed Central

Abuljadayel, Ilham Saleh; Mohanty, Dipika; Suri, Rajendar K.

2012-01-01

Background & objectives: Aplastic anaemia is a life threatening rare bone marrow failure disorder. The underlying haematopoietic cellular deficit leads to haemorrhage, infection and severe anaemia. The treatment of choice for this haematological condition is allogeneic bone marrow transplantation from fully matched HLA sibling. Though this procedure is curative in the majority of young patients with aplastic anaemia, extending this benefit to older patients or those lacking a family donor remains a major challenge. Herein, the safety and efficacy of infusing autologous retrodifferentiated haematopoietic stem cells (RHSC) into four patients with aplastic anaemia without the use of any pre- or post-conditioning regimen including immunosuppression is described. Methods: Un-mobilized, mononuclear cells were harvested from four patients with acquired aplastic anaemia by aphaeresis. Mononuclear cells of patients were cultured with purified monoclonal antibody against the monomorphic regions of the beta chain of MHC class II antigens (Clone CR3/43) for 3 h, to obtain autologous RHSC. Autologous RHSC were washed and infused into the four patients without the use of any pre- or post-conditioning regimen. Thereafter, the efficacy (engraftment) of autologous RHSC was assessed in these patients. Results: Following single infusion of the autologous RHSC, two of the four patients with aplastic anaemia become transfusion independent for more than seven years. Karyotyping and G-banding analysis prior and post-procedure in all patients remained the same. Interpretation & conclusions: The findings of this pilot study demonstrated the functional utility of reprogrammed fully differentiated adult cells into pluripotent stem cells with extensive repopulation potentials in a human setting and without any pre- or post-conditioning regimen, including immunosuppression. This autologous approach of stem cell creation may broaden the curative potentials of stem cell therapy to a wider population of patients with aplastic anaemia, including many patients suffering from other haematological and non-haematological disorders. PMID:22825605

A novel autologous stem cell procedure for the treatment of aplastic anaemia using reprogrammed mature adult cells: a pilot study.

PubMed

Abuljadayel, Ilham Saleh; Mohanty, Dipika; Suri, Rajendar K

2012-06-01

Aplastic anaemia is a life threatening rare bone marrow failure disorder. The underlying haematopoietic cellular deficit leads to haemorrhage, infection and severe anaemia. The treatment of choice for this haematological condition is allogeneic bone marrow transplantation from fully matched HLA sibling. Though this procedure is curative in the majority of young patients with aplastic anaemia, extending this benefit to older patients or those lacking a family donor remains a major challenge. Herein, the safety and efficacy of infusing autologous retrodifferentiated haematopoietic stem cells (RHSC) into four patients with aplastic anaemia without the use of any pre- or post-conditioning regimen including immunosuppression is described. Un-mobilized, mononuclear cells were harvested from four patients with acquired aplastic anaemia by aphaeresis. Mononuclear cells of patients were cultured with purified monoclonal antibody against the monomorphic regions of the beta chain of MHC class II antigens (Clone CR3/43) for 3 h, to obtain autologous RHSC. Autologous RHSC were washed and infused into the four patients without the use of any pre- or post-conditioning regimen. Thereafter, the efficacy (engraftment) of autologous RHSC was assessed in these patients. Following single infusion of the autologous RHSC, two of the four patients with aplastic anaemia become transfusion independent for more than seven years. Karyotyping and G-banding analysis prior and post-procedure in all patients remained the same. The findings of this pilot study demonstrated the functional utility of reprogrammed fully differentiated adult cells into pluripotent stem cells with extensive repopulation potentials in a human setting and without any pre- or post-conditioning regimen, including immunosuppression. This autologous approach of stem cell creation may broaden the curative potentials of stem cell therapy to a wider population of patients with aplastic anaemia, including many patients suffering from other haematological and non-haematological disorders.

microRNA-150 Regulates Mobilization and Migration of Bone Marrow-Derived Mononuclear Cells by Targeting Cxcr4

PubMed Central

Tano, Nobuko; Kim, Ha Won; Ashraf, Muhammad

2011-01-01

The interaction between chemokine receptor type 4 (CXCR4) and its ligand, stromal cell-derived factor (SDF)-1, plays an important role in stem cell mobilization and migration in ischemic tissues. MicroRNAs (miRs) are key regulators of stem cell function and are involved in regulation of stem cell survival and differentiation to adopt different cell lineages. In this study, we show that ischemia inhibits the expression of miR-150 in BM-derived mononuclear cells (MNC) and activates its target Cxcr4 gene. Our results show that miR-150/CXCR4 cascade enhances MNC mobilization and migration. By using mouse acute myocardial infarction (MI) model, we found that MNCs in peripheral blood (PB) were increased significantly at day 5 after AMI as compared to control group and the number of CXCR4 positive MNCs both in bone marrow (BM) and PB was also markedly increased after MI. Analysis by microarray-based miRNA profiling and real-time PCR revealed that the expression of miR-150 which targets Cxcr4 gene as predicted was significantly downregulated in BM-MNCs after MI. Abrogation of miR-150 markedly increased CXCR4 protein expression suggesting its target gene. To show that miR-150 regulates MNC mobilization, knockdown of miR-150 in BM-MNCs by specific antisense inhibitor resulted in their higher migration ability in vitro as compared to scramble-transfected MNCs. Furthermore, in vivo BM transplantation of MNCs lacking miR-150 expression by lentiviral vector into the irradiated wild type mice resulted in the increased number of MNCs in PB after AMI as compared to control. In conclusion, this study demonstrates that ischemia mobilizes BM stem cells via miR-150/CXCR4 dependent mechanism and miR-150 may be a novel therapeutic target for stem cell migration to the ischemic tissue for neovascularization and repair. PMID:22039399

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

PubMed Central

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A.; de Boer, Jan; Watt, Fiona M.

2016-01-01

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation. PMID:26757610

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells.

PubMed

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A; de Boer, Jan; Watt, Fiona M

2016-01-13

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation.

Lineage mapping and characterization of the native progenitor population in cellular allograft.

PubMed

Neman, Josh; Duenas, Vincent; Kowolik, Claudia; Hambrecht, Amanda; Chen, Mike; Jandial, Rahul

2013-02-01

The gold standard for bone grafting remains the autograft. However, the attractiveness of autograft is counterbalanced by donor site morbidity. To mimic autograft-and its fundamental properties of osteoconductivity, osteoinductivity, and osteogenicity-novel bone grafting materials such as cellular allograft (Osteocel Plus) are composed of allograft in which the progenitor cells are preserved. However, the true identity of these cells remains obscure largely due to the lack of specific bona fide antigenic markers for stem versus progenitor cells. To characterize the stem and progenitor population in cellular allograft, Osteocel Plus. To determine whether cells endogenous to a cellular allograft undergo extensive self-renewal (a functional hallmark of stem cells), we employed a novel use of lineage mapping using a modern and refined replication incompetent lentiviral library with high complexity to uniquely label single cells with indelible genetic tags faithfully passed on to all progeny, allowing identification of highly proliferative clones. We used genetic and proteomic profiling as well as functional assays to show that these cells are capable of multipotential differentiation (the second functional hallmark of stem cells). Use of these two functional hallmarks enabled us to establish the existence of a stem and progenitor cell population in cellular allografts. Specifically, we employed (1) cellular dissociation and (2) in vitro expansion and differentiation capacity of cells released from cellular allograft. We determined differential gene expression profiling of a bona fide human mesenchymal stem cell line and cells from cellular allograft using focused PCR arrays mesenchymal stem cell (MSC) and osteogenesis associated. Proteomic profiling of cells from cellular allograft was performed using (1) immunofluorescence for BMP-2, Runx2 SMADs, CD44, Stro-1, Collagen, RANKL, Osterix Osteocalcin, and Ki67; (2) flow cytometry for Ki67, CD44, Stro-1, Thy1, CD146, and Osteocalcin; and (3) enzyme-linked immunosorbent assays (ELISA) for BMP-2, Osteocalcin, RANKL, Osteoprotegrin, and Osteocalcin. Clonal analysis of cells from cellular allograft was performed utilizing advance lentivirus lineage mapping techniques and massive parallel sequencing. Alizarin Red, Alcian Blue, and Oil red O staining assessed tripotential differentiation capacity. Serial trypsinization of allograft cellular bone matrix yielded approximately 1×105 cells per mL with viability greater than 90%. Cells expressed a panel of 84 MSC-associated genes in a pattern similar to but not identical to pure MSCs; specifically, 59 of 84 genes showed less than a 2.5-fold change in both cell types. Protein analysis showed that cellular allograft -derived cells maintained in nondifferentiation media expressed the early osteo-progenitor markers BMP-2, SMADs, and Runx2. Corresponding flow cytometry data for MSC markers revealed the presence of Stro-1 (49%), CD44 (99%), CD90 (42%), and CD146 (97%). Lineage mapping indicated that 62% of clones persisted and generated progeny through 10 passages, strongly suggesting the presence of bona fide stem cells. Passage 10 clones also exhibited tri-lineage differentiation capacity into osteogenic (Alizarin Red with H&E counterstain), chondrogenic (Alcian Blue), and adipogenic (Oil red O). Cells that did not proliferate through 10 passages presumably differentiated along an osteo-progenitor lineage. These data indicate that cellular allograft (Osteocel Plus) contains a heterogeneous population of cells with most cells demonstrating the capacity for extensive self-renewal and multipotential differentiation, which are hallmarks of stem cells. Whether stem cell-enriched allografts function comparably to autograft will require further studies, and their efficacy in facilitating arthrodesis will depend on randomized clinical studies. Copyright © 2013 Elsevier Inc. All rights reserved.

5-Azacitidine Monotherapy Followed by Related Haploidentical Hematopoietic Stem Cell Transplantation Achieves Durable Remission in a Pediatric Patient With Acute Undifferentiated Leukemia Refractory to High-Dose Chemotherapy.

PubMed

Polishchuk, Veronika; Khazal, Sajad; Berulava, Giorgi; Roth, Michael; Mahadeo, Kris M

2016-06-01

Patients with acute leukemias of undifferentiated lineage (AUL) generally have guarded prognosis. Here, we describe the first reported pediatric patient with AUL refractory to high-dose chemotherapy who achieved clinical remission with ALL maintenance therapy and 5-azacitidine. His induction remission was followed by consolidation with reduced toxicity haploidentical hematopoietic stem cell transplant (HSCT). At 9 months post-HSCT, the patient is alive and in remission. This combination therapy of remission induction with ALL maintenance therapy and 5-azacitidine and consolidation with reduced toxicity haploidentical HSCT is novel and promising for patients who lack conventional donors and are not candidates for myeloablative therapy. © 2016 Wiley Periodicals, Inc.

Co-Seeding Human Endothelial Cells with Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells on Calcium Phosphate Scaffold Enhances Osteogenesis and Vascularization in Rats.

PubMed

Liu, Xian; Chen, Wenchuan; Zhang, Chi; Thein-Han, Wahwah; Hu, Kevin; Reynolds, Mark A; Bao, Chongyun; Wang, Ping; Zhao, Liang; Xu, Hockin H K

2017-06-01

A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CPC-HUVEC); (3) hiPSC-MSC-seeded CPC (CPC-hiPSC-MSC); and (4) HUVECs co-cultured with hiPSC-MSCs on CPC scaffolds (co-culture group). After 12 weeks, the co-culture group achieved the greatest new bone area percentage of 46.38% ± 3.8% among all groups (p < 0.05), which was more than four folds of the 10.61% ± 1.43% of CPC control. In conclusion, HUVECs co-cultured with hiPSC-MSCs substantially promoted bone regeneration. The novel construct of HUVECs co-cultured with hiPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.

Isolation and hepatocyte differentiation of mesenchymal stem cells from porcine bone marrow--"surgical waste" as a novel MSC source.

PubMed

Brückner, S; Tautenhahn, H-M; Winkler, S; Stock, P; Jonas, S; Dollinger, M; Christ, B

2013-06-01

Mesenchymal stem cells (MSC) isolated from bone marrow and differentiated into hepatocyte-like cells have increasingly gained attention for clinical cell therapy of liver diseases because of their high regenerative capacity. They are available from bone marrow aspirates of the os coxae after puncture of the crista iliaca or from bone marrow "surgical waste" gained from amputations or knee and hip operations. Thus, the aim of the study was to demonstrate whether these pBM-MSC (porcine bone marrow-derived mesenchymal stem cells) displayed mesenchymal features and hepatocyte differentiation potential. MSC were isolated either from crista iliaca punctures or after sampling and collagenase digestion of bone marrow from the os femoris. Mesenchymal features were assessed by flow cytometry for specific surface antigens and their ability to differentiate into at least 3 lineages. Functional properties, such as urea or glycogen synthesis and cytochrome P450 activity, as well as the cell morphology were examined during hepatocyte differentiation. pBM-MSC from both sources lacked the hematopoietic markers CD14 and CD45 but expressed the typical mesenchymal markers CD44, CD29, CD90, and CD105. Both cell types could differentiate into adipocyte, osteocyte, and hepatocyte lineages. After hepatocyte differentiation, CD105 expression decreased significantly and cells changed morphology from fibroblastoid into polygonal, displaying significantly increased glycogen storage, urea synthesis, and cytochrome activity. pBM-MSC from various sources were identical in respect to their mesenchymal features and their hepatocyte differentiation potential. Hence, long bones might be a particularly useful resource to isolate bone marrow mesenchymal stem cells for transplantation. Copyright © 2013 Elsevier Inc. All rights reserved.

Design of magnetic gene complexes as effective and serum resistant gene delivery systems for mesenchymal stem cells.

PubMed

Zhang, Tian-Yuan; Wu, Jia-He; Xu, Qian-Hao; Wang, Xia-Rong; Lu, Jingxiong; Hu, Ying; Jo, Jun-Ichiro; Yamamoto, Masaya; Ling, Daishun; Tabata, Yasuhiko; Gao, Jian-Qing

2017-03-30

Gene engineered mesenchymal stem cells (MSCs) have been proposed as promising tools for their various applications in biomedicine. Nevertheless, the lack of an effective and safe way to genetically modify these stem cells is still a major obstacle in the current studies. Herein, we designed novel magnetic complexes by assembling cationized pullulan derivatives with magnetic iron oxide nanoparticles for delivering target genes to MSCs. Results showed that this complexes achieved effective gene expression with the assistance of external magnetic field, and resisted the adverse effect induced by serum proteins on the gene delivery. Moreover, neither significant cytotoxicity nor the interference on the osteogenic differentiation to MSCs were observed after magnetofection. Further studies revealed that this effective and serum resistant gene transfection was partly due to the accelerated and enhanced intracellular uptake process driven by external magnetic field. To conclude, the current study presented a novel option for genetic modification of MSCs in an effective, relatively safe and serum compatible way. Copyright © 2017 Elsevier B.V. All rights reserved.

Concise Review: Cardiac Disease Modeling Using Induced Pluripotent Stem Cells.

PubMed

Yang, Chunbo; Al-Aama, Jumana; Stojkovic, Miodrag; Keavney, Bernard; Trafford, Andrew; Lako, Majlinda; Armstrong, Lyle

2015-09-01

Genetic cardiac diseases are major causes of morbidity and mortality. Although animal models have been created to provide some useful insights into the pathogenesis of genetic cardiac diseases, the significant species differences and the lack of genetic information for complex genetic diseases markedly attenuate the application values of such data. Generation of induced pluripotent stem cells (iPSCs) from patient-specific specimens and subsequent derivation of cardiomyocytes offer novel avenues to study the mechanisms underlying cardiac diseases, to identify new causative genes, and to provide insights into the disease aetiology. In recent years, the list of human iPSC-based models for genetic cardiac diseases has been expanding rapidly, although there are still remaining concerns on the level of functionality of iPSC-derived cardiomyocytes and their ability to be used for modeling complex cardiac diseases in adults. This review focuses on the development of cardiomyocyte induction from pluripotent stem cells, the recent progress in heart disease modeling using iPSC-derived cardiomyocytes, and the challenges associated with understanding complex genetic diseases. To address these issues, we examine the similarity between iPSC-derived cardiomyocytes and their ex vivo counterparts and how this relates to the method used to differentiate the pluripotent stem cells into a cardiomyocyte phenotype. We progress to examine categories of congenital cardiac abnormalities that are suitable for iPSC-based disease modeling. © AlphaMed Press.

Icariin protects rats against 5/6 nephrectomy-induced chronic kidney failure by increasing the number of renal stem cells.

PubMed

Huang, Zhongdi; He, Liqun; Huang, Di; Lei, Shi; Gao, Jiandong

2015-10-21

Chronic kidney disease poses a serious health problem worldwide with increasing prevalence and lack of effective treatment. This study aimed to investigate the mechanism of icariin in alleviating chronic renal failure induced by 5/6 nephrectomy in rats. The chronic renal failure model was established by a two-phased 5/6 nephrectomy procedure. The model rats were given daily doses of water or icariin for 8 weeks. The kidney morphology was checked by HE staining. The levels of blood urea nitrogen, serum creatinine, and serum uric acid were measured by colometric methods. The expression of specified genes was analyzed by quantitative real-time PCR and immunohistochemical staining. The number of renal stem/progenitor cells was analyzed by CD133 and CD24 immunohistochemical staining. Icariin protected against CDK-caused damages to kidney histology and improved renal function, significantly reduced levels of BUN, creatinine, and uric acid. Icariin inhibited the expression level of TGF-β1 whereas upregulated HGF, BMP-7, WT-1, and Pax2 expression. Moreover, ccariin significantly increased the expression of CD24, CD133, Osr1, and Nanog in remnant kidney and the numbers of CD133(+)/CD24(+) renal stem/progenitor cells. These data demonstrated that icariin effectively alleviated 5/6 nephrectomy induced chronic renal failure through increasing renal stem/progenitor cells.

Egg donation for stem cell research: ideas of surplus and deficit in Australian IVF patients' and reproductive donors' accounts.

PubMed

Waldby, Catherine; Carroll, Katherine

2012-05-01

We report on a study undertaken with an Australian in vitro fertilisation (IVF) clinic to understand IVF patients' and reproductive donors' perceptions of oocyte (egg) donation for stem cell research. Such perspectives are particularly valuable because IVF patients form a major recruitment group for oocyte donation for research, and because patients and donors have direct experience of the medical procedures involved. Similar studies of oocyte donation have been carried out elsewhere in the world, but to date very little social science research has been published that reports on donation for research, as distinct from donation for reproduction. Our respondents expressed a distinct unwillingness to donate viable oocytes for stem cell research. In our analysis we consider a number of factors that explain this unwillingness. These include the labour of oocyte production, the inscrutability of oocytes (the lack of a test to identify degrees of fertility) and the extent to which the oocytes' fertility sets the parameters for all downstream reproductive possibilities. We draw on the science studies literature on affordances to make sense of the social intractability of oocytes, and compare them with the respondents' much greater willingness to donate frozen embryos for human embryonic stem cells research. © 2011 The Authors. Sociology of Health & Illness © 2011 Foundation for the Sociology of Health & Illness/Blackwell Publishing Ltd.

The effect of mesenchymal stem cells delivered via hydrogel-based tissue engineered periosteum on bone allograft healing.

PubMed

Hoffman, Michael D; Xie, Chao; Zhang, Xinping; Benoit, Danielle S W

2013-11-01

Allografts remain the clinical "gold standard" for treatment of critical sized bone defects despite minimal engraftment and ∼60% long-term failure rates. Therefore, the development of strategies to improve allograft healing and integration are necessary. The periosteum and its associated stem cell population, which are lacking in allografts, coordinate autograft healing. Herein we utilized hydrolytically degradable hydrogels to transplant and localize mesenchymal stem cells (MSCs) to allograft surfaces, creating a periosteum mimetic, termed a 'tissue engineered periosteum'. Our results demonstrated that this tissue engineering approach resulted in increased graft vascularization (∼2.4-fold), endochondral bone formation (∼2.8-fold), and biomechanical strength (1.8-fold), as compared to untreated allografts, over 16 weeks of healing. Despite this enhancement in healing, the process of endochondral ossification was delayed compared to autografts, requiring further modifications for this approach to be clinically acceptable. However, this bottom-up biomaterials approach, the engineered periosteum, can be augmented with alternative cell types, matrix cues, growth factors, and/or other small molecule drugs to expedite the process of ossification. Copyright © 2013 Elsevier Ltd. All rights reserved.

Digital gene expression approach over multiple RNA-Seq data sets to detect neoblast transcriptional changes in Schmidtea mediterranea.

PubMed

Rodríguez-Esteban, Gustavo; González-Sastre, Alejandro; Rojo-Laguna, José Ignacio; Saló, Emili; Abril, Josep F

2015-05-08

The freshwater planarian Schmidtea mediterranea is recognised as a valuable model for research into adult stem cells and regeneration. With the advent of the high-throughput sequencing technologies, it has become feasible to undertake detailed transcriptional analysis of its unique stem cell population, the neoblasts. Nonetheless, a reliable reference for this type of studies is still lacking. Taking advantage of digital gene expression (DGE) sequencing technology we compare all the available transcriptomes for S. mediterranea and improve their annotation. These results are accessible via web for the community of researchers. Using the quantitative nature of DGE, we describe the transcriptional profile of neoblasts and present 42 new neoblast genes, including several cancer-related genes and transcription factors. Furthermore, we describe in detail the Smed-meis-like gene and the three Nuclear Factor Y subunits Smed-nf-YA, Smed-nf-YB-2 and Smed-nf-YC. DGE is a valuable tool for gene discovery, quantification and annotation. The application of DGE in S. mediterranea confirms the planarian stem cells or neoblasts as a complex population of pluripotent and multipotent cells regulated by a mixture of transcription factors and cancer-related genes.

Improved bone marrow stromal cell adhesion on micropatterned titanium surfaces.

PubMed

Iskandar, Maria E; Cipriano, Aaron F; Lock, Jaclyn; Gott, Shannon C; Rao, Masaru P; Liu, Huinan

2012-01-01

Implant longevity is desired for all bone replacements and fixatives. Titanium (Ti) implants fail due to lack of juxtaposed bone formation, resulting in implant loosening. Implant surface modifications have shown to affect the interactions between the implant and bone. In clinical applications, it is crucial to improve osseointegration and implant fixation at the implant and bone interface. Moreover, bone marrow derived cells play a significant role for implant and tissue integration. Therefore, the objective of this study is to investigate how surface micropatterning on Ti influences its interactions with bone marrow derived cells containing mesenchymal and hematopoietic stem cells. Bone marrow derived mesenchymal stem cells (BMSC) have the capability of differentiating into osteoblasts that contribute to bone growth, and therefore implant/bone integration. Hematopoietic stem cell derivatives are precursor cells that contribute to inflammatory response. By using all three cells naturally contained within bone marrow, we mimic the physiological environment to which an implant is exposed. Primary rat bone marrow derived cells were seeded onto Ti with surfaces composed of arrays of grooves of equal width and spacing ranging from 0.5 to 50 µm, fabricated using a novel plasma-based dry etching technique. Results demonstrated enhanced total cell adhesion on smaller micrometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, after 24-hr culture. Further studies are needed to determine bone marrow derived cell proliferation and osteogenic differentiation potential on micropatterned Ti, and eventually nanopatterned Ti.

Human cardiomyocyte generation from pluripotent stem cells: A state-of-art.

PubMed

Talkhabi, Mahmood; Aghdami, Nasser; Baharvand, Hossein

2016-01-15

The human heart is considered a non-regenerative organ. Worldwide, cardiovascular diseases continue to be the leading cause of death. Despite advances in cardiac treatment, myocardial repair remains severely limited by the lack of an appropriate source of viable cardiomyocytes (CMs) to replace damaged tissue. Human pluripotent stem cells (hPSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can efficiently be differentiated into functional CMs necessary for cell replacement therapy and other potential applications. The number of protocols that derive CMs from hPSCs has increased exponentially over the past decade following observation of the first human beating CMs. A number of highly efficient, chemical based protocols have been developed to generate human CMs (hCMs) in small-scale and large-scale suspension systems. To reduce the heterogeneity of hPSC-derived CMs, the differentiation protocols were modulated to exclusively generate atrial-, ventricular-, and nodal-like CM subtypes. Recently, remarkable advances have been achieved in hCM generation including chemical-based cardiac differentiation, cardiac subtype specification, large-scale suspension culture differentiation, and development of chemically defined culture conditions. These hCMs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues are overcome. Herein we review recent progress in the in vitro generation of CMs and cardiac subtypes from hPSCs and discuss their potential applications and current limitations. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization and Application of a Disposable Rotating Bed Bioreactor for Mesenchymal Stem Cell Expansion.

PubMed

Neumann, Anne; Lavrentieva, Antonina; Heilkenbrinker, Alexandra; Loenne, Maren; Kasper, Cornelia

2014-11-27

Recruitment of mesenchymal stromal cells (MSC) into the field of tissue engineering is a promising development since these cells can be expanded vivo to clinically relevant numbers and, after expansion, retain their ability to differentiate into various cell lineages. Safety requirements and the necessity to obtain high cell numbers without frequent subcultivation of cells raised the question of the possibility of expanding MSC in one-way (single-use) disposable bioreactors. In this study, umbilical cord-derived MSC (UC-MSC) were expanded in a disposable Z 2000 H bioreactor under dynamic conditions. Z was characterized regarding residence time and mixing in order to evaluate the optimal bioreactor settings, enabling optimal mass transfer in the absence of shear stress, allowing an reproducible expansion of MSC, while maintaining their stemness properties. Culture of the UC-MSC in disposable Z 2000 H bioreactor resulted in a reproducible 8-fold increase of cell numbers after 5 days. Cells were shown to maintain specific MSC surface marker expression as well as trilineage differentiation potential and lack stress-induced premature senescence.

Effective regeneration of dystrophic muscle using autologous iPSC-derived progenitors with CRISPR-Cas9 mediated precise correction.

PubMed

Hagan, Mackenzie; Ashraf, Muhammad; Kim, Il-Man; Weintraub, Neal L; Tang, Yaoliang

2018-01-01

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by a lack of dystrophin, which eventually leads to apoptosis of muscle cells and impaired muscle contractility. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) gene editing of induced pluripotent stem cells (IPSC) offers the potential to correct the DMD gene defect and create healthy IPSC for autologous cell transplantation without causing immune activation. However, IPSC carry a risk of tumor formation, which can potentially be mitigated by differentiation of IPSC into myogenic progenitor cells (MPC). We hypothesize that precise genetic editing in IPSC using CRISPR-Cas9 technology, coupled with MPC differentiation and autologous transplantation, can lead to safe and effective muscle repair. With future research, our hypothesis may provide an optimal autologous stem cell-based approach to treat the dystrophic pathology and improve the quality of life for patients with DMD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Replication of human noroviruses in stem cell-derived human enteroids

USDA-ARS?s Scientific Manuscript database

The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report successful cultivation of multiple HuNoV strains in...

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

PubMed Central

Suo, Zhenhe; Munthe, Else; Solberg, Steinar; Ma, Liwei; Wang, Mengyu; Westerdaal, Nomdo Anton Christiaan; Kvalheim, Gunnar; Gaudernack, Gustav

2013-01-01

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population. PMID:23469181

Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating

PubMed Central

Engler, Adam J.; Carag-Krieger, Christine; Johnson, Colin P.; Raab, Matthew; Tang, Hsin-Yao; Speicher, David W.; Sanger, Joseph W.; Sanger, Jean M.; Discher, Dennis E.

2009-01-01

Summary Fibrotic rigidification following a myocardial infarct is known to impair cardiac output, and it is also known that cardiomyocytes on rigid culture substrates show a progressive loss of rhythmic beating. Here, isolated embryonic cardiomyocytes cultured on a series of flexible substrates show that matrices that mimic the elasticity of the developing myocardial microenvironment are optimal for transmitting contractile work to the matrix and for promoting actomyosin striation and 1-Hz beating. On hard matrices that mechanically mimic a post-infarct fibrotic scar, cells overstrain themselves, lack striated myofibrils and stop beating; on very soft matrices, cells preserve contractile beating for days in culture but do very little work. Optimal matrix leads to a strain match between cell and matrix, and suggests dynamic differences in intracellular protein structures. A ‘cysteine shotgun’ method of labeling the in situ proteome reveals differences in assembly or conformation of several abundant cytoskeletal proteins, including vimentin, filamin and myosin. Combined with recent results, which show that stem cell differentiation is also highly sensitive to matrix elasticity, the methods and analyses might be useful in the culture and assessment of cardiogenesis of both embryonic stem cells and induced pluripotent stem cells. The results described here also highlight the need for greater attention to fibrosis and mechanical microenvironments in cell therapy and development. PMID:18957515

Induced pluripotent stem cells (iPSC)-derived retinal cells in disease modeling and regenerative medicine.

PubMed

Rathod, Reena; Surendran, Harshini; Battu, Rajani; Desai, Jogin; Pal, Rajarshi

2018-02-12

Retinal degenerative disorders are a leading cause of the inherited, irreversible and incurable vision loss. While various rodent model systems have provided crucial information in this direction, lack of disease-relevant tissue availability and species-specific differences have proven to be a major roadblock. Human induced pluripotent stem cells (iPSC) have opened up a whole new avenue of possibilities not just in understanding the disease mechanism but also potential therapeutic approaches towards a cure. In this review, we have summarized recent advances in the methods of deriving retinal cell types from iPSCs which can serve as a renewable source of disease-relevant cell population for basic as well as translational studies. We also provide an overview of the ongoing efforts towards developing a suitable in vitro model for modeling retinal degenerative diseases. This basic understanding in turn has contributed to advances in translational goals such as drug screening and cell-replacement therapies. Furthermore we discuss gene editing approaches for autologous repair of genetic disorders and allogeneic transplantation of stem cell-based retinal derivatives for degenerative disorders with an ultimate goal to restore vision. It is pertinent to note however, that these exciting new developments throw up several challenges that need to be overcome before their full clinical potential can be realized. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and Function of Myeloid-Derived Suppressor Cells Generated From Mouse Embryonic and Hematopoietic Stem Cells

PubMed Central

Zhou, Zuping; French, Deborah L.; Ma, Ge; Eisenstein, Samuel; Chen, Ying; Divino, Celia M.; Keller, Gordon; Chen, Shu-Hsia; Pan, Ping-Ying

2015-01-01

Emerging evidence suggests that myeloid-derived suppressor cells (MDSCs) have great potential as a novel immune intervention modality in the fields of transplantation and autoimmune diseases. Thus far, efforts to develop MDSC-based therapeutic strategies have been hampered by the lack of a reliable source of MDSCs. Here we show that functional MDSCs can be efficiently generated from mouse embryonic stem (ES) cells and bone marrow hematopoietic stem (HS) cells. In vitro-derived MDSCs encompass two homogenous subpopulations: CD115+Ly-6C+ and CD115+Ly-6C− cells. The CD115+Ly-6C+ subset is equivalent to the monocytic Gr-1+CD115+F4/80+ MDSCs found in tumor-bearing mice. In contrast, the CD115+Ly-6C− cells, a previously unreported population of MDSCs, resemble the granulocyte/macrophage progenitors developmentally. In vitro, ES- and HS-MDSCs exhibit robust suppression against T-cell proliferation induced by polyclonal stimuli or alloantigens via multiple mechanisms involving nitric oxide synthase-mediated NO production and interleukin (IL)-10. Impressively, they display even stronger suppressive activity and significantly enhance ability to induce CD4+CD25+Foxp3+ regulatory T-cell development compared with tumor-derived MDSCs. Furthermore, adoptive transfer of ES-MDSCs can effectively prevent alloreactive T-cell-mediated lethal graft-versus-host disease, leading to nearly 82% long-term survival among treated mice. The successful in vitro generation of MDSCs may represent a critical step toward potential clinical application of MDSCs. PMID:20073041

Mesenchymal stem cells from the Wharton's jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture.

PubMed

Bakhshi, Tiki; Zabriskie, Ryan C; Bodie, Shamanique; Kidd, Shannon; Ramin, Susan; Paganessi, Laura A; Gregory, Stephanie A; Fung, Henry C; Christopherson, Kent W

2008-12-01

Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow-derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton's jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated. Umbilical cord-derived MSCs (UC-MSCs) were cultured from the Wharton's jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay. Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays. These data suggest the potential therapeutic application of Wharton's jelly-derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation.

Reduction of Movement in Neurological Diseases: Effects on Neural Stem Cells Characteristics.

PubMed

Adami, Raffaella; Pagano, Jessica; Colombo, Michela; Platonova, Natalia; Recchia, Deborah; Chiaramonte, Raffaella; Bottinelli, Roberto; Canepari, Monica; Bottai, Daniele

2018-01-01

Both astronauts and patients affected by chronic movement-limiting pathologies face impairment in muscle and/or brain performance. Increased patient survival expectations and the expected longer stays in space by astronauts may result in prolonged motor deprivation and consequent pathological effects. Severe movement limitation can influence not only the motor and metabolic systems but also the nervous system, altering neurogenesis and the interaction between motoneurons and muscle cells. Little information is yet available about the effect of prolonged muscle disuse on neural stem cells characteristics. Our in vitro study aims to fill this gap by focusing on the biological and molecular properties of neural stem cells (NSCs). Our analysis shows that NSCs derived from the SVZ of HU mice had shown a reduced proliferation capability and an altered cell cycle. Furthermore, NSCs obtained from HU animals present an incomplete differentiation/maturation. The overall results support the existence of a link between reduction of exercise and muscle disuse and metabolism in the brain and thus represent valuable new information that could clarify how circumstances such as the absence of load and the lack of movement that occurs in people with some neurological diseases, may affect the properties of NSCs and contribute to the negative manifestations of these conditions.

Modeling Human Bone Marrow Failure Syndromes Using Pluripotent Stem Cells and Genome Engineering.

PubMed

Jung, Moonjung; Dunbar, Cynthia E; Winkler, Thomas

2015-12-01

The combination of epigenetic reprogramming with advanced genome editing technologies opened a new avenue to study disease mechanisms, particularly of disorders with depleted target tissue. Bone marrow failure syndromes (BMFS) typically present with a marked reduction of peripheral blood cells due to a destroyed or dysfunctional bone marrow compartment. Somatic and germline mutations have been etiologically linked to many cases of BMFS. However, without the ability to study primary patient material, the exact pathogenesis for many entities remained fragmentary. Capturing the pathological genotype in induced pluripotent stem cells (iPSCs) allows studying potential developmental defects leading to a particular phenotype. The lack of hematopoietic stem and progenitor cells in these patients can also be overcome by differentiating patient-derived iPSCs into hematopoietic lineages. With fast growing genome editing techniques, such as CRISPR/Cas9, correction of disease-causing mutations in iPSCs or introduction of mutations in cells from healthy individuals enable comparative studies that may identify other genetic or epigenetic events contributing to a specific disease phenotype. In this review, we present recent progresses in disease modeling of inherited and acquired BMFS using reprogramming and genome editing techniques. We also discuss the challenges and potential shortcomings of iPSC-based models for hematological diseases.

Membrane protein stoichiometry studied in intact mammalian cells using liquid-phase electron microscopy.

PubMed

DE Jonge, N

2018-02-01

Receptor membrane proteins in the plasma membranes of cells respond to extracellular chemical signals by conformational changes, spatial redistribution, and (re-)assembly into protein complexes, for example, into homodimers (pairs of the same protein type). The functional state of the proteins can be determined from information about how subunits are assembled into protein complexes. Stoichiometric information about the protein complex subunits, however, is generally not obtained from intact cells but from pooled material extracted from many cells, resulting in a lack of fundamental knowledge about the functioning of membrane proteins. First, functional states may dramatically differ from cell to cell on account of cell heterogeneity. Second, extracting the membrane proteins from the plasma membrane may lead to many artefacts. Liquid-phase scanning transmission electron microscopy (STEM), in short liquid STEM, is a new technique capable of determining the locations of individual membrane proteins within the intact plasma membranes of cells in liquid. Many tens of whole cells can readily be imaged. It is possible to analyse the stoichiometry of membrane proteins in single cells while accounting for heterogenic cell populations. Liquid STEM was used to image epidermal growth factor receptors in whole COS7 cells. A study of the dimerisation of the HER2 protein in breast cancer cells revealed the presence of rare cancer cells in which HER2 was in a different functional state than in the bulk cells. Stoichiometric information about receptors is essential not only for basic science but also for biomedical application because they present many important pharmaceutical targets. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Chitosan derived co-spheroids of neural stem cells and mesenchymal stem cells for neural regeneration.

PubMed

Han, Hao-Wei; Hsu, Shan-Hui

2017-10-01

Chitosan has been considered as candidate biomaterials for neural applications. The effective treatment of neurodegeneration or injury to the central nervous system (CNS) is still in lack nowadays. Adult neural stem cells (NSCs) represents a promising cell source to treat the CNS diseases but they are limited in number. Here, we developed the core-shell spheroids of NSCs (shell) and mesenchymal stem cells (MSCs, core) by co-culturing cells on the chitosan surface. The NSCs in chitosan derived co-spheroids displayed a higher survival rate than those in NSC homo-spheroids. The direct interaction of NSCs with MSCs in the co-spheroids increased the Notch activity and differentiation tendency of NSCs. Meanwhile, the differentiation potential of MSCs in chitosan derived co-spheroids was significantly enhanced toward neural lineages. Furthermore, NSC homo-spheroids and NSC/MSC co-spheroids derived on chitosan were evaluated for their in vivo efficacy by the embryonic and adult zebrafish brain injury models. The locomotion activity of zebrafish receiving chitosan derived NSC homo-spheroids or NSC/MSC co-spheroids was partially rescued in both models. Meanwhile, the higher survival rate was observed in the group of adult zebrafish implanted with chitosan derived NSC/MSC co-spheroids as compared to NSC homo-spheroids. These evidences indicate that chitosan may provide an extracellular matrix-like environment to drive the interaction and the morphological assembly between NSCs and MSCs and promote their neural differentiation capacities, which can be used for neural regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

The fused in sarcoma protein forms cytoplasmic aggregates in motor neurons derived from integration-free induced pluripotent stem cells generated from a patient with familial amyotrophic lateral sclerosis carrying the FUS-P525L mutation.

PubMed

Liu, Xinxiu; Chen, Jiayu; Liu, Wenchao; Li, Xiaogang; Chen, Qi; Liu, Tao; Gao, Shaorong; Deng, Min

2015-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons (MNs) and has no effective treatment. Mutations in the fused in sarcoma (FUS) gene and abnormal aggregation of FUS protein have been reported in ALS. However, the mechanisms involved in ALS are poorly understood. Clinical drug trails have failed due to a lack of appropriate disease models, including a lack of access to MNs from ALS patients. Induced pluripotent stem (iPS) cells derived from patients with ALS provide an indispensable resource for in vitro mechanistic studies and for future patient-specific cell-based therapies. Previous reports demonstrated that viral-based ALS-iPS cells generated from fibroblasts harvested from Caucasian populations are ideal for basic research; however, ALS-iPS cells are precluded from cell-based therapeutic applications because of the risks associated with the integration of viral sequences into the genome and inconvenience associated with dermal biopsies. To establish a model for use in clinical applications, using episomal vectors, we generated an integration-free iPS cell line from peripheral blood mononuclear cells (PBMCs) harvested from a familial ALS (FALS) patient carrying the FUS-P525L mutation and a healthy control. Furthermore, we successfully differentiated ALS patient-specific iPS cells into MNs and subsequently detected cytoplasmic mislocalization and formation of FUS protein aggregates in MNs due to the FUS-P525L mutation. Our findings offer a cell-based disease model for use in further elucidating ALS pathogenesis and provide a tool for exploring gene repair coupled with cell replacement therapy.

The human CD94 gene encodes multiple, expressible transcripts including a new partner of NKG2A/B.

PubMed

Lieto, L D; Maasho, K; West, D; Borrego, F; Coligan, J E

2006-01-01

CD94/NKG2A is an inhibitory receptor expressed by natural killer (NK) cells and a subset of CD8+ T cells. Ligation of CD94/NKG2A by its ligand HLA-E results in tyrosine phosphorylation of the NKG2A immunoreceptor tyrosine-based inhibitory motifs, and recruitment and activation of the SH2 domain-bearing tyrosine phosphatase-1, which in turn suppresses activation signals. The nkg2a gene encodes two isoforms, NKG2A and NKG2B, with the latter lacking the stem region. We identified three new alternative transcripts of the cd94 gene in addition to the originally described canonical CD94Full. One of the transcripts, termed CD94-T4, lacks the portion that encodes the stem region. CD94-T4 associates with both NKG2A and NKG2B, but preferentially associates with the latter. This is probably due to the absence of a stem region in both CD94-T4 and NKG2B. CD94-T4/NKG2B is capable of binding HLA-E and, when expressed in E6-1 Jurkat T cells, inhibits TCR mediated signals, demonstrating that this heterodimer is functional. Coevolution of stemless isoforms of CD94 and NKG2A that preferentially pair with each other to produce a functional heterodimer indicates that this may be more than a serendipitous event. CD94-T4/NKG2B may contribute to the plasticity of the NK immunological synapse by insuring an adequate inhibitory signal.

A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.

PubMed

Hadland, Brandon K; Varnum-Finney, Barbara; Mandal, Pankaj K; Rossi, Derrick J; Poulos, Michael G; Butler, Jason M; Rafii, Shahin; Yoder, Mervin C; Yoshimoto, Momoko; Bernstein, Irwin D

2017-06-06

Recent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs). However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC) potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress

PubMed Central

Ohashi, Wakana; Kimura, Shunsuke; Iwanaga, Toshihiko; Furusawa, Yukihiro; Irié, Tarou; Izumi, Hironori; Watanabe, Takashi; Hara, Takafumi; Ohara, Osamu; Koseki, Haruhiko; Sato, Toshiro; Robine, Sylvie; Mori, Hisashi; Hattori, Yuichi; Mishima, Kenji; Ohno, Hiroshi; Hase, Koji; Fukada, Toshiyuki

2016-01-01

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders. PMID:27736879

Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution.

PubMed

Pratap Sahi, Vaidurya; Cifrová, Petra; García-González, Judith; Kotannal Baby, Innu; Mouillé, Gregory; Gineau, Emilie; Müller, Karel; Baluška, František; Soukup, Aleš; Petrášek, Jan; Schwarzerová, Katerina

2017-12-25

The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport. © The Author(s) 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.

PubMed

Sampaziotis, Fotios; de Brito, Miguel Cardoso; Madrigal, Pedro; Bertero, Alessandro; Saeb-Parsy, Kourosh; Soares, Filipa A C; Schrumpf, Elisabeth; Melum, Espen; Karlsen, Tom H; Bradley, J Andrew; Gelson, William Th; Davies, Susan; Baker, Alastair; Kaser, Arthur; Alexander, Graeme J; Hannan, Nicholas R F; Vallier, Ludovic

2015-08-01

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.

Chondroitin Sulfate Is Indispensable for Pluripotency and Differentiation of Mouse Embryonic Stem Cells

NASA Astrophysics Data System (ADS)

Izumikawa, Tomomi; Sato, Ban; Kitagawa, Hiroshi

2014-01-01

Chondroitin sulfate (CS) proteoglycans are present on the surfaces of virtually all cells and in the extracellular matrix and are required for cytokinesis at early developmental stages. Studies have shown that heparan sulfate (HS) is essential for maintaining mouse embryonic stem cells (ESCs) that are primed for differentiation, whereas the function of CS has not yet been elucidated. To clarify the role of CS, we generated glucuronyltransferase-I-knockout ESCs lacking CS. We found that CS was required to maintain the pluripotency of ESCs and promoted initial ESC commitment to differentiation compared with HS. In addition, CS-A and CS-E polysaccharides, but not CS-C polysaccharides, bound to E-cadherin and enhanced ESC differentiation. Multiple-lineage differentiation was inhibited in chondroitinase ABC-digested wild-type ESCs. Collectively, these results suggest that CS is a novel determinant in controlling the functional integrity of ESCs via binding to E-cadherin.

[Mesenchymal stem/stroma cells : Therapeutic potential in the treatment of autoimmune diseases].

PubMed

Schäfer, R; Daikeler, T

2016-10-01

Mesenchymal stem and stromal cells (MSC) are propagated for the treatment of autoimmune and autoinflammatory processes. These cells can be relatively easily obtained from various tissues. The MSC feature anti-inflammatory and immunosuppressive properties in vitro as well as in animal models. Initial reports on the clinical application of MSC for various diseases are available, some with promising results and so far no reported toxicity; however, data from phase III studies are still lacking and crucial questions are still unanswered. The MSC preparations used are heterogeneous and also differ depending on the source and it is unclear whether autologous (own) or allogeneic (foreign) MSC are more suitable for therapeutic use. Long-term consequences, such as possible malignant transformation and possible endogenous tumor growth stimulation cannot be completely excluded. Ultimately, these questions can only be answered through randomized controlled trials for defined clinical indications with defined MSC.

Haploidentical Hematopoietic Stem Cell Transplantation as Platform for Post-transplant Cellular Therapy

PubMed Central

Kongtim, Piyanuch; Lee, Dean A.; Cooper, Laurence J. N.; Kebriaei, Partow; Champlin, Richard E.; Ciurea, Stefan O.

2016-01-01

Haploidentical transplantation can extend the opportunity for transplantation to almost all patients who lack an HLA-matched donor. Advances in the field of haploidentical transplantation have led to a marked decrease in treatment-related mortality, allowing investigators to focus on developing rationale pre- and peri-remission therapies aimed at preventing disease relapse post-transplant. Due to widespread availability, low treatment-related mortality and cost, haploidentical donors may become the preferred “alternative” donors for allogeneic hematopoietic stem cell transplantation. One of the major advantages of using a related donor is the possibility to collect or generate additional cellular products from the same immediate available donor, which will not be rejected. Infusion of these cells in the peri-transplant period, derived from the same immune system, is opening the possibility to markedly enhance the anti-tumor effects of the graft and hasten immunologic reconstitution post-transplant. PMID:26172479

Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

NASA Astrophysics Data System (ADS)

Salick, Max R.

The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

PubMed Central

Horiguchi, Ikki; Sakai, Yasuyuki

2015-01-01

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 µm because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors. PMID:26168084

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence

PubMed Central

Winiecka-Klimek, Marta; Smolarz, Maciej; Walczak, Maciej P.; Zieba, Jolanta; Hulas-Bigoszewska, Krystyna; Kmieciak, Blazej; Piaskowski, Sylwester; Rieske, Piotr; Grzela, Dawid P.; Stoczynska-Fidelus, Ewelina

2015-01-01

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods—direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc—in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-β-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols. PMID:26535892

Cell cycle regulatory gene abnormalities are important determinants of leukemogenesis and disease biology in adult acute lymphoblastic leukemia.

PubMed

Stock, W; Tsai, T; Golden, C; Rankin, C; Sher, D; Slovak, M L; Pallavicini, M G; Radich, J P; Boldt, D H

2000-04-01

To test the hypothesis that cell cycle regulatory gene abnormalities are determinants of clinical outcome in adult acute lymphoblastic leukemia (ALL), we screened lymphoblasts from patients on a Southwest Oncology Group protocol for abnormalities of the genes, retinoblastoma (Rb), p53, p15(INK4B), and p16(INK4A). Aberrant expression occurred in 33 (85%) patients in the following frequencies: Rb, 51%; p16(INK4A), 41%; p53, 26%. Thirteen patients (33%) had abnormalities in 2 or more genes. Outcomes were compared in patients with 0 to 1 abnormality versus patients with multiple abnormalities. The 2 groups did not differ in a large number of clinical and laboratory characteristics. The CR rates for patients with 0 to 1 and multiple abnormalities were similar (69% and 54%, respectively). Patients with 0 to 1 abnormality had a median survival time of 25 months (n = 26; 95% CI, 13-46 months) versus 8 months (n = 13; 95% CI, 4-12 months) for those with multiple abnormalities (P <.01). Stem cells (CD34+lin-) were isolated from adult ALL bone marrows and tested for p16(INK4A) expression by immunocytochemistry. In 3 of 5 patients lymphoblasts and sorted stem cells lacked p16(INK4A) expression. In 2 other patients only 50% of sorted stem cells expressed p16(INK4A). By contrast, p16 expression was present in the CD34+ lin- compartment in 95% (median) of 9 patients whose lymphoblasts expressed p16(INK4A). Therefore, cell cycle regulatory gene abnormalities are frequently present in adult ALL lymphoblasts, and they may be important determinants of disease outcome. The presence of these abnormalities in the stem compartment suggests that they contribute to leukemogenesis. Eradication of the stem cell subset harboring these abnormalities may be important to achieve cure.

Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth.

PubMed

Yao, Shenglian; Liu, Xi; Yu, Shukui; Wang, Xiumei; Zhang, Shuming; Wu, Qiong; Sun, Xiaodan; Mao, Haiquan

2016-05-21

The development of novel biomaterials that deliver precise regulatory signals to direct stem cell fate for nerve regeneration is the focus of current intensive research efforts. In this study, a hierarchically aligned fibrillar fibrin hydrogel (AFG) that was fabricated through electrospinning and the concurrent molecular self-assembly process mimics both the soft and oriented features of nerve tissue, thus providing hybrid biophysical cues to instruct cell behavior in vitro and in vivo. The electrospun hydrogels were examined by scanning electron microscopy (SEM), polarized light microscopy, small angle X-ray scattering assay and atomic force microscopy (AFM), showing a hierarchically linear-ordered structure from the nanoscale to the macroscale with a soft elastic character (elasticity ∼1 kPa). We found that this low elasticity and aligned topography of AFG exhibit co-effects on promoting the neurogenic differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) in comparison to random fibrin hydrogel (RFG) and tissue culture plate (TCP) control after two week cell culture in growth medium lacking supplementation with soluble neurogenic induction factors. In addition, AFG also induces dorsal root ganglion (DRG) neurons to rapidly project numerous long neurite outgrowths longitudinally along the AFG fibers for a total neurite extension distance of 1.96 mm in three days in the absence of neurotrophic factor supplementation. Moreover, the AFG implanted in a rat T9 dorsal hemisection spinal cord injury model was found to promote endogenous neural cell fast migration and axonal invasion along AFG fibers, resulting in aligned tissue cables in vivo. Our results suggest that matrix stiffness and aligned topography may instruct stem cell neurogenic differentiation and rapid neurite outgrowth, providing great promise for biomaterial design for applications in nerve regeneration.

Targeted myocardial delivery of GDF11 gene rejuvenates the aged mouse heart and enhances myocardial regeneration after ischemia-reperfusion injury.

PubMed

Du, Guo-Qing; Shao, Zheng-Bo; Wu, Jie; Yin, Wen-Juan; Li, Shu-Hong; Wu, Jun; Weisel, Richard D; Tian, Jia-Wei; Li, Ren-Ke

2017-01-01

Ischemic cardiac injury is the main contributor to heart failure, and the regenerative capacity of intrinsic stem cells plays an important role in tissue repair after injury. However, stem cells in aged individuals have reduced regenerative potential and aged tissues lack the capacity to renew. Growth differentiation factor 11 (GDF11), from the activin-transforming growth factor β superfamily, has been shown to promote stem cell activity and rejuvenation. We carried out non-invasive targeted delivery of the GDF11 gene to the heart using ultrasound-targeted microbubble destruction (UTMD) and cationic microbubble (CMB) to investigate the ability of GDF11 to rejuvenate the aged heart and improve tissue regeneration after injury. Young (3 months) and old (21 months) mice were used to evaluate the expression of GDF11 mRNA in the myocardium at baseline and after ischemia/reperfusion (I/R) and myocardial infarction. GDF11 expression decreased with age and following myocardial injury. UTMD-mediated delivery of the GDF11 plasmid to the aged heart after I/R injury effectively and selectively increased GDF11 expression in the heart, and improved cardiac function and reduced infarct size. Over-expression of GDF11 decreased senescence markers, p16 and p53, as well as the number of p16 + cells in old mouse hearts. Furthermore, increased proliferation of cardiac stem cell antigen 1 (Sca-1 + ) cells and increased homing of endothelial progenitor cells and angiogenesis in old ischemic hearts occurred after GDF11 over-expression. Repetitive targeted delivery of the GDF11 gene via UTMD can rejuvenate the aged mouse heart and protect it from I/R injury.

Testicular receptor 2, Nr2c1, is associated with stem cells in the developing olfactory epithelium and other cranial sensory and skeletal structures.

PubMed

Baker, Jennifer L; Wood, Bernard; Karpinski, Beverly A; LaMantia, Anthony-S; Maynard, Thomas M

2016-01-01

Comparative genomic analysis of the nuclear receptor family suggests that the testicular receptor 2, Nr2c1, undergoes positive selection in the human-chimpanzee clade based upon a significant increase in nonsynonymous compared to synonymous substitutions. Previous in situ analyses of Nr2c1 lacked the temporal range and spatial resolution necessary to characterize cellular expression of this gene from early to mid gestation, when many nuclear receptors are key regulators of tissue specific stem or progenitor cells. Thus, we asked whether Nr2c1 protein is associated with stem cell populations in the mid-gestation mouse embryo. Nr2c1 is robustly expressed in the developing olfactory epithelium. Its expression in the olfactory epithelium shifts from multiple progenitor classes at early stages to primarily transit amplifying cells later in olfactory epithelium development. In the early developing central nervous system, Nr2c1 is limited to the anterior telencephalon/olfactory bulb anlagen, coincident with Nestin-positive neuroepithelial stem cells. Nr2c1 is also seen in additional cranial sensory specializations including cells surrounding the mystacial vibrissae, the retinal pigment epithelium and Scarpa's ganglion. Nr2c1 was also detected in a subset of mesenchymal cells in developing teeth and cranial bones. The timing and distribution of embryonic expression suggests that Nr2c1 is primarily associated with the early genesis of mammalian cranial sensory neurons and craniofacial skeletal structures. Thus, Nr2c1 may be a candidate for mediating parallel adaptive changes in cranial neural sensory specializations such as the olfactory epithelium, retina and mystacial vibrissae and in non-neural craniofacial features including teeth. Copyright © 2015 Elsevier B.V. All rights reserved.

Using low-risk factors to generate non-integrated human induced pluripotent stem cells from urine-derived cells.

PubMed

Wang, Linli; Chen, Yuehua; Guan, Chunyan; Zhao, Zhiju; Li, Qiang; Yang, Jianguo; Mo, Jian; Wang, Bin; Wu, Wei; Yang, Xiaohui; Song, Libing; Li, Jun

2017-11-02

Because the lack of an induced pluripotent stem cell (iPSC) induction system with optimal safety and efficiency limits the application of these cells, development of such a system is important. To create such an induction system, we screened a variety of reprogrammed plasmid combinations and multiple compounds and then verified the system's feasibility using urine cells from different individuals. We also compared large-scale iPSC chromosomal variations and expression of genes associated with genomic stability between this system and the traditional episomal system using karyotype and quantitative reverse transcription polymerase chain reaction analyses. We developed a high-efficiency episomal system, the 6F/BM1-4C system, lacking tumorigenic factors for human urine-derived cell (hUC) reprogramming. This system includes six low-risk factors (6F), Oct4, Glis1, Klf4, Sox2, L-Myc, and the miR-302 cluster. Transfected hUCs were treated with four compounds (4C), inhibitor of lysine-demethylase1, methyl ethyl ketone, glycogen synthase kinase 3 beta, and histone deacetylase, within a short time period. Comparative analysis revealed significantly decreased chromosomal variation in iPSCs and significantly increased Sirt1 expression compared with iPSCs induced using the traditional episomal system. The 6F/BM1-4C system effectively induces reprogramming of urine cells in samples obtained from different individuals. iPSCs induced using the 6F/BM1-4C system are more stable at the cytogenetic level and have potential value for clinical application.

Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacMillan, Colleen P.; Birke, Hannah; Chuah, Aaron

Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a ‘typical’ lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre.

Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls

DOE PAGES

MacMillan, Colleen P.; Birke, Hannah; Chuah, Aaron; ...

2017-07-18

Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a ‘typical’ lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre.

Levofloxacin for the treatment of severe refractory BK virus-associated hemorrhagic cystitis in hematopoietic stem cell transplantation recipients: A report of three cases

PubMed Central

TOPTAS, TAYFUR; KAYGUSUZ-ATAGUNDUZ, ISIK; KANI, HALUK TARIK; ADIGUZEL, CAFER; FIRATLI-TUGLULAR, TULIN

2014-01-01

BK-virus (BKV) is an important etiological agent for late-onset hemorrhagic cystitis (HC) in patients undergoing hematopoietic stem cell transplantation. Late-onset HC causes significant morbidity among these patients. Therapeutic approaches remain predominantly symptomatic. Several treatment options have been used with variable success rates. Cidofovir has the highest specificity against BKV; however, its lack of availability in the majority of countries, high costs and potential nephrotoxic effects limit its use. The present study reports three cases of severe and prolonged BKV-associated HC (BKHC). HC was resolved in all three of the patients using oral levofloxacin. Thus, levofloxacin may be an effective treatment modality for achieving complete clinical and molecular response in patients with refractory, severe BKHC. PMID:25202408

Levofloxacin for the treatment of severe refractory BK virus-associated hemorrhagic cystitis in hematopoietic stem cell transplantation recipients: A report of three cases.

PubMed

Toptas, Tayfur; Kaygusuz-Atagunduz, Isik; Kani, Haluk Tarik; Adiguzel, Cafer; Firatli-Tuglular, Tulin

2014-10-01

BK-virus (BKV) is an important etiological agent for late-onset hemorrhagic cystitis (HC) in patients undergoing hematopoietic stem cell transplantation. Late-onset HC causes significant morbidity among these patients. Therapeutic approaches remain predominantly symptomatic. Several treatment options have been used with variable success rates. Cidofovir has the highest specificity against BKV; however, its lack of availability in the majority of countries, high costs and potential nephrotoxic effects limit its use. The present study reports three cases of severe and prolonged BKV-associated HC (BKHC). HC was resolved in all three of the patients using oral levofloxacin. Thus, levofloxacin may be an effective treatment modality for achieving complete clinical and molecular response in patients with refractory, severe BKHC.

GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells.

PubMed

Yu, Jiong; Su, Xiaoru; Zhu, Chengxing; Pan, Qiaoling; Yang, Jinfeng; Ma, Jing; Shen, Leyao; Cao, Hongcui; Li, Lanjuan

2015-01-01

Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP(+) hPMSCs. GFP(+) hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP(+) hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. GFP(+) hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP(+) hPMSCs showed typical hepatic phenotypes. GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP(+) hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy. © 2015 S. Karger AG, Basel.

Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations.

PubMed

Petit Cocault, Laurence; Fleury, Maud; Clay, Denis; Larghero, Jérôme; Vanneaux, Valérie; Souyri, Michèle

2016-04-01

Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Generating induced pluripotent stem cell derived endothelial cells and induced endothelial cells for cardiovascular disease modelling and therapeutic angiogenesis.

PubMed

Clayton, Z E; Sadeghipour, S; Patel, S

2015-10-15

Standard therapy for atherosclerotic coronary and peripheral arterial disease is insufficient in a significant number of patients because extensive disease often precludes effective revascularization. Stem cell therapy holds promise as a supplementary treatment for these patients, as pre-clinical and clinical research has shown transplanted cells can promote angiogenesis via direct and paracrine mechanisms. Induced pluripotent stem cells (iPSCs) are a novel cell type obtained by reprogramming somatic cells using exogenous transcription factor cocktails, which have been introduced to somatic cells via viral or plasmid constructs, modified mRNA or small molecules. IPSCs are now being used in disease modelling and drug testing and are undergoing their first clinical trial, but despite recent advances, the inefficiency of the reprogramming process remains a major limitation, as does the lack of consensus regarding the optimum transcription factor combination and delivery method and the uncertainty surrounding the genetic and epigenetic stability of iPSCs. IPSCs have been successfully differentiated into vascular endothelial cells (iPSC-ECs) and, more recently, induced endothelial cells (iECs) have also been generated by direct differentiation, which bypasses the pluripotent intermediate. IPSC-ECs and iECs demonstrate endothelial functionality in vitro and have been shown to promote neovessel growth and enhance blood flow recovery in animal models of myocardial infarction and peripheral arterial disease. Challenges remain in optimising the efficiency, safety and fidelity of the reprogramming and endothelial differentiation processes and establishing protocols for large-scale production of clinical-grade, patient-derived cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mesenchymal stem cells for cartilage regeneration in osteoarthritis

PubMed Central

Kristjánsson, Baldur; Honsawek, Sittisak

2017-01-01

Osteoarthritis (OA) is a slowly progressive disease where cartilage of the synovial joint degenerates. It is most common in the elderly where patients experience pain and reduce physical activity. In combination with lack of conventional treatment, patients are often left with no other choices than arthroplasty. Over the last years, multipotent stromal cells have been used in efforts to treat OA. Mesenchymal stem/progenitor cells (MSCs) are stromal cells that can differentiate into bone, fat, and cartilage cells. They reside within bone marrow and fat. MSCs can also be found in synovial joints where they affect the progression of OA. They can be isolated and proliferated in an incubator before being applied in clinical trials. When it comes to treatment, emphasis has hitherto been on autologous MSCs, but allogenic cells from healthy donors are emerging as another source of the cells. The first adaptations of MSCs revolved in the use of cell-rich matrix, delivered as invasive surgical procedure, which resulted in production of hyaline cartilage and fibrocartilage. However, the demand for less invasive delivery of cells has prompted the use of direct intra-articular injections, wherein a large amount of suspended cells are implanted in the cartilage defect. PMID:28979850

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

PubMed

Giustacchini, Alice; Thongjuea, Supat; Barkas, Nikolaos; Woll, Petter S; Povinelli, Benjamin J; Booth, Christopher A G; Sopp, Paul; Norfo, Ruggiero; Rodriguez-Meira, Alba; Ashley, Neil; Jamieson, Lauren; Vyas, Paresh; Anderson, Kristina; Segerstolpe, Åsa; Qian, Hong; Olsson-Strömberg, Ulla; Mustjoki, Satu; Sandberg, Rickard; Jacobsen, Sten Eirik W; Mead, Adam J

2017-06-01

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

PubMed

Heslop, James A; Kia, Richard; Pridgeon, Christopher S; Sison-Young, Rowena L; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W; Mills, John S; Kitteringham, Neil R; Goldring, Chris E; Park, Bong K

2017-05-01

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Types of Stem Cells

MedlinePlus

... Cell Glossary Search Toggle Nav Types of Stem Cells Stem cells are the foundation from which all ... About Stem Cells > Types of Stem Cells Stem cells Stem cells are the foundation for every organ ...

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PubMed

Morgan, Joshua T; Wood, Joshua A; Walker, Naomi J; Raghunathan, Vijay Krishna; Borjesson, Dori L; Murphy, Christopher J; Russell, Paul

2014-01-01

To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Human Trabecular Meshwork Cells Exhibit Several Characteristics of, but Are Distinct from, Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Morgan, Joshua T.; Wood, Joshua A.; Walker, Naomi J.; Raghunathan, Vijay Krishna; Borjesson, Dori L.; Murphy, Christopher J.

2014-01-01

Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities. PMID:24456002

Placental Stem Cell Correction of Murine Intermediate Maple Syrup Urine Disease

PubMed Central

Skvorak, Kristen J.; Dorko, Kenneth; Marongiu, Fabio; Tahan, Veysel; Hansel, Marc C.; Gramignoli, Roberto; Gibson, K. Michael; Strom, Stephen C.

2012-01-01

We previously reported improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model post allogenic hepatocyte transplant, confirming that a small number of enzyme proficient liver-engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAEC) share stem cell characteristics while lacking many safety and ethical concerns, and differentiate to hepatocyte-like cells. Eight direct hepatic hAEC transplants were administered to iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at days 35 and 100. Treatment at the neonatal stage is clinically relevant for MSUD, and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplants compared to iMSUD (severely cachectic; dead ≤28 days). Branched chain α-keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. Branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in the sera and brain, as were other large neutral amino acids. Conclusion: Placental-derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver-based metabolic diseases. PMID:23175463

Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiotherapy response prediction

PubMed Central

Jeong, Youngtae; Hoang, Ngoc T.; Lovejoy, Alexander; Stehr, Henning; Newman, Aaron M.; Gentles, Andrew J.; Kong, William; Truong, Diana; Martin, Shanique; Chaudhuri, Aadel; Heiser, Diane; Zhou, Li; Say, Carmen; Carter, Justin N.; Hiniker, Susan M.; Loo, Billy W.; West, Robert B.; Beachy, Philip; Alizadeh, Ash A.; Diehn, Maximilian

2016-01-01

Lung squamous cell carcinomas (LSCC) pathogenesis remains incompletely understood and biomarkers predicting treatment response remain lacking. Here we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histological and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in non-small lung cancer (NSCLC) patients and could be non-invasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs. PMID:27663899

Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.

PubMed

Stebbins, Matthew J; Wilson, Hannah K; Canfield, Scott G; Qian, Tongcheng; Palecek, Sean P; Shusta, Eric V

2016-05-15

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties, the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease, yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently, in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here, we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Stress-triggered atavistic reprogramming (STAR) addiction: driving force behind head and neck cancer?

PubMed Central

Masuda, Muneyuki; Wakasaki, Takahiro; Toh, Satoshi

2016-01-01

Recent results of the Cancer Genome Atlas on head and neck squamous cell carcinoma (HNSCC) revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role for epigenetics in oncogenesis has become apparent. In parallel, it has gained general acceptance that cancer is considered as complex adaptive system, which evolves responding environmental selective pressures. This somatic evolution appears to proceed concurrently with the acquisition of an atavistic pluripotent state (i.e., “stemness”), which is inducible by intrinsic epigenetic reprogramming program as demonstrated by induced pluripotent stem (iPS) cells. This Nobel prize-winning discovery has markedly accelerated and expanded cancer stem cell research from the point of epigenetic reprogramming. Taken together, we hypothesize that stress-triggered atavistic reprogramming (STAR) may be the major driving force of HNSCC evolution. In this perspective, we discuss the possible mechanisms of STAR in HNSCC, focusing on recent topics of epigenetic reprogramming in developmental and cancer cell biology. PMID:27429838

β-Arrestin2 mediates progression of murine primary myelofibrosis

PubMed Central

Rein, Lindsay A.M.; Wisler, James W.; Kim, Jihee; Theriot, Barbara; Huang, LiYin; Price, Trevor; Yang, Haeyoon; Chen, Wei; Sipkins, Dorothy; Fedoriw, Yuri; Walker, Julia K.L.; Premont, Richard T.; Lefkowitz, Robert J.

2017-01-01

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. β-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, β-arrestin2 (βarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between βarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor βarr2-knockout (βarr2–/–) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted βarr2–/– cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of βarr2–/– cells. In order to assess the effect of acute loss of βarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional βarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic βarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that βarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease. PMID:29263312

miRNA-regulated cancer stem cells: understanding the property and the role of miRNA in carcinogenesis.

PubMed

Chakraborty, Chiranjib; Chin, Kok-Yong; Das, Srijit

2016-10-01

Over the last few years, microRNAs (miRNA)-controlled cancer stem cells have drawn enormous attention. Cancer stem cells are a small population of tumor cells that possess the stem cell property of self-renewal. Recent data shows that miRNA regulates this small population of stem cells. In the present review, we explained different characteristics of cancer stem cells as well as miRNA regulation of self-renewal and differentiation in cancer stem cells. We also described the migration and tumor formation. Finally, we described the different miRNAs that regulate various types of cancer stem cells, such as prostate cancer stem cells, head and neck cancer stem cells, breast cancer stem cells, colorectal cancer stem cells, lung cancer stem cells, gastric cancer stem cells, pancreatic cancer stem cells, etc. Extensive research is needed in order to employ miRNA-based therapeutics to control cancer stem cell population in various cancers in the future.

Epigenetic Mechanisms Regulate MHC and Antigen Processing Molecules in Human Embryonic and Induced Pluripotent Stem Cells

PubMed Central

Suárez-Álvarez, Beatriz; Rodriguez, Ramón M.; Calvanese, Vincenzo; Blanco-Gelaz, Miguel A.; Suhr, Steve T.; Ortega, Francisco; Otero, Jesus; Cibelli, Jose B.; Moore, Harry; Fraga, Mario F.; López-Larrea, Carlos

2010-01-01

Background Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored. Methodology/Principal Findings We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of β2-microglobulin (β2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and β2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation. Conclusions/Significance Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance. PMID:20419139

What is a stem cell?

PubMed

Slack, Jonathan M W

2018-05-15

The historical roots of the stem cell concept are traced with respect to its usage in embryology and in hematology. The modern consensus definition of stem cells, comprising both pluripotent stem cells in culture and tissue-specific stem cells in vivo, is explained and explored. Methods for identifying stem cells are discussed with respect to cell surface markers, telomerase, label retention and transplantability, and properties of the stem cell niche are explored. The CreER method for identifying stem cells in vivo is explained, as is evidence in favor of a stochastic rather than an obligate asymmetric form of cell division. In conclusion, it is found that stem cells do not possess any unique and specific molecular markers; and stem cell behavior depends on the environment of the cell as well as the stem cell's intrinsic qualities. Furthermore, the stochastic mode of division implies that stem cell behavior is a property of a cell population not of an individual cell. In this sense, stem cells do not exist in isolation but only as a part of multicellular system. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Methods and Principles Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells. © 2018 Wiley Periodicals, Inc.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

PubMed

Moore, Finola E; Garcia, Elaine G; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C; Garcia, Amaris J; Mylvaganam, Ravi; Yoder, Jeffrey A; Blackburn, Jessica S; Sadreyev, Ruslan I; Ceol, Craig J; North, Trista E; Langenau, David M

2016-05-30

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. © 2016 Moore et al.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

PubMed Central

Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

2016-01-01

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

Advances in generation of functional β-cells from induced pluripotent stem cells as a cure for diabetes mellitus.

PubMed

Kalra, Kunal; Chandrabose, Srijaya Thekkeparambil; Ramasamy, Thamil Selvee; Kasim, Noor Hayaty Binti Abu

2018-06-04

Diabetes mellitus is one of the leading cause for death worldwide. Loss and functional failure of pancreatic β-cells, the parenchyma cells in the islets of Langerhans onsets and progresses diabetes mellitus. The increasing incidence of this metabolic disorder necessitates efficient strategies to produce functional β-cells for treating diabetes mellitus. Human induced pluripotent stem cells (hiPSC), holds potential for treating diabetes owning to their self-renewal capacity and ability to differentiate into β-cells. iPSC technology also provides unlimited starting material to generate differentiated cells for regenerative applications. Progress has also been made in establishing in-vitro culture protocols to yield definitive endoderm, pancreatic endoderm progenitor cells and β-cells via different reprogramming strategies and growth factor supplementation. However, these generated β-cells are still immature, lack functional characteristics and exhibit lower capability in reversing the diseases conditions. Current methods employed to generate mature and functional β-cells include; use of small and large molecules to enhance the reprogramming and differentiation efficiency, 3D culture systems to improve the functional properties and heterogeneity of differentiated cells. This review details recent advancements in the generation of mature β-cells by reprogramming stem cells into iPSCs that is further programmed to β-cells. It also provides deeper insight of current reprogramming protocols and their efficacy, focusing on the underlying mechanism of chemical based approach to generate iPSCs. Furthermore, we have highlighted the recent differentiation strategies both in-vitro and in-vivo to date and the future prospects in generation of mature β-cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

PubMed

Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

2014-10-01

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

Novel insights into the pathways regulating the canine hair cycle and their deregulation in alopecia X.

PubMed

Brunner, Magdalena A T; Jagannathan, Vidhya; Waluk, Dominik P; Roosje, Petra; Linek, Monika; Panakova, Lucia; Leeb, Tosso; Wiener, Dominique J; Welle, Monika M

2017-01-01

Alopecia X is a hair cycle arrest disorder in Pomeranians. Histologically, kenogen and telogen hair follicles predominate, whereas anagen follicles are sparse. The induction of anagen relies on the activation of hair follicle stem cells and their subsequent proliferation and differentiation. Stem cell function depends on finely tuned interactions of signaling molecules and transcription factors, which are not well defined in dogs. We performed transcriptome profiling on skin biopsies to analyze altered molecular pathways in alopecia X. Biopsies from five affected and four non-affected Pomeranians were investigated. Differential gene expression revealed a downregulation of key regulator genes of the Wnt (CTNNB1, LEF1, TCF3, WNT10B) and Shh (SHH, GLI1, SMO, PTCH2) pathways. In mice it has been shown that Wnt and Shh signaling results in stem cell activation and differentiation Thus our findings are in line with the lack of anagen hair follicles in dogs with Alopecia X. We also observed a significant downregulation of the stem cell markers SOX9, LHX2, LGR5, TCF7L1 and GLI1 whereas NFATc1, a quiescence marker, was upregulated in alopecia X. Moreover, genes coding for enzymes directly involved in the sex hormone metabolism (CYP1A1, CYP1B1, HSD17B14) were differentially regulated in alopecia X. These findings are in agreement with the so far proposed but not yet proven deregulation of the sex hormone metabolism in this disease.

Bone Marrow Stem Cells and Ear Framework Reconstruction.

PubMed

Karimi, Hamid; Emami, Seyed-Abolhassan; Olad-Gubad, Mohammad-Kazem

2016-11-01

Repair of total human ear loss or congenital lack of ears is one of the challenging issues in plastic and reconstructive surgery. The aim of the present study was 3D reconstruction of the human ear with cadaveric ear cartilages seeded with human mesenchymal stem cells. We used cadaveric ear cartilages with preserved perichondrium. The samples were divided into 2 groups: group A (cartilage alone) and group B (cartilage seeded with a mixture of fibrin powder and mesenchymal stem cell [1,000,000 cells/cm] used and implanted in back of 10 athymic rats). After 12 weeks, the cartilages were removed and shape, size, weight, flexibility, and chondrocyte viability were evaluated. P value <0.05 was considered significant. In group A, size and weight of cartilages clearly reduced (P < 0.05) and then shape and flexibility (torsion of cartilages in clockwise and counterclockwise directions) were evaluated, which were found to be significantly reduced (P > 0.05). After staining with hematoxylin and eosin and performing microscopic examination, very few live chondrocytes were found in group A. In group B, size and weight of samples were not changed (P < 0.05); the shape and flexibility of samples were well maintained (P < 0.05) and on performing microscopic examination of cartilage samples, many live chondrocytes were found in cartilage (15-20 chondrocytes in each microscopic field). In samples with human stem cell, all variables (size, shape, weight, and flexibility) were significantly maintained and abundant live chondrocytes were found on performing microscopic examination. This method may be used for reconstruction of full defect of auricles in humans.

Prognostic roles of pathology markers immunoexpression and clinical parameters in Hepatoblastoma.

PubMed

Wu, Jia-Feng; Chang, Hsiu-Hao; Lu, Meng-Yao; Jou, Shiann-Tarng; Chang, Kai-Chi; Ni, Yen-Hsuan; Chang, Mei-Hwei

2017-08-29

Hepatoblastoma, a leading primary hepatic malignant tumor in children, is originated from primitive hepatic stem cells. We aimed to elucidate the relationships between the histological distribution of β-catenin and hepatic stem cell markers with the clinical outcomes of hepatoblastoma. Immunohistochemistry was applied to detect β-catenin and hepatic stem cell markers expression in 31 hepatoblastoma tumors. We analyzed the relationship between the stem cell markers and the clinical course of hepatoblastoma. Thirty-one hepatoblastoma patients were diagnosed at a mean age of 2.58 ± 3.78 years, and 7 (22.58%) died. A lack of anticipated decrease in alpha-fetal protein levels after neoadjuvant chemotherapy indicated a higher mortality rate. Nuclear β-catenin expression was significantly associated with membranous epithelial cell adhesion molecule (EpCAM) expression in hepatoblastoma tumor specimens. The co-expression of nuclear β-catenin and membranous EpCAM together with an age at diagnosis ≤1.25 years were predictive of an alpha-fetoprotein level < 1200 ng/mL after neoadjuvant chemotherapy (P < 0.05). An alpha-fetoprotein level < 1200 ng/mL after neoadjuvant chemotherapy and age at hepatoblastoma diagnosis ≤1.25 years are both predictors of better overall and native liver survival in hepatoblastoma patients. Presence of membranous EpCAM with nuclear β-catenin and younger diagnostic age of hepatoblastoma are predictive of serum alpha-fetoprotein levels drop after chemotherapy. Younger diagnostic age and lower alpha-fetoprotein levels after neoadjuvant chemotherapy and are predictive of better overall and native liver survival in hepatoblastoma patients.

Isolation and gene expression analysis of single potential human spermatogonial stem cells.

PubMed

von Kopylow, K; Schulze, W; Salzbrunn, A; Spiess, A-N

2016-04-01

It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Acquired aplastic anemia.

PubMed

Keohane, Elaine M

2004-01-01

Acquired aplastic anemia (AA) is a disorder characterized by a profound deficit of hematopoietic stem and progenitor cells, bone marrow hypocellularity, and peripheral blood pancytopenia. It primarily affects children, young adults, and those over 60 years of age. The majority of cases are idiopathic; however, idiosyncratic reactions to some drugs, chemicals, and viruses have been implicated in its etiology. An autoimmune T-cell reaction likely causes the stem cell depletion, but the precise mechanism, as well as the eliciting and target antigens, is unknown. Symptoms vary from severe life-threatening cytopenias to moderate or non-severe disease that does not require transfusion support. The peripheral blood typically exhibits pancytopenia, reticulocytopenia, and normocytic or macrocytic erythrocytes. The bone marrow is hypocellular and may exhibit dysplasia of the erythrocyte precursors. First line treatment for severe AA consists of hematopoietic stem cell transplantation in young patients with HLA identical siblings, while immunosuppression therapy is used for older patients and for those of any age who lack a HLA matched donor. Patients with AA have an increased risk of developing paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndrome (MDS), or acute leukemia. Further elucidation of the pathophysiology of this disease will result in a better understanding of the interrelationship among AA, PNH, and MDS, and may lead to novel targeted therapies.

Lessons we learned from high-throughput and top-down systems biology analyses about glioma stem cells.

PubMed

Mock, Andreas; Chiblak, Sara; Herold-Mende, Christel

2014-01-01

A growing body of evidence suggests that glioma stem cells (GSCs) account for tumor initiation, therapy resistance, and the subsequent regrowth of gliomas. Thus, continuous efforts have been undertaken to further characterize this subpopulation of less differentiated tumor cells. Although we are able to enrich GSCs, we still lack a comprehensive understanding of GSC phenotypes and behavior. The advent of high-throughput technologies raised hope that incorporation of these newly developed platforms would help to tackle such questions. Since then a couple of comparative genome-, transcriptome- and proteome-wide studies on GSCs have been conducted giving new insights in GSC biology. However, lessons had to be learned in designing high-throughput experiments and some of the resulting conclusions fell short of expectations because they were performed on only a few GSC lines or at one molecular level instead of an integrative poly-omics approach. Despite these shortcomings, our knowledge of GSC biology has markedly expanded due to a number of survival-associated biomarkers as well as glioma-relevant signaling pathways and therapeutic targets being identified. In this article we review recent findings obtained by comparative high-throughput analyses of GSCs. We further summarize fundamental concepts of systems biology as well as its applications for glioma stem cell research.

Mesenchymal Stem Cell Therapy in Intracerebral Haemorrhagic Stroke.

PubMed

Bedini, Gloria; Bersano, Anna; Zanier, Elisa R; Pischiutta, Francesca; Parati, Eugenio A

2018-01-10

Spontaneous intracerebral haemorrhage (ICH) is a relatively common fatal disease, with an overall global incidence estimated at 24.6 per 100,000 person-years. Given the high degree of morbidity and mortality associated with ICH, therapies that may have neuroprotective effects are of increasing interest to clinicians. In this last context, cell therapies offer the promise of improving the disease course which cannot be addressed adequately by existing treatments. The aim of this review is to evaluate the protective effects and molecular mechanisms of mesenchymal stem cells (MSCs) on haemorrhagic brain following ICH. We also discuss possible emerging therapeutic approaches worth of further research. The available literature on the therapeutic potential of MSCs in ICH animal models clearly demonstrated that MSCs enhance the functional recovery and reduce the volume of the infarct size exerting anti-inflammatory and angiogenic properties. However, the quality of the original articles investigating the efficacy of stem cell therapies in ICH animal models is still poor and the lack of ICH clinical trial does not permit to reach any relevant conclusions. Further studies have to be implemented in order to achieve standardized methods of MSCs isolation, characterization and administration to improve ICH treatments with MSCs or MSC-derived products. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Transplantation of spinal cord-derived neural stem cells for ALS: Analysis of phase 1 and 2 trials.

PubMed

Glass, Jonathan D; Hertzberg, Vicki S; Boulis, Nicholas M; Riley, Jonathan; Federici, Thais; Polak, Meraida; Bordeau, Jane; Fournier, Christina; Johe, Karl; Hazel, Tom; Cudkowicz, Merit; Atassi, Nazem; Borges, Lawrence F; Rutkove, Seward B; Duell, Jayna; Patil, Parag G; Goutman, Stephen A; Feldman, Eva L

2016-07-26

To test the safety of spinal cord transplantation of human stem cells in patients with amyotrophic lateral sclerosis (ALS) with escalating doses and expansion of the trial to multiple clinical centers. This open-label trial included 15 participants at 3 academic centers divided into 5 treatment groups receiving increasing doses of stem cells by increasing numbers of cells/injection and increasing numbers of injections. All participants received bilateral injections into the cervical spinal cord (C3-C5). The final group received injections into both the lumbar (L2-L4) and cervical cord through 2 separate surgical procedures. Participants were assessed for adverse events and progression of disease, as measured by the ALS Functional Rating Scale-Revised, forced vital capacity, and quantitative measures of strength. Statistical analysis focused on the slopes of decline of these phase 2 trial participants alone or in combination with the phase 1 participants (previously reported), comparing these groups to 3 separate historical control groups. Adverse events were mostly related to transient pain associated with surgery and to side effects of immunosuppressant medications. There was one incident of acute postoperative deterioration in neurologic function and another incident of a central pain syndrome. We could not discern differences in surgical outcomes between surgeons. Comparisons of the slopes of decline with the 3 separate historical control groups showed no differences in mean rates of progression. Intraspinal transplantation of human spinal cord-derived neural stem cells can be safely accomplished at high doses, including successive lumbar and cervical procedures. The procedure can be expanded safely to multiple surgical centers. This study provides Class IV evidence that for patients with ALS, spinal cord transplantation of human stem cells can be safely accomplished and does not accelerate the progression of the disease. This study lacks the precision to exclude important benefit or safety issues. © 2016 American Academy of Neurology.

Non-myeloablative autologous haematopoietic stem cell transplantation expands regulatory cells and depletes IL-17 producing mucosal-associated invariant T cells in multiple sclerosis

PubMed Central

Abrahamsson, Sofia V.; Angelini, Daniela F.; Dubinsky, Amy N.; Morel, Esther; Oh, Unsong; Jones, Joanne L.; Carassiti, Daniele; Reynolds, Richard; Salvetti, Marco; Calabresi, Peter A.; Coles, Alasdair J.; Battistini, Luca; Martin, Roland; Burt, Richard K.

2013-01-01

Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive conditioning regimen, and re-infusing the autologous haematopoietic cell product. ‘Non-myeloablative’ conditioning regimens to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years. We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multi-colour fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution post-transplantation we observed significant though transient increases in the proportion of CD4+FoxP3+ T cells and CD56high natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+CD57+ cytotoxic T cells were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a CD161high proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization revealed that the CD161highCD8+ T cells were mucosal-associated invariant T cells, a novel cell population originating in the gut mucosa but expressing the central nervous system-homing receptor CCR6. Detection of mucosal-associated invariant T cells in post-mortem multiple sclerosis brain white matter active lesions confirmed their involvement in the disease pathology. Intracellular cytokine staining demonstrated interferon γ and interleukin 17 production and lack of interleukin 10 production, a pro-inflammatory profile. Mucosal-associated invariant T cell frequency did not change in patients treated with interferon β; and was more depleted after autologous haematopoietic stem cell transplantation than in patients who had received high-dose cyclophosphamide (n = 7) or alemtuzumab (n = 21) treatment alone, suggesting an additive or synergistic effect of the conditioning regime components. We propose that a favourably modified balance of regulatory and pro-inflammatory lymphocytes underlies the suppression of central nervous system inflammation in patients with multiple sclerosis following non-myeloablative autologous haematopoietic stem cell transplantation with a conditioning regimen consisting of cyclophosphamide and alemtuzumab. PMID:23864273

Synaptic Regulator α-Synuclein in Dopaminergic Fibers Is Essentially Required for the Maintenance of Subependymal Neural Stem Cells.

PubMed

Perez-Villalba, Ana; Sirerol-Piquer, M Salomé; Belenguer, Germán; Soriano-Cantón, Raúl; Muñoz-Manchado, Ana Belén; Villadiego, Javier; Alarcón-Arís, Diana; Soria, Federico N; Dehay, Benjamin; Bezard, Erwan; Vila, Miquel; Bortolozzi, Analía; Toledo-Aral, Juan José; Pérez-Sánchez, Francisco; Fariñas, Isabel

2018-01-24

Synaptic protein α-synuclein (α-SYN) modulates neurotransmission in a complex and poorly understood manner and aggregates in the cytoplasm of degenerating neurons in Parkinson's disease. Here, we report that α-SYN present in dopaminergic nigral afferents is essential for the normal cycling and maintenance of neural stem cells (NSCs) in the brain subependymal zone of adult male and female mice. We also show that premature senescence of adult NSCs into non-neurogenic astrocytes in mice lacking α-SYN resembles the effects of dopaminergic fiber degeneration resulting from chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine or intranigral inoculation of aggregated toxic α-SYN. Interestingly, NSC loss in α-SYN-deficient mice can be prevented by viral delivery of human α-SYN into their sustantia nigra or by treatment with l-DOPA, suggesting that α-SYN regulates dopamine availability to NSCs. Our data indicate that α-SYN, present in dopaminergic nerve terminals supplying the subependymal zone, acts as a niche component to sustain the neurogenic potential of adult NSCs and identify α-SYN and DA as potential targets to ameliorate neurogenic defects in the aging and diseased brain. SIGNIFICANCE STATEMENT We report an essential role for the protein α-synuclein present in dopaminergic nigral afferents in the regulation of adult neural stem cell maintenance, identifying the first synaptic regulator with an implication in stem cell niche biology. Although the exact role of α-synuclein in neural transmission is not completely clear, our results indicate that it is required for stemness and the preservation of neurogenic potential in concert with dopamine. Copyright © 2018 the authors 0270-6474/18/380815-12$15.00/0.

Efficient programming of human eye conjunctiva-derived induced pluripotent stem (ECiPS) cells into definitive endoderm-like cells.

PubMed

Massumi, Mohammad; Hoveizi, Elham; Baktash, Parvaneh; Hooti, Abdollah; Ghazizadeh, Leili; Nadri, Samad; Pourasgari, Farzaneh; Hajarizadeh, Athena; Soleimani, Masoud; Nabiuni, Mohammad; Khorramizadeh, Mohammad R

2014-03-10

Due to pluripotency of induced pluripotent stem (iPS) cells, and the lack of immunological incompatibility and ethical issues, iPS cells have been considered as an invaluable cell source for future cell replacement therapy. This study was aimed first at establishment of novel iPS cells, ECiPS, which directly reprogrammed from human Eye Conjunctiva-derived Mesenchymal Stem Cells (EC-MSCs); second, comparing the inductive effects of Wnt3a/Activin A biomolecules to IDE1 small molecule in derivation of definitive endoderm (DE) from the ECiPS cells. To that end, first, the EC-MSCs were transduced by SOKM-expressing lentiviruses and characterized for endogenous expression of embryonic markers Then the established ECiPS cells were induced to DE formation by Wnt3a/Activin A or IDE1. Quantification of GSC, Sox17 and Foxa2 expression, as DE-specific markers, in both mRNA and protein levels revealed that induction of ECiPS cells by either Wnt3a/Activin A or IDE1 could enhance the expression level of the genes; however the levels of increase were higher in Wnt3a/Activin A induced ECiPS-EBs than IDE1 induced cells. Furthermore, the flow cytometry analyses showed no synergistic effect between Activin A and Wnt3a to derive DE-like cells from ECiPS cells. The comparative findings suggest that although both Wnt3a/Activin A signaling and IDE1 molecule could be used for differentiation of iPS into DE cells, the DE-inducing effect of Wnt3a/Activin A was statistically higher than IDE1. Copyright © 2014 Elsevier Inc. All rights reserved.

On-chip immobilization of planarians for in vivo imaging.

PubMed

Dexter, Joseph P; Tamme, Mary B; Lind, Christine H; Collins, Eva-Maria S

2014-09-17

Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians. We have developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra.

On-chip immobilization of planarians for in vivo imaging

PubMed Central

Dexter, Joseph P.; Tamme, Mary B.; Lind, Christine H.; Collins, Eva-Maria S.

2014-01-01

Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians. We have developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra. PMID:25227263

Concise Review: Workshop Review: Understanding and Assessing the Risks of Stem Cell-Based Therapies

PubMed Central

Heslop, James A.; Hammond, Thomas G.; Santeramo, Ilaria; Tort Piella, Agnès; Hopp, Isabel; Zhou, Jing; Baty, Roua; Graziano, Enrique I.; Proto Marco, Bernabé; Caron, Alexis; Sköld, Patrik; Andrews, Peter W.; Baxter, Melissa A.; Hay, David C.; Hamdam, Junnat; Sharpe, Michaela E.; Patel, Sara; Jones, David R.; Reinhardt, Jens; Danen, Erik H.J.; Ben-David, Uri; Stacey, Glyn; Björquist, Petter; Piner, Jacqueline; Mills, John; Rowe, Cliff; Pellegrini, Giovanni; Sethu, Swaminathan; Antoine, Daniel J.; Cross, Michael J.; Murray, Patricia; Williams, Dominic P.; Kitteringham, Neil R.; Park, B. Kevin

2015-01-01

The field of stem cell therapeutics is moving ever closer to widespread application in the clinic. However, despite the undoubted potential held by these therapies, the balance between risk and benefit remains difficult to predict. As in any new field, a lack of previous application in man and gaps in the underlying science mean that regulators and investigators continue to look for a balance between minimizing potential risk and ensuring therapies are not needlessly kept from patients. Here, we attempt to identify the important safety issues, assessing the current advances in scientific knowledge and how they may translate to clinical therapeutic strategies in the identification and management of these risks. We also investigate the tools and techniques currently available to researchers during preclinical and clinical development of stem cell products, their utility and limitations, and how these tools may be strategically used in the development of these therapies. We conclude that ensuring safety through cutting-edge science and robust assays, coupled with regular and open discussions between regulators and academic/industrial investigators, is likely to prove the most fruitful route to ensuring the safest possible development of new products. PMID:25722427

Generation of Human Liver Chimeric Mice with Hepatocytes from Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

PubMed

Yang, Jiayin; Wang, Yu; Zhou, Ting; Wong, Lai-Yung; Tian, Xiao-Yu; Hong, Xueyu; Lai, Wing-Hon; Au, Ka-Wing; Wei, Rui; Liu, Yuqing; Cheng, Lai-Hung; Liang, Guichan; Huang, Zhijian; Fan, Wenxia; Zhao, Ping; Wang, Xiwei; Ibañez, David P; Luo, Zhiwei; Li, Yingying; Zhong, Xiaofen; Chen, Shuhan; Wang, Dongye; Li, Li; Lai, Liangxue; Qin, Baoming; Bao, Xichen; Hutchins, Andrew P; Siu, Chung-Wah; Huang, Yu; Esteban, Miguel A; Tse, Hung-Fat

2017-03-14

Familial hypercholesterolemia (FH) causes elevation of low-density lipoprotein cholesterol (LDL-C) in blood and carries an increased risk of early-onset cardiovascular disease. A caveat for exploration of new therapies for FH is the lack of adequate experimental models. We have created a comprehensive FH stem cell model with differentiated hepatocytes (iHeps) from human induced pluripotent stem cells (iPSCs), including genetically engineered iPSCs, for testing therapies for FH. We used FH iHeps to assess the effect of simvastatin and proprotein convertase subtilisin/kexin type 9 (PCSK9) antibodies on LDL-C uptake and cholesterol lowering in vitro. In addition, we engrafted FH iHeps into the liver of Ldlr -/- /Rag2 -/- /Il2rg -/- mice, and assessed the effect of these same medications on LDL-C clearance and endothelium-dependent vasodilation in vivo. Our iHep models recapitulate clinical observations of higher potency of PCSK9 antibodies compared with statins for reversing the consequences of FH, demonstrating the utility for preclinical testing of new therapies for FH patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Chondrogenically primed tonsil-derived mesenchymal stem cells encapsulated in riboflavin-induced photocrosslinking collagen-hyaluronic acid hydrogel for meniscus tissue repairs.

PubMed

Koh, Rachel H; Jin, Yinji; Kang, Byung-Jae; Hwang, Nathaniel S

2017-04-15

Current meniscus tissue repairing strategies involve partial or total meniscectomy, followed by allograft transplantation or synthetic material implantation. However, allografts and synthetic implants have major drawbacks such as the limited supply of grafts and lack of integration into host tissue, respectively. In this study, we investigated the effects of conditioned medium (CM) from meniscal fibrochondrocytes and TGF-β3 on tonsil-derived mesenchymal stem cells (T-MSCs) for meniscus tissue engineering. CM-expanded T-MSCs were encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogels and cultured in chondrogenic medium containing TGF-β3. In vitro results indicate that CM-expanded cells followed by TGF-β3 exposure stimulated the expression of fibrocartilage-related genes (COL2, SOX9, ACAN, COL1) and production of extracellular matrix components. Histological assessment of in vitro and subcutaneously implanted in vivo constructs demonstrated that CM-expanded cells followed by TGF-β3 exposure resulted in highest cell proliferation, GAG accumulation, and collagen deposition. Furthermore, when implanted into meniscus defect model, CM treatment amplified the potential of TGF-β3 and induced complete regeneration. Conditioned medium derived from chondrocytes have been reported to effectively prime mesenchymal stem cells toward chondrogenic lineage. Type I collagen is the main component of meniscus extracellular matrix and hyaluronic acid is known to promote meniscus regeneration. In this manuscript, we investigated the effects of conditioned medium (CM) and transforming growth factor-β3 (TGF-β3) on tonsil-derived mesenchymal stem cells (T-MSCs) encapsulated in riboflavin-induced photocrosslinked collagen-hyaluronic acid (COL-RF-HA) hydrogel. We employed a novel source of conditioned medium, derived from meniscal fibrochondrocytes. Our in vitro and in vivo results collectively illustrate that CM-expanded cells followed by TGF-β3 exposure have the best potential for meniscus regeneration. This manuscript highlights a novel stem cell commitment strategy combined with biomaterials designs for meniscus regeneration. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

From Early Embryonic to Adult Stage: Comparative Study of Action Potentials of Native and Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Peinkofer, Gabriel; Burkert, Karsten; Urban, Katja; Krausgrill, Benjamin; Hescheler, Jürgen; Saric, Tomo; Halbach, Marcel

2016-10-01

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPS-CMs) are promising candidates for cell therapy, drug screening, and developmental studies. It is known that iPS-CMs possess immature electrophysiological properties, but an exact characterization of their developmental stage and subtype differentiation is hampered by a lack of knowledge of electrophysiological properties of native CMs from different developmental stages and origins within the heart. Thus, we sought to systematically investigate action potential (AP) properties of native murine CMs and to establish a database that allows classification of stem cell-derived CMs. Hearts from 129S2PasCrl mice were harvested at days 9-10, 12-14, and 16-18 postcoitum, as well as 1 day, 3-4 days, 1-2 weeks, 3-4 weeks, and 6 weeks postpartum. AP recordings in left and right atria and at apical, medial, and basal left and right ventricles were performed with sharp glass microelectrodes. Measurements revealed significant changes in AP morphology during pre- and postnatal murine development and significant differences between atria and ventricles, enabling a classification of developmental stage and subtype differentiation of stem cell-derived CMs based on their AP properties. For iPS-CMs derived from cell line TiB7.4, a typical ventricular phenotype was demonstrated at later developmental stages, while there were electrophysiological differences from atrial as well as ventricular native CMs at earlier stages. This finding supports that iPS-CMs can develop AP properties similar to native CMs, but points to differences in the maturation process between iPS-CMs and native CMs, which may be explained by dissimilar conditions during in vitro differentiation and in vivo development.

Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion.

PubMed

Gorin, Caroline; Rochefort, Gael Y; Bascetin, Rumeyza; Ying, Hanru; Lesieur, Julie; Sadoine, Jérémy; Beckouche, Nathan; Berndt, Sarah; Novais, Anita; Lesage, Matthieu; Hosten, Benoit; Vercellino, Laetitia; Merlet, Pascal; Le-Denmat, Dominique; Marchiol, Carmen; Letourneur, Didier; Nicoletti, Antonino; Vital, Sibylle Opsahl; Poliard, Anne; Salmon, Benjamin; Muller, Laurent; Chaussain, Catherine; Germain, Stéphane

2016-03-01

Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF. ©AlphaMed Press.

Defining adipose tissue-derived stem cells in tissue and in culture.

PubMed

Lin, Ching-Shwun; Xin, Zhong-Cheng; Deng, Chun-Hua; Ning, Hongxiu; Lin, Guiting; Lue, Tom F

2010-06-01

Adipose tissue-derived stem cells (ADSC) are routinely isolated from the stromal vascular fraction (SVF) of homogenized adipose tissue. Similar to other types of mesenchymal stem cells (MSC), ADSC remain difficult to define due to the lack of definitive cellular markers. Still, many types of MSC, including ADSC, have been shown to reside in a perivascular location, and increasing evidence shows that both MSC and ADSC may in fact be vascular stem cells (VSC). Locally, these cells differentiate into smooth muscle and endothelial cells that are assembled into newly formed blood vessels during angiogenesis and neovasculogenesis. Additionally, MSC or ADSC can also differentiate into tissue cells such as adipocytes in the adipose tissue. Systematically, MSC or ADSC are recruited to injury sites where they participate in the repair/regeneration of the injured tissue. Due to the vasculature's dynamic capacity for growth and multipotential nature for diversification, VSC in tissue are individually at various stages and on different paths of differentiation. Therefore, when isolated and put in culture, these cells are expected to be heterogeneous in marker expression, renewal capacity, and differentiation potential. Although this heterogeneity of VSC does impose difficulties and cause confusions in basic science studies, its impact on the development of VSC as a therapeutic cell source has not been as apparent, as many preclinical and clinical trials have reported favorable outcomes. With this understanding, ADSC are generally defined as CD34+CD31- although loss of CD34 expression in culture is well documented. In adipose tissue, CD34 is localized to the intima and adventitia of blood vessels but not the media where cells expressing alpha-smooth muscle actin (SMA) exist. By excluding the intima, which contains the CD34+CD31+ endothelial cells, and the media, which contains the CD34-CD31- smooth muscle cells, it leaves the adventitia as the only possible location for the CD34+ ADSC. In the capillary, CD34 and CD140b (a pericyte marker) are mutually exclusively expressed, thus suggesting that pericytes are not the CD34+ ADSC. Many other cellular markers for vascular cells, stem cells, and stem cell niche have also been investigated as possible ADSC markers. Particularly the best-known MSC marker STRO-1 has been found either expressed or not expressed in cultured ADSC. In the adipose tissue, STRO-1 appears to be expressed exclusively in the endothelium of certain but not all blood vessels, and thus not associated with the CD34+ ADSC. In conclusion, we believe that ADSC exist as CD34+CD31-CD104b-SMA- cells in the capillary and in the adventitia of larger vessels. In the capillary these cells coexist with pericytes and endothelial cells, both of which are possibly progenies of ADSC (or more precisely VSC). In the larger vessels, these ADSC or VSC exist as specialized fibroblasts (having stem cell properties) in the adventitia.

Changing Nuclear Landscape and Unique PML Structures During Early Epigenetic Transitions of Human Embryonic Stem Cells

PubMed Central

Butler, John T.; Hall, Lisa L.; Smith, Kelly P.; Lawrence, Jeanne B.

2010-01-01

The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different “nuclear landscape” in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell-type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML-defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display ~1–3 large PML structures of two morphological types: long linear “rods” or elaborate “rosettes”, which lack substantial SUMO-1, Daxx, and Sp100.These occur primarily between Day 0–2 of differentiation and become rare thereafter. PML rods may be “taut” between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a “gap” in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML-defined structures. PMID:19449340

Differential marker expression by cultures rich in mesenchymal stem cells

PubMed Central

2013-01-01

Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

Interactions of Human Endothelial and Multipotent Mesenchymal Stem Cells in Cocultures

PubMed Central

Ern, Christina; Krump-Konvalinkova, Vera; Docheva, Denitsa; Schindler, Stefanie; Rossmann, Oliver; Böcker, Wolfgang; Mutschler, Wolf; Schieker, Matthias

2010-01-01

Current strategies for tissue engineering of bone rely on the implantation of scaffolds, colonized with human mesenchymal stem cells (hMSC), into a recipient. A major limitation is the lack of blood vessels. One approach to enhance the scaffold vascularisation is to supply the scaffolds with endothelial cells (EC). The main goal of this study was to establish a coculture system of hMSC and EC for the purposes of bone tissue engineering. Therefore, the cell behaviour, proliferation and differentiation capacity in various cell culture media as well as cell interactions in the cocultures were evaluated. The differentiation capacity of hMSC along osteogenic, chondrogenic, and adipogenic lineage was impaired in EC medium while in a mixed EC and hMSC media, hMSC maintained osteogenic differentiation. In order to identify and trace EC in the cocultures, EC were transduced with eGFP. Using time-lapse imaging, we observed that hMSC and EC actively migrated towards cells of their own type and formed separate clusters in long term cocultures. The scarcity of hMSC and EC contacts in the cocultures suggest the influence of growth factor-mediated cell interactions and points to the necessity of further optimization of the coculture conditions. PMID:21625373