Chick stem cells: Current progress and future prospects

PubMed Central

Intarapat, Sittipon; Stern, Claudio D.

2013-01-01

Chick embryonic stem cells (cESCs) can be derived from cells obtained from stage X embryos (blastoderm stage); these have the ability to contribute to all somatic lineages in chimaeras, but not to the germ line. However, lines of stem cells that are able to contribute to the germ line can be established from chick primordial germ cells (cPGCs) and embryonic germ cells (cEGCs). This review provides information on avian stem cells, emphasizing different sources of cells and current methods for derivation and culture of pluripotent cells from chick embryos. We also review technologies for isolation and derivation of chicken germ cells and the production of transgenic birds. PMID:24103496

Patently controversial: markets, morals, and the President's proposal for embryonic stem cell research.

PubMed

Fins, Joseph J; Schachter, Madeleine

2002-09-01

This essay considers the implications of President George W. Bush's proposal for human embryonic stem cell research. Through the perspective of patent law, privacy, and informed consent, we elucidate the ongoing controversy about the moral standing of human embryonic stem cells and their derivatives and consider how the inconsistencies in the president's proposal will affect clinical practice and research.

Modeling TSC and LAM Using Patient Derived Induced Pluripotent Stem Cells

DTIC Science & Technology

2016-10-01

lentiviral knockdown, and CRISPR /Cas9 genome editing in embryonic stem cells (ESCs). We have characterized the iPSCs extensively and found that they display...induced pluripotent stem cells (iPSCs) embryonic stem cells (ESCs) reprogramming CRISPR /Cas9 genome editing neural stem cells (NSCs) neural crest... CRISPR /cas9 in two additional human pluripotent stem cell lines (WA07 (H7) – female cell line registry #0061; and a control male iPSC lines generated

Human embryonic stem cell lines: socio-legal concerns and therapeutic promise.

PubMed

McLaren, Anne

2002-10-01

Stem cell lines would be very valuable for the repair of diseased or damaged organs. Stem cells derived from adult tissues raise few ethical problems, and would not be rejected if derived from the patient. They show considerable plasticity and might be appropriate for some clinical conditions, but they tend not to grow well in culture. Stem cells derived from the early human embryo proliferate indefinitely in culture and can give rise to many different tissues, but their derivation requires destruction of the embryo, which is not ethically acceptable in some countries. Other countries allow strictly regulated destructive research on human embryos, usually those that have been produced for infertile couples in infertility clinics. Embryos that are no longer required for the couple's own reproductive project could be donated for research rather than just discarded. Different approaches are being developed to avoid immunological rejection of embryonic stem cells used for therapy. Derivation of embryonic stem cell lines by somatic cell nuclear transfer ('cloning') from the patients themselves might be one possible approach, but is unlikely to be used in routine clinical practice if more cost-effective methods are available.

Parthenogenesis-derived Multipotent Stem Cells Adapted for Tissue Engineering Applications

PubMed Central

Koh, Chester J.; Delo, Dawn M.; Lee, Jang Won; Siddiqui, M. Minhaj; Lanza, Robert P.; Soker, Shay; Yoo, James J.; Atala, Anthony

2009-01-01

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications. PMID:18799133

[Embryonic stem cells and therapeutic cloning].

PubMed

Sunde, A; Eftedal, I

2001-08-30

Increased interest in the therapeutic use of human stem cells has emerged following significant progress in ongoing research. The cloning of a sheep, the isolation of human embryonic stem cells, and the discovery that adult stem cells may be reprogrammed taken together give substance to hopes that novel principles of treatment may be developed for a variety of serious conditions. Embryonic stem cells are derived from pre-embryos at the blastocyst stage and may give rise to all bodily tissues and cells. Animal models have demonstrated that embryonic stem cells when transplanted into adult hosts may differentiate and develop into cells and tissues applicable for treatment of a variety of conditions, including Parkinson's disease, multiple sclerosis, spinal injuries, cardiac stroke and cancer. Transplanted embryonic stem cells are exposed to immune reactions similar to those acting on organ transplants, hence immunosuppression of the recipient is generally required. It is, however, possible to obtain embryonic stem cells that are genetically identical to the patient's own cells by means of therapeutic cloning techniques. The nucleus from a somatic cell is transferred into an egg after removal of the egg's own genetic material. Under specific condition the egg will use genetic information from the somatic cell in organising the formation of a blastocyst which in turn generates embryonic stem cells. These cells have a genetic composition identical to that of the patient and are suitable for stem cell therapy.

[Cell therapy for Parkinson's disease: III. Neonatal, fetal and embryonic stem cell-based applications].

PubMed

Anisimov, S V

2009-01-01

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Shrink-film configurable multiscale wrinkles for functional alignment of human embryonic stem cells and their cardiac derivatives.

PubMed

Chen, Aaron; Lieu, Deborah K; Freschauf, Lauren; Lew, Valerie; Sharma, Himanshu; Wang, Jiaxian; Nguyen, Diep; Karakikes, Ioannis; Hajjar, Roger J; Gopinathan, Ajay; Botvinick, Elliot; Fowlkes, Charless C; Li, Ronald A; Khine, Michelle

2011-12-22

A biomimetic substrate for cell-culture is fabricated by plasma treatment of a prestressed thermoplastic shrink film to create tunable multiscaled alignment "wrinkles". Using this substrate, the functional alignment of human embryonic stem cell derived cardiomyocytes is demonstrated. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis.

PubMed

Lee, Seung Tae; Choi, Mun Hwan; Lee, Eun Ju; Gong, Seung Pyo; Jang, Mi; Park, Sang Hyun; Jee, Hyang; Kim, Dae Yong; Han, Jae Yong; Lim, Jeong Mook

2008-11-01

To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. Prospective model study. Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. F1 hybrid B6D2F1 mice. Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. Preimplantation development and stem cell characterization. More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.

Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

EPA Science Inventory

Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

PubMed

Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

2006-07-01

Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

PubMed

Murdoch, Alison; Braude, Peter; Courtney, Aidan; Brison, Daniel; Hunt, Charles; Lawford-Davies, James; Moore, Harry; Stacey, Glyn; Sethe, Sebastian

2012-03-01

The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.

Human embryonic stem cells and therapeutic cloning.

PubMed

Hwang, Woo Suk; Lee, Byeong Chun; Lee, Chang Kyu; Kang, Sung Keun

2005-06-01

The remarkable potential of embryonic stem (ES) cells is their ability to develop into many different cell types. ES cells make it possible to treat patients by transplanting specialized healthy cells derived from them to repair damaged and diseased cells or tissues, known as "stem cell therapy". However, the issue of immunocompatibility is one of considerable significance in ES cell transplantation. One approach to overcome transplant rejection of human ES (hES) cells is to derive hES cells from nuclear transfer of the patient's own cells. This concept is known as "therapeutic cloning". In this review, we describe the derivations of ES cells and cloned ES cells by somatic cell nuclear transfer, and their potential applications in transplantation medicine.

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells.

PubMed

Shi, Cheng; Shen, Huan; Jiang, Wei; Song, Zhi-Hua; Wang, Cheng-Yan; Wei, Li-Hui

2011-04-01

Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability. Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells. We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line. This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.

Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017.

PubMed

Akyash, Fatemeh; Sadeghian-Nodoushan, Fatemeh; Tahajjodi, Somayyeh Sadat; Nikukar, Habib; Farashahi Yazd, Ehsan; Azimzadeh, Mostafa; D Moore, Harry; Aflatoonian, Behrouz

2017-05-01

This report explains briefly the minutes of a 1-day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27 th April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.

[Embryonic stem cells - a scientific by-product of the assisted reproduction technology?].

PubMed

Sterthaus, Oliver; Zhang, Hong; De Geyter, Christian

2009-12-01

The differentiation potential of embryonic stem (ES) cells seems to be higher when compared to adult stem cells, which mainly differentiate into certain tissue types only. ES cells have the potential to play an important role in regenerative medicine as demonstrated with murine ES cells. However, with human embryonic stem cells (hESC) several obstacles still have to be overcome, when these are to be used in clinical applications. The expansion of hESC, safety issues as well as the immune-tolerance after transplantation are all problems that still have to be solved. Since 2005 the derivation of hESC lines from super-numerous embryos has become permitted in Switzerland, albeit under strictly restrictive guidelines. In 2008 the Basler hESC laboratory was successful in derivating the first hESC line with a normal chromosome complement in Switzerland (CHES2). Now, new applications allow the personalized establishment of immune-tolerant stem cells, which lead to the replacement of therapeutic cloning by induced pluripotent stem cells (iPS).

Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

2017-03-09

Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

Recent Advances towards the Clinical Application of Stem Cells for Retinal Regeneration

PubMed Central

Becker, Silke; Jayaram, Hari; Limb, G. Astrid

2012-01-01

Retinal degenerative diseases constitute a major cause of irreversible blindness in the world. Stem cell-based therapies offer hope for these patients at risk of or suffering from blindness due to the deterioration of the neural retina. Various sources of stem cells are currently being investigated, ranging from human embryonic stem cells to adult-derived induced pluripotent stem cells as well as human Müller stem cells, with the first clinical trials to investigate the safety and tolerability of human embryonic stem cell-derived retinal pigment epithelium cells having recently commenced. This review aims to summarize the latest advances in the development of stem cell strategies for the replacement of retinal neurons and their supportive cells, the retinal pigment epithelium (RPE) affected by retinal degenerative conditions. Particular emphasis will be given to the advances in stem cell transplantation and the challenges associated with their translation into clinical practice. PMID:24710533

Pluripotent stem cells and reprogrammed cells in farm animals.

PubMed

Nowak-Imialek, Monika; Kues, Wilfried; Carnwath, Joseph W; Niemann, Heiner

2011-08-01

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

PubMed

Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

2015-09-01

Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. Copyright © 2015. Published by Elsevier B.V.

Stem cell potency and the ability to contribute to chimeric organisms.

PubMed

Polejaeva, Irina; Mitalipov, Shoukhrat

2013-03-01

Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining two or more preimplantation embryos or by introducing into host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for the generation of knockout or knockin mice. Although the derivation of ESCs or ESC-like cells has been reported for other species, only mouse and rat pluripotent stem cells have been shown to contribute to germline-competent chimeras, which is the defining feature of ESCs. Herein, we describe different approaches employed for the generation of embryonic chimeras, define chimera-competent cell types, and describe cases of spontaneous chimerism in humans. We also review the current state of derivation of pluripotent stem cells in several species and discuss outcomes of various chimera studies when such cells are used.

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro.

PubMed

Ishiwata, Isamu; Tamagawa, Tomoharu; Tokieda, Yuko; Iguchi, Megumi; Sato, Kahei; Ishikawa, Hiroshi

2003-03-01

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.

Adult bone marrow-derived stem cells for organ regeneration and repair.

PubMed

Tögel, Florian; Westenfelder, Christof

2007-12-01

Stem cells have been recognized as a potential tool for the development of innovative therapeutic strategies. There are in general two types of stem cells, embryonic and adult stem cells. While embryonic stem cell therapy has been riddled with problems of allogeneic rejection and ethical concerns, adult stem cells have long been used in the treatment of hematological malignancies. With the recognition of additional, potentially therapeutic characteristics, bone marrow-derived stem cells have become a tool in regenerative medicine. The bone marrow is an ideal source of stem cells because it is easily accessible and harbors two types of stem cells. Hematopoietic stem cells give rise to all blood cell types and have been shown to exhibit plasticity, while multipotent marrow stromal cells are the source of osteocytes, chondrocytes, and fat cells and have been shown to support and generate a large number of different cell types. This review describes the general characteristics of these stem cell populations and their current and potential future applications in regenerative medicine. 2007 Wiley-Liss, Inc

Integration of immunological aspects in the European Human Embryonic Stem Cell Registry.

PubMed

Borstlap, Joeri; Kurtz, Andreas

2008-05-01

The immunological properties of stem cells are of increasing importance in regenerative medicine. Immunomodulatory mechanisms seem to play an important role not only with respect to the understanding of underlying mechanisms of autologous versus allogenic therapeutic approaches, but also for endogeneous tissue regeneration. The newly established European human embryonic stem cell registry (hESCreg) offers an international database for the registration, documentation and characterisation of human embryonic stem cells (hESC) and their use. By doing so, hESCreg aims to develop a model procedure for further standardisation efforts in the field of stem cell research and regenerative medicine, and eventually the registry may lead to a repository of therapy-related information. Currently the stem cell characterisation data acquired by the registry are divided into several categories such as cell derivation, culture conditions, genetic constitution, stem cell marker expression and degree of modification. This article describes immunological aspects of stem cell characterisation and explores the layout and relevance of a possible additional section to the hESCreg repository to include immunological characteristics of human embryonic stem cells.

In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells.

PubMed

Ishikura, Yukiko; Yabuta, Yukihiro; Ohta, Hiroshi; Hayashi, Katsuhiko; Nakamura, Tomonori; Okamoto, Ikuhiro; Yamamoto, Takuya; Kurimoto, Kazuki; Shirane, Kenjiro; Sasaki, Hiroyuki; Saitou, Mitinori

2016-12-06

The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Effect of allo- and xenotransplantation of embryonic nervous tissue and umbilical cord blood-derived stem cells on structural and functional state of cerebral cortex of albino rats in posttraumatic period.

PubMed

Ereniev, S I; Semchenko, V V; Sysheva, E V; Bogdashin, I V; Shapovalova, V V; Khizhnyak, A S; Gasanenko, L N

2005-11-01

Comparative study of the structural and functional state of cerebral cortex of adult albino rats after intracerebral allo- and xenotransplantation of embryonic nervous tissue and intravenous injection of umbilical cord blood-derived stem cells at different terms after diffuse-focal cerebral trauma revealed the best cerebroprotective effect on day 7 of posttraumatic period in animals receiving embryonic nervous tissue.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

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

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth ofmore » undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.« less

Stem cell research in cell transplantation: sources, geopolitical influence, and transplantation.

PubMed

Eve, David J; Fillmore, Randolph W; Borlongan, Cesar V; Sanberg, Paul R

2010-01-01

If the rapidly progressing field of stem cell research reaches its full potential, successful treatments and enhanced understanding of many diseases are the likely results. However, the full potential of stem cell science will only be reached if all possible avenues can be explored and on a worldwide scale. Until 2009, the US had a highly restrictive policy on obtaining cells from human embryos and fetal tissue, a policy that pushed research toward the use of adult-derived cells. Currently, US policy is still in flux, and retrospective analysis does show the US lagging behind the rest of the world in the proportional increase in embryonic/fetal stem cell research. The majority of US studies being on either a limited number of cell lines, or on cells derived elsewhere (or funded by other sources than Federal) rather than on freshly isolated embryonic or fetal material. Neural, mesenchymal, and the mixed stem cell mononuclear fraction are the most commonly investigated types, which can generally be classified as adult-derived stem cells, although roughly half of the neural stem cells are fetal derived. Other types, such as embryonic and fat-derived stem cells, are increasing in their prominence, suggesting that new types of stem cells are still being pursued. Sixty percent of the reported stem cell studies involved transplantation, of which over three quarters were allogeneic transplants. A high proportion of the cardiovascular systems articles were on allogeneic transplants in a number of different species, including several autologous studies. A number of pharmaceutical grade stem cell products have also recently been tested and reported on. Stem cell research shows considerable promise for the treatment of a number of disorders, some of which have entered clinical trials; over the next few years it will be interesting to see how these treatments progress in the clinic.

'New embryos' - new challenges for the ethics of stem cell research.

PubMed

Holm, Søren

2008-01-01

Among the many ethical issues raised by human embryonic stem cell research (in the following all references to 'stem cells' should be read as references to human embryonic stem cells), two have gained specific prominence: (1) whether stem cell research is ethically problematic because it entails the destruction of human embryos and (2) what kind of control embryo donors should have over the stem cell lines derived from their embryos. In the present paper, I will analyse how these two issues are engaged by various attempts to derive stem cells from anomalous embryos (e.g. embryos in cleavage arrest, embryos not implanted following pre-implantation genetic diagnosis or embryos created by altered nuclear transfer) or in ways that are claimed to be non-destructive for the embryo (e.g. blastocyst or blastomere biopsy). Copyright 2008 S. Karger AG, Basel.

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

PubMed

Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

2016-06-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552. © 2016 AlphaMed Press.

Cell differentiation: therapeutical challenges in diabetes.

PubMed

Roche, Enrique; Vicente-Salar, Nestor; Arribas, Maribel; Paredes, Beatriz

2012-01-01

Stem cells, derived from either embryonic or adult tissues, are considered to be potential sources of insulin-secreting cells to be transplanted into type 1 and advanced stages of type 2 diabetic patients. Many laboratories have considered this possibility, resulting in a large amount of published protocols, with a wide degree of complexity among them. Our group was the first to report that it was possible to obtain insulin-secreting cells from mouse embryonic stem cells, proving the feasibility of this new challenge. The same observation was immediately reported using human embryonic stem cells. However, the resulting cell product was not properly characterised, affecting the reproducibility of the protocol by other groups. A more elaborated protocol was developed by Lumelsky and co-workers, demonstrating that neuroectodermal cells could be an alternative source for insulin-producing cells. However, the resulting cells of this protocol produced low amounts of the hormone. This aimed other groups to perform key changes in order to improve the insulin content of the resulting cells. Recently, Baetge's group has published a new protocol based on the knowledge accumulated in pancreatic development. In this protocol, human embryonic stem cells were differentiated into islet-like structures through a five step protocol, emulating the key steps during embryonic development of the endocrine pancreas. The final cell product, however, seemed to be in an immature state, thus further improvement is required. Despite this drawback, the protocol represents the culmination of work performed by different groups and offers new research challenges for the investigators in this exciting field. Concerning adult stem cells, the possibility of identifying pancreatic precursors or of reprogramming extrapancreatic derived cells are key possibilities that may circumvent the problems that appear when using embryonic stem cells, such as immune rejection and tumour formation.

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.

PubMed

Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

2017-04-14

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos. Copyright © 2017, American Association for the Advancement of Science.

[Cell therapy for Parkinson's disease: IV. Risks and future trends].

PubMed

Anisimov, S V

2009-01-01

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells

PubMed Central

West, Michael D.; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B.; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-01

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1, encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro-derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition. PMID:29487692

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells.

PubMed

West, Michael D; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-30

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1 , encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro -derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition.

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

PubMed Central

Sehgal, Poonam; Chaturvedi, Pankaj; Kumaran, R. Ileng; Kumar, Satish; Parnaik, Veena K.

2013-01-01

Background Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. Methodology and Principal Findings We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells. Conclusions We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development. PMID:23451281

Single cell RNA sequencing of stem cell-derived retinal ganglion cells.

PubMed

Daniszewski, Maciej; Senabouth, Anne; Nguyen, Quan H; Crombie, Duncan E; Lukowski, Samuel W; Kulkarni, Tejal; Sluch, Valentin M; Jabbari, Jafar S; Chamling, Xitiz; Zack, Donald J; Pébay, Alice; Powell, Joseph E; Hewitt, Alex W

2018-02-13

We used single cell sequencing technology to characterize the transcriptomes of 1,174 human embryonic stem cell-derived retinal ganglion cells (RGCs) at the single cell level. The human embryonic stem cell line BRN3B-mCherry (A81-H7), was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells showed increased expression of genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature RGC phenotype.

Production of cloned and transgenic embryos using buffalo (Bubalus bubalis) embryonic stem cell-like cells isolated from in vitro fertilized and cloned blastocysts.

PubMed

George, Aman; Sharma, Ruchi; Singh, Karn P; Panda, Sudeepta K; Singla, Suresh K; Palta, Prabhat; Manik, Radhaysham; Chauhan, Manmohan S

2011-06-01

Here, we report the isolation and characterization of embryonic stem (ES) cell-like cells from cloned blastocysts, generated using fibroblasts derived from an adult buffalo (BAF). These nuclear transfer embryonic stem cell-like cells (NT-ES) grew in well-defined and dome-shaped colonies. The expression pattern of pluripotency marker genes was similar in both NT-ES and in vitro fertilization (IVF) embryo-derived embryonic stem cell-like cells (F-ES). Upon spontaneous differentiation via embryoid body formation, cells of different morphology were observed, among which predominant were endodermal-like and epithelial-like cell types. The ES cell-like cells could be passaged only mechanically and did not form colonies when plated as single cell suspension at different concentrations. When F-ES cell-like, NT-ES cell-like, and BAF cells of same genotype were used for hand-made cloning (HMC), no significant difference (p > 0.05) was observed in cleavage and blastocyst rate. Following transfer of HMC embryos to synchronized recipients, pregnancies were established only with F-ES cell-like and BAF cell-derived embryos, and one live calf was born from F-ES cell-like cells. Further, when transfected NT-ES cell-like cells and BAF were used for HMC, no significant difference (p > 0.05) was observed between cleavage and blastocyst rate. In conclusion, here we report for the first time the derivation of ES cell-like cells from an adult buffalo, and its genetic modification. We also report the birth of a live cloned calf from buffalo ES cell-like cells.

Skeletal tissue engineering using mesenchymal or embryonic stem cells: clinical and experimental data.

PubMed

Gamie, Zakareya; MacFarlane, Robert J; Tomkinson, Alicia; Moniakis, Alexandros; Tran, Gui Tong; Gamie, Yehya; Mantalaris, Athanasios; Tsiridis, Eleftherios

2014-11-01

Mesenchymal stem cells (MSCs) can be obtained from a wide variety of tissues for bone tissue engineering such as bone marrow, adipose, birth-associated, peripheral blood, periosteum, dental and muscle. MSCs from human fetal bone marrow and embryonic stem cells (ESCs) are also promising cell sources. In vitro, in vivo and clinical evidence was collected using MEDLINE® (1950 to January 2014), EMBASE (1980 to January 2014) and Google Scholar (1980 to January 2014) databases. Enhanced results have been found when combining bone marrow-derived mesenchymal stem cells (BMMSCs) with recently developed scaffolds such as glass ceramics and starch-based polymeric scaffolds. Preclinical studies investigating adipose tissue-derived stem cells and umbilical cord tissue-derived stem cells suggest that they are likely to become promising alternatives. Stem cells derived from periosteum and dental tissues such as the periodontal ligament have an osteogenic potential similar to BMMSCs. Stem cells from human fetal bone marrow have demonstrated superior proliferation and osteogenic differentiation than perinatal and postnatal tissues. Despite ethical concerns and potential for teratoma formation, developments have also been made for the use of ESCs in terms of culture and ideal scaffold.

Cryopreservation of Human Stem Cells for Clinical Application: A Review

PubMed Central

Hunt, Charles J.

2011-01-01

Summary Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell. PMID:21566712

Cryopreservation of Human Stem Cells for Clinical Application: A Review.

PubMed

Hunt, Charles J

2011-01-01

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

PubMed

Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

2012-05-01

In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

Review article: stem cells in human reproduction.

PubMed

Gargett, Caroline E

2007-07-01

The derivation of human embryonic stem (hES) cells heralds a new era in stem cell research, generating excitement for their therapeutic potential in regenerative medicine. Pioneering work of embryologists, developmental biologists, and reproductive medicine practitioners in in vitro fertilization clinics has facilitated hES cell research. This review summarizes current research focused on optimizing hES cell culture conditions for good manufacturing practice, directing hES cell differentiation toward trophectoderm and germ cells, and approaches used to reprogram cells for pluripotent cell derivation. The identification of germ stem cells in the testis and the recent controversy over their existence in the ovary raise the possibility of harnessing them for treating young cancer survivors. There is also the potential to harvest fetal stem cells with pluripotent cell-like properties from discarded placental tissues. The recent identification of adult stem/progenitor cell activity in the human endometrium offers a new understanding of common gynecological diseases. Discoveries resulting from research into embryonic, germ, fetal, and adult stem cells are highly relevant to human reproduction.

Stem cell sources for clinical islet transplantation in type 1 diabetes: embryonic and adult stem cells.

PubMed

Miszta-Lane, Helena; Mirbolooki, Mohammadreza; James Shapiro, A M; Lakey, Jonathan R T

2006-01-01

Lifelong immunosuppressive therapy and inadequate sources of transplantable islets have led the islet transplantation benefits to less than 0.5% of type 1 diabetics. Whereas the potential risk of infection by animal endogenous viruses limits the uses of islet xeno-transplantation, deriving islets from stem cells seems to be able to overcome the current problems of islet shortages and immune compatibility. Both embryonic (derived from the inner cell mass of blastocysts) and adult stem cells (derived from adult tissues) have shown controversial results in secreting insulin in vitro and normalizing hyperglycemia in vivo. ESCs research is thought to have much greater developmental potential than adult stem cells; however it is still in the basic research phase. Existing ESC lines are not believed to be identical or ideal for generating islets or beta-cells and additional ESC lines have to be established. Research with ESCs derived from humans is controversial because it requires the destruction of a human embryo and/or therapeutic cloning, which some believe is a slippery slope to reproductive cloning. On the other hand, adult stem cells are already in some degree specialized, recipients may receive their own stem cells. They are flexible but they have shown mixed degree of availability. Adult stem cells are not pluripotent. They may not exist for all organs. They are difficult to purify and they cannot be maintained well outside the body. In order to draw the future avenues in this field, existent discrepancies between the results need to be clarified. In this study, we will review the different aspects and challenges of using embryonic or adult stem cells in clinical islet transplantation for the treatment of type 1 diabetes.

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

PubMed

Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Stem cells: sources and applications.

PubMed

Vats, A; Tolley, N S; Polak, J M; Buttery, L D K

2002-08-01

Tissue engineering is a multidisciplinary area of research aimed at regeneration of tissues and restoration of function of organs through implantation of cells/tissues grown outside the body, or stimulating cells to grow into implanted matrix. In this short review, some of the most recent developments in the use of stem cells for tissue repair and regeneration will be discussed. There is no doubt that stem cells derived from adult and embryonic sources hold great therapeutic potential but it is clear that there is still much research required before their use is commonplace. There is much debate over adult versus embryonic stem cells and whether both are required. It is probably too early to disregard one or other of these cell sources. With regard to embryonic stem cells, the major concern relates to the ethics of their creation and the proposed practice of therapeutic cloning.

Concentration dependent survival and neural differentiation of murine embryonic stem cells cultured on polyethylene glycol dimethacrylate hydrogels possessing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle.

PubMed

Lim, Hyun Ju; Mosley, Matthew C; Kurosu, Yuki; Smith Callahan, Laura A

2017-07-01

N-cadherin cell-cell signaling plays a key role in the structure and function of the nervous system. However, few studies have incorporated bioactive signaling from n-cadherin into tissue engineering matrices. The present study uses a continuous gradient approach in polyethylene glycol dimethacrylate hydrogels to identify concentration dependent effects of n-cadherin peptide, His-Ala-Val-Asp-Lle (HAVDI), on murine embryonic stem cell survival and neural differentiation. The n-cadherin peptide was found to affect the expression of pluripotency marker, alkaline phosphatase, in murine embryonic stem cells cultured on n-cadherin peptide containing hydrogels in a concentration dependent manner. Increasing n-cadherin peptide concentrations in the hydrogels elicited a biphasic response in neurite extension length and mRNA expression of neural differentiation marker, neuron-specific class III β-tubulin, in murine embryonic stem cells cultured on the hydrogels. High concentrations of n-cadherin peptide in the hydrogels were found to increase the expression of apoptotic marker, caspase 3/7, in murine embryonic stem cells compared to that of murine embryonic stem cell cultures on hydrogels containing lower concentrations of n-cadherin peptide. Increasing the n-cadherin peptide concentration in the hydrogels facilitated greater survival of murine embryonic stem cells exposed to increasing oxidative stress caused by hydrogen peroxide exposure. The combinatorial approach presented in this work demonstrates concentration dependent effects of n-cadherin signaling on mouse embryonic stem cell behavior, underscoring the need for the greater use of systematic approaches in tissue engineering matrix design in order to understand and optimize bioactive signaling in the matrix for tissue formation. Single cell encapsulation is common in tissue engineering matrices. This eliminates cellular access to cell-cell signaling. N-cadherin, a cell-cell signaling molecule, plays a vital role in the development of neural tissues, but has not been well studied as a bioactive signaling element in neural tissue engineering matrices. The present study uses a systematic continuous gradient approach to identify concentration dependent effects of n-cadherin derived peptide, HAVDI, on the survival and neural differentiation of murine embryonic stem cells. This work underscores the need for greater use to combinatorial strategies to understand the effect complex bioactive signaling, such as n-cadherin, and the need to optimize the concentration of such bioactive signaling within tissue engineering matrices for maximal cellular response. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Multipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System

PubMed Central

Hasegawa, Kouichi; Menheniott, Trevelyan; Rollo, Ben; Zhang, Dongcheng; Hough, Shelley; Alshawaf, Abdullah; Febbraro, Fabia; Ighaniyan, Samiramis; Leung, Jessie; Elliott, David A.; Newgreen, Donald F.; Pera, Martin F.

2015-01-01

Abstract The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named “caudal neural progenitors” (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube. Stem Cells 2015;33:1759–1770 PMID:25753817

Reprogrammed mouse astrocytes retain a "memory" of tissue origin and possess more tendencies for neuronal differentiation than reprogrammed mouse embryonic fibroblasts.

PubMed

Tian, Changhai; Wang, Yongxiang; Sun, Lijun; Ma, Kangmu; Zheng, Jialin C

2011-02-01

Direct reprogramming of a variety of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2 with either Klf4 and Myc or Lin28 and Nanog generates the induced pluripotent stem cells (iPSCs) with marker similarity to embryonic stem cells. However, the difference between iPSCs derived from different origins is unclear. In this study, we hypothesized that reprogrammed cells retain a "memory" of their origins and possess additional potential of related tissue differentiation. We reprogrammed primary mouse astrocytes via ectopic retroviral expression of OCT3/4, Sox2, Klf4 and Myc and found the iPSCs from mouse astrocytes expressed stem cell markers and formed teratomas in SCID mice containing derivatives of all three germ layers similar to mouse embryonic stem cells besides semblable morphologies. To test our hypothesis, we compared embryonic bodies (EBs) formation and neuronal differentiation between iPSCs from mouse embryonic fibroblasts (MEFsiPSCs) and iPSCs from mouse astrocytes (mAsiPSCs). We found that mAsiPSCs grew slower and possessed more potential for neuronal differentiation compared to MEFsiPSCs. Our results suggest that mAsiPSCs retain a "memory" of the central nervous system, which confers additional potential upon neuronal differentiation.

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

PubMed

Sternberg, Hal; Kidd, Jennifer; Murai, James T; Jiang, Jianjie; Rinon, Ariel; Erickson, Isaac E; Funk, Walter D; Wang, Qian; Chapman, Karen B; Vangsness, C Thomas; West, Michael D

2013-03-01

The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

PubMed

Morizane, Ryuji; Monkawa, Toshiaki; Fujii, Shizuka; Yamaguchi, Shintaro; Homma, Koichiro; Matsuzaki, Yumi; Okano, Hideyuki; Itoh, Hiroshi

2014-01-01

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

Human induced pluripotent stem cells: a review of the US patent landscape.

PubMed

Georgieva, Bilyana P; Love, Jane M

2010-07-01

Human induced pluripotent stem (iPS) cells and human embryonic stem cells are cells that have the ability to differentiate into a variety of cell types. Embryonic stem cells are derived from human embryos; however, by contrast, human iPS cells can be obtained from somatic cells that have undergone a process of 'reprogramming' via genetic manipulation such that they develop pluripotency. Since iPS cells are not derived from human embryos, they are a less complicated source of human pluripotent cells and are considered valuable research tools and potentially useful in therapeutic applications in regenerative medicine. Worldwide, there are only three issued patents concerning iPS cells. Therefore, the patent landscape in this field is largely undefined. This article provides an overview of the issued patents as well as the pending published patent applications in the field.

Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation.

PubMed

Kress, C; Vandormael-Pournin, S; Baldacci, P; Cohen-Tannoudji, M; Babinet, C

1998-12-01

The inbred mouse strain DDK carries a conditional early embryonic lethal mutation that is manifested when DDK females are crossed to males of other inbred strains but not in the corresponding reciprocal crosses. It has been shown that embryonic lethality could be assigned to a single genetic locus called Ovum mutant (Om), on Chromosome (Chr) 11 near Syca 1. In the course of our study of the molecular mechanisms underlying the embryonic lethality, we were interested in deriving an embryonic stem cell bearing the Om mutation in the homozygous state (Omd/Omd). However, it turned out that DDK is nonpermissive for ES cell establishment, with a standard protocol. Here we show that permissiveness could be obtained using Omd/Omd blastocysts with a 75% 129/Sv and 25% DDK genetic background. Several germline-competent Omd/Omd ES cell lines have been derived from blastocysts of this genotype. Such a scenario could be extended to the generation of ES cell lines bearing any mutation present in an otherwise nonpermissive mouse strain.

Human embryonic stem cell research: an intercultural perspective.

PubMed

Walters, LeRoy

2004-03-01

In 1998, researchers discovered that embryonic stem cells could be derived from early human embryos. This discovery has raised a series of ethical and public-policy questions that are now being confronted by multiple international organizations, nations, cultures, and religious traditions. This essay surveys policies for human embryonic stem cell research in four regions of the world, reports on the recent debate at the United Nations about one type of such research, and reviews the positions that various religious traditions have adopted regarding this novel type of research. In several instances the religious traditions seem to have influenced the public-policy debates.

Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent.

PubMed

Rao, Yang; Cui, Jihong; Yin, Lu; Liu, Wei; Liu, Wenguang; Sun, Mei; Yan, Xingrong; Wang, Ling; Chen, Fulin

2016-10-22

Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

Embryonic Stem Cell-Derived Mesenchymal Stem Cells (MSCs) Have a Superior Neuroprotective Capacity Over Fetal MSCs in the Hypoxic-Ischemic Mouse Brain.

PubMed

Hawkins, Kate E; Corcelli, Michelangelo; Dowding, Kate; Ranzoni, Anna M; Vlahova, Filipa; Hau, Kwan-Leong; Hunjan, Avina; Peebles, Donald; Gressens, Pierre; Hagberg, Henrik; de Coppi, Paolo; Hristova, Mariya; Guillot, Pascale V

2018-05-01

Human mesenchymal stem cells (MSCs) have huge potential for regenerative medicine. In particular, the use of pluripotent stem cell-derived mesenchymal stem cells (PSC-MSCs) overcomes the hurdle of replicative senescence associated with the in vitro expansion of primary cells and has increased therapeutic benefits in comparison to the use of various adult sources of MSCs in a wide range of animal disease models. On the other hand, fetal MSCs exhibit faster growth kinetics and possess longer telomeres and a wider differentiation potential than adult MSCs. Here, for the first time, we compare the therapeutic potential of PSC-MSCs (ES-MSCs from embryonic stem cells) to fetal MSCs (AF-MSCs from the amniotic fluid), demonstrating that ES-MSCs have a superior neuroprotective potential over AF-MSCs in the mouse brain following hypoxia-ischemia. Further, we demonstrate that nuclear factor (NF)-κB-stimulated interleukin (IL)-13 production contributes to an increased in vitro anti-inflammatory potential of ES-MSC-conditioned medium (CM) over AF-MSC-CM, thus suggesting a potential mechanism for this observation. Moreover, we show that induced pluripotent stem cell-derived MSCs (iMSCs) exhibit many similarities to ES-MSCs, including enhanced NF-κB signaling and IL-13 production in comparison to AF-MSCs. Future studies should assess whether iMSCs also exhibit similar neuroprotective potential to ES-MSCs, thus presenting a potential strategy to overcome the ethical issues associated with the use of embryonic stem cells and providing a potential source of cells for autologous use against neonatal hypoxic-ischemic encephalopathy in humans. Stem Cells Translational Medicine 2018;7:439-449. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Present state and future perspectives of using pluripotent stem cells in toxicology research

PubMed Central

Löser, Peter

2011-01-01

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed. PMID:21225242

Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone disease.

PubMed

Christou, Y A; Moore, H D; Shaw, P J; Monk, P N

2007-10-01

Human embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to valuable insights into the principles of developmental and cell biology and to the proposed use of human embryonic stem cells or their differentiated progeny in regenerative medicine. This review focuses on the prospects for the use of embryonic stem cells in cell-based therapy for motor neurone disease or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that specifically affects upper and lower motor neurones and leads ultimately to death from respiratory failure. Stem cell-derived motor neurones could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurones, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro by embryonic stem cells. Here we discuss the need for new therapeutic strategies in the treatment of motor neurone disease, the developmental processes that result in motor neurone formation in vivo, a number of experimental approaches to motor neurone production in vitro and recent progress in the application of stem cells to the treatment and understanding of motor neurone disease.

Survival of priceless cells: active and passive protection of embryonic stem cells against immune destruction.

PubMed

Utermöhlen, Olaf; Krönke, Martin

2007-06-15

This review focuses on our current knowledge of the mechanisms employed by embryonic stem (ES) cells to avoid destruction by cell-mediated immune responses. Recently, ES cells have been found to shield themselves against cytotoxic effector cells by expressing CD95L and serine protease inhibitor SPI-6 mediating apoptosis of the cytotoxic cells and inactivation of granzyme B, respectively. These findings are discussed in view of their implications for using ES cell-derived transplants in regenerative medicine as well as for our understanding of early embryonic stages during invasion and implantation.

Gene screening of Wharton's jelly derived stem cells.

PubMed

Mechiche Alami, S; Velard, F; Draux, F; Siu Paredes, F; Josse, J; Lemaire, F; Gangloff, S C; Graesslin, O; Laurent-Maquin, D; Kerdjoudj, H

2014-01-01

Stem cells are the most powerful candidate for the treatment of various diseases. Suitable stem cell source should be harvested with minimal invasive procedure, found in great quantity, and transplanted with no risk of immune response and tumor formation. Fetal derived stem cells have been introduced as an excellent alternative to adult and embryonic stem cells use, but unfortunately, their degree of "stemness" and molecular characterization is still unclear. Several studies have been performed deciphering whether fetal stem cells meet the needs of regenerative medicine. We believe that a transcriptomic screening of Wharton's jelly stem cells will bring insights on cell population features.

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

Blastocyst-like structures generated solely from stem cells.

PubMed

Rivron, Nicolas C; Frias-Aldeguer, Javier; Vrij, Erik J; Boisset, Jean-Charles; Korving, Jeroen; Vivié, Judith; Truckenmüller, Roman K; van Oudenaarden, Alexander; van Blitterswijk, Clemens A; Geijsen, Niels

2018-05-01

The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells 1 . From mouse blastocysts, it is possible to derive both trophoblast 2 and embryonic stem-cell lines 3 , which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

PubMed

Weber, Marlen; Apostolova, Galina; Widera, Darius; Mittelbronn, Michel; Dechant, Georg; Kaltschmidt, Barbara; Rohrer, Hermann

2015-02-01

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage. © 2014 AlphaMed Press.

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Cell Therapy From Bench to Bedside Translation in CNS Neurorestoratology Era

PubMed Central

Huang, Hongyun; Chen, Lin; Sanberg, Paul

2010-01-01

Recent advances in cell biology, neural injury and repair, and the progress towards development of neurorestorative interventions are the basis for increased optimism. Based on the complexity of the processes of demyelination and remyelination, degeneration and regeneration, damage and repair, functional loss and recovery, it would be expected that effective therapeutic approaches will require a combination of strategies encompassing neuroplasticity, immunomodulation, neuroprotection, neurorepair, neuroreplacement, and neuromodulation. Cell-based restorative treatment has become a new trend, and increasing data worldwide have strongly proven that it has a pivotal therapeutic value in CNS disease. Moreover, functional neurorestoration has been achieved to a certain extent in the CNS clinically. Up to now, the cells successfully used in preclinical experiments and/or clinical trial/treatment include fetal/embryonic brain and spinal cord tissue, stem cells (embryonic stem cells, neural stem/progenitor cells, hematopoietic stem cells, adipose-derived adult stem/precursor cells, skin-derived precursor, induced pluripotent stem cells), glial cells (Schwann cells, oligodendrocyte, olfactory ensheathing cells, astrocytes, microglia, tanycytes), neuronal cells (various phenotypic neurons and Purkinje cells), mesenchymal stromal cells originating from bone marrow, umbilical cord, and umbilical cord blood, epithelial cells derived from the layer of retina and amnion, menstrual blood-derived stem cells, Sertoli cells, and active macrophages, etc. Proof-of-concept indicates that we have now entered a new era in neurorestoratology. PMID:21359168

Productive vs non-productive infection by cell-free Varicella zoster virus of human neurons derived from embryonic stem cells is dependent upon infectious viral dose

PubMed Central

Sloutskin, Anna; Kinchington, Paul R.; Goldstein, Ronald S.

2013-01-01

Varicella Zoster virus (VZV) productively infects humans causing varicella upon primary infection and herpes zoster upon reactivation from latency in neurons. In-vitro studies using cell-associated VZV infection have demonstrated productive VZV-infection, while a few recent studies of human neurons derived from stem cells incubated with cell-free, vaccine-derived VZV did not result in generation of infectious virus. In the present study, 90%-pure human embryonic stem cell-derived neurons were incubated with recombinant cell-free pOka-derived made with an improved method or with VZV vaccine. We found that cell-free pOka and vOka at higher multiplicities of infection elicited productive infection in neurons followed by spread of infection, cytopathic effect and release of infectious virus into the medium. These results further validate the use of this unlimited source of human neurons for studying unexplored aspects of VZV interaction with neurons such as entry, latency and reactivation. PMID:23769240

Adipose-derived mesenchymal stem cells and regenerative medicine.

PubMed

Konno, Masamitsu; Hamabe, Atsushi; Hasegawa, Shinichiro; Ogawa, Hisataka; Fukusumi, Takahito; Nishikawa, Shimpei; Ohta, Katsuya; Kano, Yoshihiro; Ozaki, Miyuki; Noguchi, Yuko; Sakai, Daisuke; Kudoh, Toshihiro; Kawamoto, Koichi; Eguchi, Hidetoshi; Satoh, Taroh; Tanemura, Masahiro; Nagano, Hiroaki; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

2013-04-01

Adipose tissue-derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β-cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos

PubMed Central

Chen, Chien-Hong; Li, Yi; Hu, Yeshu; An, Li-You; Yang, Lan; Zhang, Jifeng; Chen, Y. Eugene

2015-01-01

Abstract The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model. PMID:26579970

Use of “MGE Enhancers” for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons

PubMed Central

Chen, Ying-Jiun J.; Vogt, Daniel; Wang, Yanling; Visel, Axel; Silberberg, Shanni N.; Nicholas, Cory R.; Danjo, Teruko; Pollack, Joshua L.; Pennacchio, Len A.; Anderson, Stewart; Sasai, Yoshiki; Baraban, Scott C.; Kriegstein, Arnold R.; Alvarez-Buylla, Arturo; Rubenstein, John L. R.

2013-01-01

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6+ cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6+ cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP+ cells, while enhancer 1056 is active in Olig2+ cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments. PMID:23658702

Towards a global human embryonic stem cell bank.

PubMed

Lott, Jason P; Savulescu, Julian

2007-08-01

An increasingly unbridgeable gap exists between the supply and demand of transplantable organs. Human embryonic stem cell technology could solve the organ shortage problem by restoring diseased or damaged tissue across a range of common conditions. However, such technology faces several largely ignored immunological challenges in delivering cell lines to large populations. We address some of these challenges and argue in favor of encouraging contribution or intentional creation of embryos from which widely immunocompatible stem cell lines could be derived. Further, we argue that current immunological constraints in tissue transplantation demand the creation of a global stem cell bank, which may hold particular promise for minority populations and other sub-groups currently marginalized from organ procurement and allocation systems. Finally, we conclude by offering a number of practical and ethically oriented recommendations for constructing a human embryonic stem cell bank that we hope will help solve the ongoing organ shortage problem.

Current status of stem cells in cardiac repair.

PubMed

Henning, Robert J

2018-03-01

One out of every two men and one out of every three women greater than the age of 40 will experience an acute myocardial infarction (AMI) at some time during their lifetime. As more patients survive their AMIs, the incidence of congestive heart failure (CHF) is increasing. 6 million people in the USA have ischemic cardiomyopathies and CHF. The search for new and innovative treatments for patients with AMI and CHF has led to investigations and use of human embryonic stem cells, cardiac stem/progenitor cells, bone marrow-derived mononuclear cells and mesenchymal stem cells for treatment of these heart conditions. This paper reviews current investigations with human embryonic, cardiac, bone marrow and mesenchymal stem cells, and also stem cell paracrine factors and exosomes.

Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells.

PubMed

Sovalat, Hanna; Scrofani, Maurice; Eidenschenk, Antoinette; Pasquet, Stéphanie; Rimelen, Valérie; Hénon, Philippe

2011-04-01

Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro.

PubMed

Tokumoto, Yasuhito; Ogawa, Shinichiro; Nagamune, Teruyuki; Miyake, Jun

2010-06-01

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), and defects in these cells can result in the loss of CNS functions. Although oligodendrocyte progenitor cells transplantation therapy is an effective cure for such symptoms, there is no readily available source of these cells. Recent studies have described the generation of induced pluripotent stem cells (iPS cells) from somatic cells, leading to anticipation of this technique as a novel therapeutic tool in regenerative medicine. In this study, we evaluated the ability of iPS cells derived from mouse embryonic fibroblasts to differentiate into oligodendrocytes and compared this with the differential ability of mouse embryonic stem cells (ES cells). Experiments using an in vitro oligodendrocyte differentiation protocol that was optimized to ES cells demonstrated that 2.3% of iPS cells differentiated into O4(+) oligodendrocytes compared with 24.0% of ES cells. However, the rate of induction of A2B5(+) oligodendrocyte precursor cell (OPC) was similar for both iPS-derived cells and ES-derived cells (14.1% and 12.6%, respectively). These findings suggest that some intracellular factors in iPS cells inhibit the terminal differentiation of oligodendrocytes from the OPC stage. (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Recurrent genomic instability of chromosome 1q in neural derivatives of human embryonic stem cells

PubMed Central

Varela, Christine; Denis, Jérôme Alexandre; Polentes, Jérôme; Feyeux, Maxime; Aubert, Sophie; Champon, Benoite; Piétu, Geneviève; Peschanski, Marc; Lefort, Nathalie

2012-01-01

Human pluripotent stem cells offer a limitless source of cells for regenerative medicine. Neural derivatives of human embryonic stem cells (hESCs) are currently being used for cell therapy in 3 clinical trials. However, hESCs are prone to genomic instability, which could limit their clinical utility. Here, we report that neural differentiation of hESCs systematically produced a neural stem cell population that could be propagated for more than 50 passages without entering senescence; this was true for all 6 hESC lines tested. The apparent spontaneous loss of evolution toward normal senescence of somatic cells was associated with a jumping translocation of chromosome 1q. This chromosomal defect has previously been associated with hematologic malignancies and pediatric brain tumors with poor clinical outcome. Neural stem cells carrying the 1q defect implanted into the brains of rats failed to integrate and expand, whereas normal cells engrafted. Our results call for additional quality controls to be implemented to ensure genomic integrity not only of undifferentiated pluripotent stem cells, but also of hESC derivatives that form cell therapy end products, particularly neural lines. PMID:22269325

Amniotic fluid stem cells: a promising therapeutic resource for cell-based regenerative therapy.

PubMed

Antonucci, Ivana; Pantalone, Andrea; Tete, Stefano; Salini, Vincenzo; Borlongan, Cesar V; Hess, David; Stuppia, Liborio

2012-01-01

Stem cells have been proposed as a powerful tool in the treatment of several human diseases, both for their ability to represent a source of new cells to replace those lost due to tissue injuries or degenerative diseases, and for the ability of produce trophic molecules able to minimize damage and promote recovery in the injured tissue. Different cell types, such as embryonic, fetal or adult stem cells, human fetal tissues and genetically engineered cell lines, have been tested for their ability to replace damaged cells and to restore the tissue function after transplantation. Amniotic fluid -derived Stem cells (AFS) are considered a novel resource for cell transplantation therapy, due to their high renewal capacity, the "in vitro" expression of embryonic cell lineage markers, and the ability to differentiate in tissues derived from all the three embryonic layers. Moreover, AFS do not produce teratomas when transplanted into animals and are characterized by a low antigenicity, which could represent an advantage for cell transplantation or cell replacement therapy. The present review focuses on the biological features of AFS, and on their potential use in the treatment of pathological conditions such as ischemic brain injury and bone damages.

[Stem cells--cloning, plasticity, bioethic].

PubMed

Pflegerl, Pamina; Keller, Thomas; Hantusch, Brigitte; Hoffmann, Thomas Sören; Kenner, Lukas

2008-01-01

Stem cells with certain characteristics have become promising tools for molecular medicine. They have the potential to self-regenerate and to differentiate into specific tissues. Besides their great potential, embryonic stem cells (ESC) run the risk of enhanced tumorigenesis. The use of human embryonic stem cells (hESC) is ethically problematic because their isolation involves the destruction of human embryos. Recently developed methods generate are able to pluripotent stem cells from fibroblasts. Alternatives for ESC are adult stem cells (ASC) derived from bone marrow, cord blood, amniotic fluid and other tissues. The following article is on the basis of testimony of Lukas Kenner for the German Bundestag about the use of ESC for research, therapy and drug development. Ethical aspects are taken into consideration.

Clinical potentials of human pluripotent stem cells in lung diseases

PubMed Central

2014-01-01

Lung possesses very limited regenerative capacity. Failure to maintain homeostasis of lung epithelial cell populations has been implicated in the development of many life-threatening pulmonary diseases leading to substantial morbidity and mortality worldwide, and currently there is no known cure for these end-stage pulmonary diseases. Embryonic stem cells (ESCs) and somatic cell-derived induced pluripotent stem cells (iPSCs) possess unlimited self-renewal capacity and great potential to differentiate to various cell types of three embryonic germ layers (ectodermal, mesodermal, and endodermal). Therapeutic use of human ESC/iPSC-derived lung progenitor cells for regeneration of injured or diseased lungs will have an enormous clinical impact. This article provides an overview of recent advances in research on pluripotent stem cells in lung tissue regeneration and discusses technical challenges that must be overcome for their clinical applications in the future. PMID:24995122

Balancing Ethical Pros and Cons of Stem Cell Derived Gametes.

PubMed

Segers, Seppe; Mertes, Heidi; de Wert, Guido; Dondorp, Wybo; Pennings, Guido

2017-07-01

In this review we aim to provide an overview of the most important ethical pros and cons of stem cell derived gametes (SCD-gametes), as a contribution to the debate about reproductive tissue engineering. Derivation of gametes from stem cells holds promising applications both for research and for clinical use in assisted reproduction. We explore the ethical issues connected to gametes derived from embryonic stem cells (both patient specific and non-patient specific) as well as those related to gametes derived from induced pluripotent stem cells. The technology of SCD-gametes raises moral concerns of how reproductive autonomy relates to issues of embryo destruction, safety, access, and applications beyond clinical infertility.

Rats, cats, and elephants, but still no unicorn: induced pluripotent stem cells from new species.

PubMed

Trounson, Alan

2009-01-09

Two independent studies in this issue of Cell Stem Cell (Liao et al., 2009; Li et al., 2009) derive rat induced pluripotent stem cells (iPSCs). In one report, the method used results in rat and human iPSCs that exhibit phenotypic traits similar to mouse embryonic stem cells.

Identification and Single-Cell Functional Characterization of an Endodermally Biased Pluripotent Substate in Human Embryonic Stem Cells.

PubMed

Allison, Thomas F; Smith, Andrew J H; Anastassiadis, Konstantinos; Sloane-Stanley, Jackie; Biga, Veronica; Stavish, Dylan; Hackland, James; Sabri, Shan; Langerman, Justin; Jones, Mark; Plath, Kathrin; Coca, Daniel; Barbaric, Ivana; Gokhale, Paul; Andrews, Peter W

2018-05-09

Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

PubMed

Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

2016-03-22

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

Modeling Niemann Pick type C1 using human embryonic and induced pluripotent stem cells.

PubMed

Ordoñez, M Paulina; Steele, John W

2017-02-01

Data generated in Niemann Pick type C1 (NPC1) human embryonic and human induced pluripotent stem cell derived neurons complement on-going studies in animal models and provide the first example, in disease-relevant human cells, of processes that underlie preferential neuronal defects in a NPC1. Our work and that of other investigators in human neurons derived from stem cells highlight the importance of performing rigorous mechanistic studies in relevant cell types to guide drug discovery and therapeutic development, alongside of existing animal models. Through the use of human stem cell-derived models of disease, we can identify and discover or repurpose drugs that revert early events that lead to neuronal failure in NPC1. Together with the study of disease pathogenesis and efficacy of therapies in animal models, these strategies will fulfill the promise of stem cell technology in the development of new treatments for human diseases. This article is part of a Special Issue entitled SI: Exploiting human neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

Stem cells to gametes: how far should we go?

PubMed

Whittaker, Peter

2007-03-01

Murine embryonic stem cells have recently been shown to be capable of differentiating in vitro into oocytes or sperm. Should these findings be duplicated using human embryonic stem cells, this would raise a number of social and ethical concerns, some specific to these particular developments, others shared with other aspects of stem cell research. This review outlines the properties of stem cells and their conversion to gametes. Concerns raised include embryo destruction, quality of gametes derived in this way, possibility for children with two male biological parents, movement towards germ line gene therapy and 'designer babies', and the future impacts on health service provisions. It is important that public discussion of some of these issues should take place.

Delivery of Differentiation Factors by Mesoporous Silica Particles Assists Advanced Differentiation of Transplanted Murine Embryonic Stem Cells

PubMed Central

Kozhevnikova, Mariya; König, Niclas; Zhou, Chunfang; Leao, Richardson; Knöpfel, Thomas; Pankratova, Stanislava; Trolle, Carl; Berezin, Vladimir; Bock, Elisabeth; Aldskogius, Håkan

2013-01-01

Stem cell transplantation holds great hope for the replacement of damaged cells in the nervous system. However, poor long-term survival after transplantation and insufficiently robust differentiation of stem cells into specialized cell types in vivo remain major obstacles for clinical application. Here, we report the development of a novel technological approach for the local delivery of exogenous trophic factor mimetics to transplanted cells using specifically designed silica nanoporous particles. We demonstrated that delivering Cintrofin and Gliafin, established peptide mimetics of the ciliary neurotrophic factor and glial cell line-derived neurotrophic factor, respectively, with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also their long-term survival in vivo. We propose that the delivery of growth factors by mesoporous nanoparticles is a potentially versatile and widely applicable strategy for efficient differentiation and functional integration of stem cell derivatives upon transplantation. PMID:24089415

Embryonic hematopoiesis in vertebrate somites gives rise to definitive hematopoietic stem cells

PubMed Central

Qiu, Juhui; Fan, Xiaoying; Wang, Yixia; Jin, Hongbin; Song, Yixiao; Han, Yang; Huang, Shenghong; Meng, Yaping; Tang, Fuchou; Meng, Anming

2016-01-01

Hematopoietic stem cells (HSCs) replenish all types of blood cells. It is debating whether HSCs in adults solely originate from the aorta-gonad-mesonephros (AGM) region, more specifically, the dorsal aorta, during embryogenesis. Here, we report that somite hematopoiesis, a previously unwitnessed hematopoiesis, can generate definitive HSCs (dHSCs) in zebrafish. By transgenic lineage tracing, we found that a subset of cells within the forming somites emigrate ventromedially and mix with lateral plate mesoderm-derived primitive hematopoietic cells before the blood circulation starts. These somite-derived hematopoietic precursors and stem cells (sHPSCs) subsequently enter the circulation and colonize the kidney of larvae and adults. RNA-seq analysis reveals that sHPSCs express hematopoietic genes with sustained expression of many muscle/skeletal genes. Embryonic sHPSCs transplanted into wild-type embryos expand during growth and survive for life time with differentiation into various hematopoietic lineages, indicating self-renewal and multipotency features. Therefore, the embryonic origin of dHSCs in adults is not restricted to the AGM. PMID:27252540

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

Stem Cells, Science, and Public Reasoning

ERIC Educational Resources Information Center

Hurlbut, J. Benjamin; Robert, Jason Scott

2012-01-01

These are interesting days in the scientific, social, and political debates about human embryonic stem cell research. Pluripotent stem cells--cells that can, in principle, give rise to the body's full range of cell types--were previously derivable only from human embryos that were destroyed in the process. Now, a variety of somatic cell types can…

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 .

Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

PubMed Central

Féraud, Olivier; Valogne, Yannick; Melkus, Michael W.; Zhang, Yanyan; Oudrhiri, Noufissa; Haddad, Rima; Daury, Aurélie; Rocher, Corinne; Larbi, Aniya; Duquesnoy, Philippe; Divers, Dominique; Gobbo, Emilie; Brunet de la Grange, Philippe; Louache, Fawzia; Bennaceur-Griscelli, Annelise; Mitjavila-Garcia, Maria Teresa

2016-01-01

Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process. PMID:26938212

Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

PubMed Central

Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

2012-01-01

Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

PubMed

Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

2016-08-01

Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. © The Author(s) 2016.

Placental-derived stem cells: Culture, differentiation and challenges

PubMed Central

Oliveira, Maira S; Barreto-Filho, João B

2015-01-01

Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice. PMID:26029347

Stem cells in retinal regeneration: past, present and future.

PubMed

Ramsden, Conor M; Powner, Michael B; Carr, Amanda-Jayne F; Smart, Matthew J K; da Cruz, Lyndon; Coffey, Peter J

2013-06-01

Stem cell therapy for retinal disease is under way, and several clinical trials are currently recruiting. These trials use human embryonic, foetal and umbilical cord tissue-derived stem cells and bone marrow-derived stem cells to treat visual disorders such as age-related macular degeneration, Stargardt's disease and retinitis pigmentosa. Over a decade of analysing the developmental cues involved in retinal generation and stem cell biology, coupled with extensive surgical research, have yielded differing cellular approaches to tackle these retinopathies. Here, we review these various stem cell-based approaches for treating retinal diseases and discuss future directions and challenges for the field.

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

PubMed Central

Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

2009-01-01

BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

Research with parthenogenetic stem cells will help decide whether a safer clinical use is possible.

PubMed

Muñoz, M; Penarossa, G; Caamaño, J N; Díez, C; Brevini, T A L; Gómez, E

2015-04-01

The derivation and use of parthenogenetic stem cells (pESCs) are envisaged as a reliable alternative to conventional embryonic stem cells. Similar to embryonic stem cells in their proliferation, expression of pluripotency markers and capacity to multilineage differentiation, pESCs are at a lower risk of immune rejection within stem cell-based therapeutics. Moreover, pESCs represent an important model system to study the effect of paternally imprinted genes on cell differentiation. However, currently available information about the genetic and epigenetic behaviour of pESCs is limited. Thus, a detailed look at the biology of parthenogenetic (PG) embryos and PG-derived cell lines would allow gaining insight into the full potential of pESC in biotechnology. In this commentary article we review some features related to the biology of PG embryos and pESCs. In addition, novel traits on bovine pESCs (bpESCs) are discussed. Copyright © 2013 John Wiley & Sons, Ltd.

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Integration-deficient lentivectors: an effective strategy to purify and differentiate human embryonic stem cell-derived hepatic progenitors.

PubMed

Yang, Guanghua; Si-Tayeb, Karim; Corbineau, Sébastien; Vernet, Rémi; Gayon, Régis; Dianat, Noushin; Martinet, Clémence; Clay, Denis; Goulinet-Mainot, Sylvie; Tachdjian, Gérard; Tachdjian, Gérard; Burks, Deborah; Vallier, Ludovic; Bouillé, Pascale; Dubart-Kupperschmitt, Anne; Weber, Anne

2013-07-19

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine. However, the safety of cell therapy using differentiated hPSC derivatives must be improved through methods that will permit the transplantation of homogenous populations of a specific cell type. To date, purification of progenitors and mature cells generated from either embryonic or induced pluripotent stem cells remains challenging with use of conventional methods. We used lentivectors encoding green fluorescent protein (GFP) driven by the liver-specific apoliprotein A-II (APOA-II) promoter to purify human hepatic progenitors. We evaluated both integrating and integration-defective lentivectors in combination with an HIV integrase inhibitor. A human embryonic stem cell line was differentiated into hepatic progenitors using a chemically defined protocol. Subsequently, cells were transduced and sorted at day 16 of differentiation to obtain a cell population enriched in hepatic progenitor cells. After sorting, more than 99% of these APOA-II-GFP-positive cells expressed hepatoblast markers such as α-fetoprotein and cytokeratin 19. When further cultured for 16 days, these cells underwent differentiation into more mature cells and exhibited hepatocyte properties such as albumin secretion. Moreover, they were devoid of vector DNA integration. We have developed an effective strategy to purify human hepatic cells from cultures of differentiating hPSCs, producing a novel tool that could be used not only for cell therapy but also for in vitro applications such as drug screening. The present strategy should also be suitable for the purification of a broad range of cell types derived from either pluripotent or adult stem cells.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

PubMed

Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

2017-05-01

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration.

PubMed

Leucht, Philipp; Kim, Jae-Beom; Amasha, Raimy; James, Aaron W; Girod, Sabine; Helms, Jill A

2008-09-01

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

In vitro fertilization, the Nobel Prize, and human embryonic stem cells.

PubMed

Gearhart, John; Coutifaris, Christos

2011-01-07

Robert Edwards was awarded the 2010 Nobel Prize in Physiology or Medicine for the development of human in vitro fertilization. His work not only provided the means to overcome many forms of infertility, but it also enabled research on early stages of human embryos and the derivation of human embryonic stem cells. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation of mouse chimeras with high contribution of tetraploid embryonic stem cells and embryonic stem cell-fibroblast hybrid cells.

PubMed

Matveeva, Natalia M; Kizilova, Elena A; Serov, Oleg L

2015-01-01

The in vitro long-term cultivation of embryonic stem (ES) cells derived from pre-implantation embryos offers the unique possibility of combining ES cells with pre-implantation embryos to generate chimeras, thus facilitating the creation of a bridge between in vitro and in vivo investigations. Genomic manipulation using ES cells and homologous recombination is one of the most outstanding scientific achievements, resulting in the generation of animals with desirable genome modifications. As such, the generation of ES cells with different ploidy via cell fusion also deserves much attention because this approach allows for the production of chimeras that contain somatic cells with various ploidy. Therefore, this is a powerful tool that can be used to study the role of polyploidy in the normal development of mammals.

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

PubMed

Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

2016-02-03

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

Hematopoietic stem cells: can old cells learn new tricks?

PubMed

Ho, Anthony D; Punzel, Michael

2003-05-01

Since the establishment of cell lines derived from human embryonic stem (ES) cells, it has been speculated that out of such "raw material," we could some day produce all sorts of replacement parts for the human body. Human pluripotent stem cells can be isolated from embryonic, fetal, or adult tissues. Enormous self-renewal capacity and developmental potential are the characteristics of ES cells. Somatic stem cells, especially those derived from hematopoietic tissues, have also been reported to exhibit developmental potential heretofore not considered possible. The initial evidences for the plasticity potential of somatic stem cells were so encouraging that the opponents of ES cell research used them as arguments for restricting ES cell research. In the past months, however, critical issues have been raised challenging the validity and the interpretation of the initial data. Whereas hematopoietic stem-cell therapy has been a clinical reality for almost 40 years, there is still a long way to go in basic research before novel therapy strategies with stem cells as replacement for other organ systems can be established. Given the present status, we should keep all options open for research in ES cells and adult stem cells to appreciate the complexity of their differentiation pathways and the relative merits of various types of stem cells for regenerative medicine.

Autonomous assembly of epithelial structures by subrenal implantation of dissociated embryonic inner-ear cells.

PubMed

Wang, Li; Zhang, Kaiqing; Zhu, Helen He; Gao, Wei-Qiang

2015-05-27

Microenvironment and cell-cell interactions play an important role during embryogenesis and are required for the stemness and differentiation of stem cells. The inner-ear sensory epithelium, containing hair cells and supporting cells, is derived from the stem cells within the otic vesicle at early embryonic stages. However, whether or not such microenvironment or cell-cell interactions within the embryonic otic tissue have the capacity to regulate the proliferation and differentiation of stem cells and to autonomously reassemble the cells into epithelial structures is unknown. Here, we report that on enzymatic digestion and dissociation to harvest all the single cells from 13.5-day-old rat embryonic (E13.5) inner-ear tissue as well as on implantation of these cells under renal capsules; the dissociated cells are able to reassemble themselves to form epithelial structures as early as 7 days after implantation. By 25 days after implantation, more mature epithelial structures are formed. Immunostaining with cell-type-specific markers reveals that hair cells and supporting cells are not only formed, but are also well aligned with the hair cells located in the apical layer surrounded by the supporting cells. These findings suggest that microenvironment and cell-cell interactions within the embryonic inner-ear tissue have the autonomous signals to induce the formation of sensory epithelial structures. This method may also provide a useful system to study the potential of stem cells to differentiate into hair cells in vivo.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

PubMed

Acuna-Mendoza, Soledad; Martin, Sabrina; Kuchler-Bopp, Sabine; Ribes, Sandy; Thalgott, Jérémy; Chaussain, Catherine; Creuzet, Sophie; Lesot, Hervé; Lebrin, Franck; Poliard, Anne

2017-12-01

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 Rosa TomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

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

Treatment of hypoxic-ischemic encephalopathy in mouse by transplantation of embryonic stem cell-derived cells.

PubMed

Ma, Jie; Wang, Yu; Yang, Jianhua; Yang, Min; Chang, Keun-A; Zhang, Linhua; Jiang, Feng; Li, Yi; Zhang, Zhonggong; Heo, Chaejeong; Suh, Yoo-Hun

2007-07-01

A 7-day-old hypoxic-ischemic encephalopathy (HIE) mouse model was used to study the effect of transplantation of embryonic stem (ES) cell-derived cells on the HIE. After the inducement in vitro, the ES cell-derived cells expressed Nestin and MAP-2, rather than GFAP mRNA. After transplantation, ES cell-derived cells can survive, migrate into the injury site, and specifically differentiate into neurons, showing improvement of the learning ability and memory of the HIE mouse at 8 months post-transplantation. The non-grafted HIE mouse brain showed typical pathological changes in the hippocampus and cerebral cortex, where the number of neurons was reduced, while in the cell graft group, number of the neurons increased in the same regions. Although further study is necessary to elucidate the precise mechanisms responsible for this functional recovery, we believe that ES cells have advantages for use as a donor source in HIE.

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Murine mesenchymal and embryonic stem cells express a similar Hox gene profile.

PubMed

Phinney, Donald G; Gray, Andrew J; Hill, Katy; Pandey, Amitabh

2005-12-30

Using degenerate oligonucleotide primers targeting the homeobox domain, we amplified by PCR and sequenced 723 clones from five murine cell populations and lines derived from embryonic mesoderm and adult bone marrow. Transcripts from all four vertebrate Hox clusters were expressed by the different populations. Hierarchical clustering of the data revealed that mesenchymal stem cells (MSCs) and the embryonic stem (ES) cell line D3 shared a similar Hox expression profile. These populations exclusively expressed Hoxb2, Hoxb5, Hoxb7, and Hoxc4, transcripts regulating self-renewal and differentiation of other stem cells. Additionally, Hoxa7 transcript quantified by real-time PCR strongly correlated (r2=0.89) with the number of Hoxa7 clones identified by sequencing, validating that data from the PCR screen reflects differences in Hox mRNA abundance between populations. This is the first study to catalogue Hox transcripts in murine MSCs and by comparative analyses identify specific Hox genes that may contribute to their stem cell character.

Conference Scene: Induced pluripotent cells: a new path for regenerative medicine. 7 October 2010, BioPark, Welwyn Garden City, Hertfordshire, UK.

PubMed

Crutzen, Hélène S G

2011-01-01

Embryonic stem cells and induced pluripotent stem (iPS) cells, which are embryonic stem-like cells derived from adult tissues, have the broadest differentiation potential. These cells are unique in their ability to self-renew, to be maintained in an undifferentiated state for long periods of culturing and to give rise to many different cell lineages including germ-line cells. They therefore represent an invaluable tool for facilitating research towards the realization of regenerative medicine. The recent developments in embryonic stem cell and iPS cell technology have allowed human cell models to be developed that will hopefully provide novel platforms for disease analysis not only at the basic science level, but also for drug discovery and screening, and other clinical applications. This 1-day conference, chaired by Professor Peter Andrews from the University of Sheffield, UK, and Dr Chris Denning from the University of Nottingham, UK, focused on generation of iPS cells, their differentiation into specific fates and applications to disease modeling. It consisted of 11 talks by UK-based and international researchers, and three posters; Ms Azra Fatima from Cologne University, Germany, won the competition for her poster on the derivation of iPS cells from a patient with arrhythmogenic right ventricular cardiomyopathy.

Can Human Embryonic Stem Cell-Derived Stromal Cells Serve a Starting Material for Myoblasts?

PubMed Central

Ando, Yu; Saito, Marie; Machida, Masakazu; Yoshida-Noro, Chikako; Akutsu, Hidenori; Takahashi, Masataka

2017-01-01

A large number of myocytes are necessary to treat intractable muscular disorders such as Duchenne muscular dystrophy with cell-based therapies. However, starting materials for cellular therapy products such as myoblasts, marrow stromal cells, menstrual blood-derived cells, and placenta-derived cells have a limited lifespan and cease to proliferate in vitro. From the viewpoints of manufacturing and quality control, cells with a long lifespan are more suitable as a starting material. In this study, we generated stromal cells for future myoblast therapy from a working cell bank of human embryonic stem cells (ESCs). The ESC-derived CD105+ cells with extensive in vitro proliferation capability exhibited myogenesis and genetic stability in vitro. These results imply that ESC-derived CD105+ cells are another cell source for myoblasts in cell-based therapy for patients with genetic muscular disorders. Since ESCs are immortal, mesenchymal stromal cells generated from ESCs can be manufactured at a large scale in one lot for pharmaceutical purposes. PMID:28706537

A PITX3-EGFP Reporter Line Reveals Connectivity of Dopamine and Non-dopamine Neuronal Subtypes in Grafts Generated from Human Embryonic Stem Cells.

PubMed

Niclis, Jonathan C; Gantner, Carlos W; Hunt, Cameron P J; Kauhausen, Jessica A; Durnall, Jennifer C; Haynes, John M; Pouton, Colin W; Parish, Clare L; Thompson, Lachlan H

2017-09-12

Development of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Embryonic and Induced Pluripotent Stem Cells: Understanding, Creating, and Exploiting the Nano-Niche for Regenerative Medicine

PubMed Central

2013-01-01

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the capacity to differentiate into any specialized cell type of the human body, and therefore, ESC/iPSC-derived cell types offer great potential for regenerative medicine. However, key to realizing this potential requires a strong understanding of stem cell biology, techniques to maintain stem cells, and strategies to manipulate cells to efficiently direct cell differentiation toward a desired cell type. As nanoscale science and engineering continues to produce novel nanotechnology platforms, which inform, infiltrate, and impinge on many aspects of everyday life, it is no surprise that stem cell research is turning toward developments in nanotechnology to answer research questions and to overcome obstacles in regenerative medicine. Here we discuss recent advances in ESC and iPSC manipulation using nanomaterials and highlight future challenges within this area of research. PMID:23414366

Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

PubMed

Cohen, Shahar; Leshansky, Lucy; Zussman, Eyal; Burman, Michael; Srouji, Samer; Livne, Erella; Abramov, Natalie; Itskovitz-Eldor, Joseph

2010-10-01

The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

PubMed

Cong, Shan; Cao, Guifang; Liu, Dongjun

2014-12-01

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

PubMed Central

Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

2015-01-01

Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

PubMed

Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

2015-07-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

PubMed

Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

2018-03-03

Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

Neural Stem Cell or Human Induced Pluripotent Stem Cell-derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy

PubMed Central

Upadhya, Dinesh; Hattiangady, Bharathi; Shetty, Geetha A.; Zanirati, Gabriele; Kodali, Maheedhar; Shetty, Ashok K.

2016-01-01

Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE-like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE-NSCs and hiPSC-derived MGE-like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post-grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft-derived cells, and the effects of grafts on the host hippocampus. PMID:27532817

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

PubMed

Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

2011-06-01

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

Hepatic differentiation of pluripotent stem cells.

PubMed

Loya, Komal; Eggenschwiler, Reto; Ko, Kinarm; Sgodda, Malte; André, Francoise; Bleidissel, Martina; Schöler, Hans R; Cantz, Tobias

2009-10-01

In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

Application of Graphene Based Nanotechnology in Stem Cells Research.

PubMed

Hu, Shanshan; Zeng, Yongxiang; Yang, Shuying; Qin, Han; Cai, He; Wang, Jian

2015-09-01

The past several years have witnessed significant advances in stem cell therapy, tissue engineering and regenerative medicine. Graphene, with its unique properties such as high electrical conductivity, elasticity and good molecule absorption, have potential for creating the next generation of biomaterials. This review summarizes the interrelationship between graphene and stem cells. The analysis of graphene when applied on mesenchymal stem cells, neural stem cells, induced pluripotent stem cells, embryonic stem cells, periodontal ligament stem cells, human adipose-derived stem cells and cancer stem cells, and how graphene influences cell behavior and differentiation are discussed in details.

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

PubMed

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes in vitro and in vivo. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.

Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state

PubMed Central

Leitch, Harry G.; Blair, Kate; Mansfield, William; Ayetey, Harold; Humphreys, Peter; Nichols, Jennifer; Surani, M. Azim; Smith, Austin

2010-01-01

Mouse and rat embryonic stem cells can be sustained in defined medium by dual inhibition (2i) of the mitogen-activated protein kinase (Erk1/2) cascade and of glycogen synthase kinase 3. The inhibitors suppress differentiation and enable self-renewal of pluripotent cells that are ex vivo counterparts of naïve epiblast cells in the mature blastocyst. Pluripotent stem cell lines can also be derived from unipotent primordial germ cells via a poorly understood process of epigenetic reprogramming. These are termed embryonic germ (EG) cells to denote their distinct origin. Here we investigate whether EG cell self-renewal and derivation are supported by 2i. We report that mouse EG cells can be established with high efficiency using 2i in combination with the cytokine leukaemia inhibitory factor (LIF). Furthermore, addition of fibroblast growth factor or stem cell factor is unnecessary using 2i-LIF. The derived EG cells contribute extensively to healthy chimaeric mice, including to the germline. Using the same conditions, we describe the first derivations of EG cells from the rat. Rat EG cells express a similar marker profile to rat and mouse ES cells. They have a diploid karyotype, can be clonally expanded and genetically manipulated, and are competent for multilineage colonisation of chimaeras. These findings lend support to the postulate of a conserved molecular ground state in pluripotent rodent cells. Future research will determine the extent to which this is maintained in other mammals and whether, in some species, primordial germ cells might be a more tractable source than epiblast for the capture of naïve pluripotent stem cells. PMID:20519324

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

PubMed Central

Martin, Colin H.; Woll, Petter S.; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos

2008-01-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs. PMID:18621931

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.

PubMed

Martin, Colin H; Woll, Petter S; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos; Kaufman, Dan S

2008-10-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34(+) cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34(+) cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34(+) cells also did not produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

A Quantitative Proteomic Analysis of Hemogenic Endothelium Reveals Differential Regulation of Hematopoiesis by SOX17

PubMed Central

Clarke, Raedun L.; Robitaille, Aaron M.; Moon, Randall T.; Keller, Gordon

2015-01-01

Summary The in vitro derivation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is complicated by the existence of multiple overlapping embryonic blood cell programs called primitive, erythromyeloid progenitor (EMP), and definitive. As HSCs are only generated during the definitive stage of hematopoiesis, deciphering the regulatory pathways that control the emergence of this program and identifying markers that distinguish it from the other programs are essential. To identify definitive specific pathways and marker sets, we used label-free proteomics to determine the proteome of embryo-derived and mouse embryonic stem cell-derived VE-CADHERIN+CD45− definitive hematopoietic progenitors. With this approach, we identified Stat1 as a marker that distinguishes the definitive erythroid lineage from the primitive- and EMP-derived lineages. Additionally, we provide evidence that the generation of the Stat1+ definitive lineage is dependent on Sox17. These findings establish an approach for monitoring the emergence of definitive hematopoiesis in the PSC differentiation cultures. PMID:26267830

Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells.

PubMed

Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

2016-04-12

Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

PubMed

Sternberg, Hal; Jiang, Jianjie; Sim, Pamela; Kidd, Jennifer; Janus, Jeffrey; Rinon, Ariel; Edgar, Ron; Shitrit, Alina; Larocca, David; Chapman, Karen B; Binette, Francois; West, Michael D

2014-01-01

The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage

PubMed Central

Qin, Mingde; Chen, Ruihua; Li, Hong; Liang, Hansi; Xue, Qun; Li, Fang; Chen, Ying; Zhang, Xueguang

2016-01-01

Amniotic fluid stem cells (AFSCs) are a type of fetal stem cell whose stemness encompasses both embryonic and adult stem cells, suggesting that they may be easily and efficiently reprogrammed into induced pluripotent stem cells (iPSCs). To further simplify the reprogramming process, the creation of AFSC-derived iPSCs using a single factor is desirable. Here we report the generation of one-factor human AFSC-iPSCs (AiPSCs) from human AFSCs by ectopic expression of the transcription factor OCT4. Just like human embryonic stem cells, AiPSCs exhibited similar epigenetic status, global gene expression profiles, teratoma formation and in vitro & in vivo pluripotency. Our results indicate that the OCT4 is necessary and sufficient to directly reprogram human AFSCs into pluripotent AiPSCs. Moreover, reflecting the similar memory characteristics of AFSCs and neural stem cells, we show that AiPSC membrane-derived vesicles (MVs) repair cerebral ischemia damage. We anticipate that the successful generation of one-factor AiPSCs will facilitate the creation of patient-specific pluripotent stem cells without the need for transgenic expression of oncogenes. Moreover, MVs from tissue-specific AiPSCs have potential in tissue repair, representing a novel application of iPSCs. PMID:27019637

Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

PubMed

Billing, Anja M; Ben Hamidane, Hisham; Bhagwat, Aditya M; Cotton, Richard J; Dib, Shaima S; Kumar, Pankaj; Hayat, Shahina; Goswami, Neha; Suhre, Karsten; Rafii, Arash; Graumann, Johannes

2017-01-06

Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

NASA Astrophysics Data System (ADS)

Shinde, Vaibhav; Brungs, Sonja; Hescheler, Jürgen; Hemmersbach, Ruth; Sachinidis, Agapios

2016-06-01

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.

Akt Suppression of TGFβ Signaling Contributes to the Maintenance of Vascular Identity in Embryonic Stem Cell-Derived Endothelial Cells

PubMed Central

Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

2016-01-01

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (EC) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells, and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs, and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs, and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. PMID:23963623

Akt suppression of TGFβ signaling contributes to the maintenance of vascular identity in embryonic stem cell-derived endothelial cells.

PubMed

Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

2014-01-01

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (ECs) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. © AlphaMed Press.

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.

Induced Pluripotent Stem Cells: A novel frontier in the study of human primary immunodeficiencies

PubMed Central

Pessach, Itai M.; Ordovas-Montanes, Jose; Zhang, Shen-Ying; Casanova, Jean-Laurent; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Manos, Philip D.; Schlaeger, Thorsten M.; Park, In-Hyun; Rucci, Francesca; Agarwal, Suneet; Mostoslavsky, Gustavo; Daley, George Q.; Notarangelo, Luigi D.

2010-01-01

Background The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells through the exogenous expression of transcription promises to revolutionize the study of human diseases. Objective Here we report on the generation of 25 induced pluripotent stem cell lines from 6 patients with various forms of Primary Immunodeficiencies, affecting adaptive and/or innate immunity. Methods Patients’ dermal fibroblasts were reprogrammed by expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC using a single excisable polycistronic lentiviral vector. Results Induced pluripotent stem cells derived from patients with primary immunodeficiencies show a stemness profile that is comparable to that observed in human embryonic stem cells. Following in vitro differentiation into embryoid bodies, pluripotency of the patient-derived indiced pluripotent stem cells lines was demonstrated by expression of genes characteristic of each of the three embryonic layers. We have confirmed the patient-specific origin of the induced pluripotent stem cell lines, and ascertained maintenance of karyotypic integrity. Conclusion By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of induced pluripotent stem cell lines from patients with primary immunodeficiencies represents a unique resource to investigate the pathophysiology of hematopoietic and extra-hematopoietic manifestations of these diseases, and may assist in the development of novel therapeutic approaches based on gene correction. PMID:21185069

Differential impact of science policy on subfields of human embryonic stem cell research.

PubMed

Moon, Seongwuk; Cho, Seong Beom

2014-01-01

In this research, we examine how restrictive policy influenced performance in human embryonic stem cell research (hESC) between 1998 and 2008. In previous research, researchers argued whether restrictive policy decreased the performance of stem cell research in some nations, especially in the US. Here, we hypothesize that this policy influenced specific subfields of the hESC research. To investigate the selective policy effects, we categorize hESC research publications into three subfields-derivation, differentiation, and medical application research. Our analysis shows that restrictive policy had different effects on different subfields. In general, the US outperformed in overall hESC research throughout these periods. In the derivation of hESC, however, the US almost lost its competence under restrictive policy. Interestingly, the US scientific community showed prominent resilience in hESC research through international collaboration. We concluded that the US resilience and performance stemmed from the wide breadth of research portfolio of US scientists across the hESC subfields, combined with their strategic efforts to collaborate internationally on derivation research.

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.

No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

PubMed Central

Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

2010-01-01

Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

Autophagy in Human Embryonic Stem Cells

PubMed Central

Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark

2011-01-01

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC. PMID:22110659

Vitrification of mouse embryo-derived ICM cells: a tool for preserving embryonic stem cell potential?

PubMed

Desai, Nina; Xu, Jing; Tsulaia, Tamara; Szeptycki-Lawson, Julia; AbdelHafez, Faten; Goldfarb, James; Falcone, Tommaso

2011-02-01

Vitrification technology presents new opportunities for preservation of embryo derived stem cells without first establishing a viable ESC line. This study tests the feasibility of cryopreserving ICM cells using vitrification. ICMs from mouse embryos were isolated and vitrified in HSV straws or on cryoloops. Upon warming, the vitrified ICMs were cultured and observed for attachment and morphology. Colonies were passaged every 3-6 days. ICMs and ICM-derived ESC colonies were tested for expression of stem cell specific markers. ICMs vitrified on both the cryoloop and the HSV straw had high survival rates. ICM derived ESCs remained undifferentiated for several passages and demonstrated expression of typical stem cell markers; SSEA-1, Sox-2, Oct 4 and alkaline phosphatase. This is the first report on successful vitrification of isolated ICMs and the subsequent derivation of ESC colonies. Vitrification of isolated ICMs is a novel approach for preservation of the "stem cell source" material.

Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.

PubMed

Veazey, Kylee J; Golding, Michael C

2011-01-01

Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.

Characterization of glial-restricted precursors from rhesus monkey embryonic stem cells.

PubMed

Chen, Hongwei; Mao, Yu; Wang, Shufen; Li, Bin; Wang, Jinhuan; Li, Jian; Ma, Yuanye

2015-01-01

Glial-restricted precursor (GRP) cells, the earliest glial progenitors for both astrocytes and oligodendrocytes, have been derived from embryos and embryonic stem cells (ESC) in rodents. However, knowledge regarding the equivalent cell type in primates is limited due to restrictions imposed by ethics and resources. Here we report successful derivation and characterization of primate GRP cells from rhesus monkey ESC. The purified monkey GRP cells were A 2 B 5 -positive and FGF2-dependent for survival and proliferation. The differentiation assays indicated that they were tri-potential in vitro and bi-potential in vivo . These newly purified GRP cells will help to facilitate understanding of the molecular mechanism of glial development in primates as well as provide a source of therapeutic donor cells for use in neuroregenerative medicine.

Producing primate embryonic stem cells by somatic cell nuclear transfer.

PubMed

Byrne, J A; Pedersen, D A; Clepper, L L; Nelson, M; Sanger, W G; Gokhale, S; Wolf, D P; Mitalipov, S M

2007-11-22

Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

PubMed

Tang, Meilin; Yin, Mengmeng; Tang, Ming; Liang, Huamin; Yu, Chong; Hu, Xinwu; Luo, Hongyan; Baudis, Birte; Haustein, Moritz; Khalil, Markus; Sarić, Tomo; Hescheler, Jürgen; Xi, Jiaoya

2013-01-01

Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to β-adrenergic and muscarinic stimulations. Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells. Copyright © 2013 S. Karger AG, Basel.

Magnetic Resonance Imaging of Iron Oxide-Labeled Human Embryonic Stem Cell-Derived Cardiac Progenitors.

PubMed

Skelton, Rhys J P; Khoja, Suhail; Almeida, Shone; Rapacchi, Stanislas; Han, Fei; Engel, James; Zhao, Peng; Hu, Peng; Stanley, Edouard G; Elefanty, Andrew G; Kwon, Murray; Elliott, David A; Ardehali, Reza

2016-01-01

Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 μg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts, highlighting its utility in future cardiac stem cell therapy trials. ©AlphaMed Press.

The promise of human embryonic stem cells in aging-associated diseases

PubMed Central

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features

PubMed Central

Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger

2016-01-01

Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP. PMID:27385131

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features.

PubMed

Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger

2016-07-07

Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.

Induced pluripotent stem (iPS) cells from human fetal stem cells.

PubMed

Guillot, Pascale V

2016-02-01

Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Somatic cell nuclear transfer in Oregon: expanding the pluripotent space and informing research ethics.

PubMed

Lomax, Geoffrey P; DeWitt, Natalie D

2013-12-01

In May, Oregon Health and Science University (OHSU) announced the successful derivation, by the Mitalipov laboratory, of embryonic stem cells by somatic cell nuclear transfer. This experiment was recognized as a "formidable technical feat" and potentially a key step toward developing cell-based therapies. The OHSU report is also an example of how a scientific breakthrough can inform research ethics. This article suggests ways that nuclear transfer embryonic stem cell lines may contribute to research ethics by adding rigor to studies addressing pressing research questions important to the development of cell-based therapies.

A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures

PubMed Central

Emirandetti, Amanda; Lewicka, Michalina; Hermanson, Ola; Fisahn, André

2010-01-01

Background Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. Methodology/Principal Findings In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. Conclusions/Significance Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture. PMID:21079795

Generation of glucose-responsive, insulin-producing cells from human umbilical cord blood-derived mesenchymal stem cells.

PubMed

Prabakar, Kamalaveni R; Domínguez-Bendala, Juan; Molano, R Damaris; Pileggi, Antonello; Villate, Susana; Ricordi, Camillo; Inverardi, Luca

2012-01-01

We sought to assess the potential of human cord blood-derived mesenchymal stem cells (CB-MSCs) to derive insulin-producing, glucose-responsive cells. We show here that differentiation protocols based on stepwise culture conditions initially described for human embryonic stem cells (hESCs) lead to differentiation of cord blood-derived precursors towards a pancreatic endocrine phenotype, as assessed by marker expression and in vitro glucose-regulated insulin secretion. Transplantation of these cells in immune-deficient animals shows human C-peptide production in response to a glucose challenge. These data suggest that human cord blood may be a promising source for regenerative medicine approaches for the treatment of diabetes mellitus.

Equivalency of Buffalo (Bubalus Bubalis) Embryonic Stem Cells Derived From Fertilized, Parthenogenetic, and Hand-Made Cloned Embryos

PubMed Central

Muzaffar, Musharifa; Selokar, Naresh L.; Singh, Karn P.; Zandi, Mohammad; Singh, Manoj K.; Shah, Riaz A.; Chauhan, Manmohan S.; Singla, Suresh K.; Palta, Prabhat

2012-01-01

Abstract This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production. PMID:22582863

Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.

PubMed

Muzaffar, Musharifa; Selokar, Naresh L; Singh, Karn P; Zandi, Mohammad; Singh, Manoj K; Shah, Riaz A; Chauhan, Manmohan S; Singla, Suresh K; Palta, Prabhat; Manik, Radheysham

2012-06-01

This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.

From embryonic stem cells to functioning germ cells: science, clinical and ethical perspectives.

PubMed

Kiatpongsan, Sorapop

2007-10-01

Embryonic stem cells have been well recognized as cells having a versatile potential to differentiate into all types of cells in the body including germ cells. There are many research studies focusing on the differentiation processes and protocols to derive various types of somatic cells from embryonic stem cells. However, germ cells have unique differentiation process and developmental pathway compared with somatic cells. Consequently, they will require different differentiation protocols and special culture techniques. More understanding and established in vitro systems for gametogenesis will greatly contribute to further progression of knowledge and technology in germ cell biology, reproductive biology and reproductive medicine. Moreover if oocytes can be efficiently produced in vitro, this will play an important role on progression in nuclear transfer and nuclear reprogramming technology. The present article will provide concise review on past important discoveries, current ongoing studies and future views of this challenging research area. An ethical perspective has also been proposed to give comprehensive summary and viewpoint for future clinical application.

Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy.

PubMed

Parrotta, Elvira; De Angelis, Maria Teresa; Scalise, Stefania; Candeloro, Patrizio; Santamaria, Gianluca; Paonessa, Mariagrazia; Coluccio, Maria Laura; Perozziello, Gerardo; De Vitis, Stefania; Sgura, Antonella; Coluzzi, Elisa; Mollace, Vincenzo; Di Fabrizio, Enzo Mario; Cuda, Giovanni

2017-11-28

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, hold enormous promise for many biomedical applications, such as regenerative medicine, drug testing, and disease modeling. Although induced pluripotent stem cells resemble embryonic stem cells both morphologically and functionally, the extent to which these cell lines are truly equivalent, from a molecular point of view, remains controversial. Principal component analysis and K-means cluster analysis of collected Raman spectroscopy data were used for a comparative study of the biochemical fingerprint of human induced pluripotent stem cells and human embryonic stem cells. The Raman spectra analysis results were further validated by conventional biological assays. Raman spectra analysis revealed that the major difference between human embryonic stem cells and induced pluripotent stem cells is due to the nucleic acid content, as shown by the strong positive peaks at 785, 1098, 1334, 1371, 1484, and 1575 cm -1 , which is enriched in human induced pluripotent stem cells. Here, we report a nonbiological approach to discriminate human induced pluripotent stem cells from their native embryonic stem cell counterparts.

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

PubMed

Xue, Fei; Ma, Yinghong; Chen, Y Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang; Xu, Jie

2012-08-01

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

In vivo differentiation of induced pluripotent stem cells into neural stem cells by chimera formation.

PubMed

Choi, Hyun Woo; Hong, Yean Ju; Kim, Jong Soo; Song, Hyuk; Cho, Ssang Gu; Bae, Hojae; Kim, Changsung; Byun, Sung June; Do, Jeong Tae

2017-01-01

Like embryonic stem cells, induced pluripotent stem cells (iPSCs) can differentiate into all three germ layers in an in vitro system. Here, we developed a new technology for obtaining neural stem cells (NSCs) from iPSCs through chimera formation, in an in vivo environment. iPSCs contributed to the neural lineage in the chimera, which could be efficiently purified and directly cultured as NSCs in vitro. The iPSC-derived, in vivo-differentiated NSCs expressed NSC markers, and their gene-expression pattern more closely resembled that of fetal brain-derived NSCs than in vitro-differentiated NSCs. This system could be applied for differentiating pluripotent stem cells into specialized cell types whose differentiation protocols are not well established.

Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Saitou, Mitinori

2013-08-01

Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

Stem cell research and transplantation: science leading ethics.

PubMed

Daar, A S; Bhatt, A; Court, E; Singer, P A

2004-10-01

One of the most exciting developments in the biological sciences in the past decade has been the discovery and characterization of human embryonic stem cells (ESCs). The interest to transplanters is the potential applications of stem cells in regenerative medicine (RM), which may involve tissue engineering, genetic engineering, and other techniques to repair, replace, or regenerate failing tissues and organs. There is little controversy surrounding human adult stem cells. However, human ESCs are surrounded by a number of ethical controversies, the extent of which is partly dependent on their source. Those derived from currently existing embryonic stem cell lines are less controversial than those derived from "excess" embryos from in vitro fertilization (IVF) clinics, while ESCs derived from IVF embryos specifically created for the purpose are not acceptable to many people arguing from religious and other moral perspectives. Somatic cell nuclear transfer, or therapeutic cloning, must be distinguished from reproductive cloning. It holds the most promise for regenerative medicine. ESCs can also be derived from gonadal ridges of aborted fetuses. The transplant community must strive to uphold societal values in its effort to find remedies for their ailing patients and address the perennial problem of organ shortage. Transplanters also have a responsibility to engage the public in their efforts to gain public understanding and support, and policy makers must take into account public opinion. Only in this way can we realize the great potential of stem cell research for organ transplantation.

Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

PubMed

Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

2015-05-15

Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

PubMed

Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Isolation and characterization of true mesenchymal stem cells derived from human term decidua capable of multilineage differentiation into all 3 embryonic layers.

PubMed

Macias, Maria I; Grande, Jesús; Moreno, Ana; Domínguez, Irene; Bornstein, Rafael; Flores, Ana I

2010-11-01

The objective of the study was to isolate and characterize a population of mesenchymal stem cells (MSCs) from human term placental membranes. We isolated an adherent cell population from extraembryonic membranes. Morphology, phenotype, growth characteristics, karyotype, and immunological and differentiation properties were analyzed. The isolated placental MSCs were from maternal origin and named as decidua-derived mesenchymal stem cells (DMSCs). DMSCs differentiated into derivatives of all germ layers. It is the first report about placental MSC differentiation into alveolar type II cells. Clonally expanded DMSCs differentiated into all embryonic layers, including pulmonary cells. DMSCs showed higher life span than placental cells from fetal origin and proliferated without genomic instability. The data suggest that DMSCs are true multipotent MSCs, distinguishing them from other placental MSCs. DMSCs could be safely used in the mother as a potential source of MSCs for pelvic floor dysfunctions and immunological diseases. Additionally, frozen DMSCs can be stored for both autologous and allogeneic tissue regeneration. Copyright © 2010 Mosby, Inc. All rights reserved.

The business of human embryonic stem cell research and an international analysis of relevant laws.

PubMed

De Trizio, Ella; Brennan, Christopher S

2004-01-01

Few sciences have held out such therapeutic promise and correspondingly stirred so much controversy in countries throughout the world as the developing science surrounding human embryonic stem cells. Since the first reported development of several lines of human embryonic stem cells in 1988, many governments around the world have attempted to address the thorny ethical issues raised by human embryonic stem cell research by the passage of laws. In some cases these laws have directly regulated governmental funding of the science; in other cases they have created a legal environment that has either encouraged or discouraged both governmental and private funding of the science. This article first differentiates human embryonic stem cells from other types of stem cells and frames the ethical controversy surrounding human embryonic stem cell research, then surveys laws governing human embryonic stem cell research in various scientifically advanced countries located throughout the Pacific Rim, Europe and North America and explains the impact these laws have had on governmental and private funding of human embryonic stem cell research.

Use of pluripotent stem cells for reproductive medicine: are we there yet?

PubMed

Duggal, Galbha; Heindryckx, Björn; Deroo, Tom; De Sutter, Petra

2014-01-01

In recent years, pluripotent stem cells have demonstrated to be exciting tools to understand embryonic development, cell lineage specification, tissue generation and repair, and various other biological processes. In addition, the identification and isolation of germ line stem cells has given more insight into germ cell biology at the molecular level and into the underlying causes of infertility which was not possible earlier. The recent derivation of in vitro derived sperm and oocytes from pluripotent stem cells in the mouse model represents a major breakthrough in the field and substantiates the critical relevance of stem cells as a potential alternative resource for treating infertility. Although the past years have yielded compelling information in understanding germ cell development via in vitro stem cell assays, extended investigative research is necessary in order to derive fully functional 'artificial gametes' in a safe way for future therapeutic applications.

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

PubMed

Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

2012-08-01

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

Derivation, propagation and differentiation of human embryonic stem cells.

PubMed

Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

2004-04-01

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

PubMed

Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

2018-01-01

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

New perspectives in human stem cell therapeutic research.

PubMed

Trounson, Alan

2009-06-11

Human stem cells are in evaluation in clinical stem cell trials, primarily as autologous bone marrow studies, autologous and allogenic mesenchymal stem cell trials, and some allogenic neural stem cell transplantation projects. Safety and efficacy are being addressed for a number of disease state applications. There is considerable data supporting safety of bone marrow and mesenchymal stem cell transplants but the efficacy data are variable and of mixed benefit. Mechanisms of action of many of these cells are unknown and this raises the concern of unpredictable results in the future. Nevertheless there is considerable optimism that immune suppression and anti-inflammatory properties of mesenchymal stem cells will be of benefit for many conditions such as graft versus host disease, solid organ transplants and pulmonary fibrosis. Where bone marrow and mesenchymal stem cells are being studied for heart disease, stroke and other neurodegenerative disorders, again progress is mixed and mostly without significant benefit. However, correction of multiple sclerosis, at least in the short term is encouraging. Clinical trials on the use of embryonic stem cell derivatives for spinal injury and macular degeneration are beginning and a raft of other clinical trials can be expected soon, for example, the use of neural stem cells for killing inoperable glioma and embryonic stem cells for regenerating beta islet cells for diabetes. The change in attitude to embryonic stem cell research with the incoming Obama administration heralds a new co-operative environment for study and evaluation of stem cell therapies. The Californian stem cell initiative (California Institute for Regenerative Medicine) has engendered global collaboration for this new medicine that will now also be supported by the US Federal Government. The active participation of governments, academia, biotechnology, pharmaceutical companies, and private investment is a powerful consortium for advances in health.

Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics.

PubMed

Kooreman, Nigel G; de Almeida, Patricia E; Stack, Jonathan P; Nelakanti, Raman V; Diecke, Sebastian; Shao, Ning-Yi; Swijnenburg, Rutger-Jan; Sanchez-Freire, Veronica; Matsa, Elena; Liu, Chun; Connolly, Andrew J; Hamming, Jaap F; Quax, Paul H A; Brehm, Michael A; Greiner, Dale L; Shultz, Leonard D; Wu, Joseph C

2017-08-22

There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Efficient Generation of Functional Hepatocytes From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells by HNF4α Transduction

PubMed Central

Takayama, Kazuo; Inamura, Mitsuru; Kawabata, Kenji; Katayama, Kazufumi; Higuchi, Maiko; Tashiro, Katsuhisa; Nonaka, Aki; Sakurai, Fuminori; Hayakawa, Takao; Kusuda Furue, Miho; Mizuguchi, Hiroyuki

2012-01-01

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity. PMID:22068426

Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

PubMed

Wang, Zhongde

2011-01-01

Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

PubMed

Quintana-Bustamante, Oscar; Segovia, Jose C

2016-01-01

Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

A method for deriving homogenous population of oligodendrocytes from mouse embryonic stem cells.

PubMed

Neman, J; de Vellis, J

2012-06-01

There is a pressing need for new therapeutics for the generation and transplantation of oligodendrocyte to the white matter to help replace and render injured cells that are lost in demyelinating disease. There are a few protocols describing a homogenous derivation of non-manipulated mouse embryonic stem cells to oligodendrocytes (ES-OL). Moreover, protocols that are successful in producing ES-OL do so with low efficiency. Therefore, we describe clear methodology for differentiation of mouse ES cells to oligodendrocyte to a high degree of homogenity and reproducibility in vitro. In addition, taking advantage of three defined media, we can generate a defined ES to oligodendrocyte lineage while selecting against neurons and astrocytes. More specifically, (1) Glial stem cell defining media (GSCDM), supplemented with appropriate combination of SHH and RA support pro-oligodendrocyte developing neural spheres from ES cells, (2) Oligodendrocyte differentiating media, induces lineage selection of oligodendrocytes progenitors from neural stem cells, and (3) Oligodendrocyte maturation media, supports oligodendrocytes progenitor maturation. Moreover, the ES cell derived oligodendrocytes display mature properites in the prescence of rat dorsal root gangila in vitro. Thus confirming thier potential for use to invesitgate developmental pathways and future potential use of cells in transplantation towards myelin repair. Copyright © 2012 Wiley Periodicals, Inc.

Synchrotron Radiation X-Ray Microfluorescence Reveals Polarized Distribution of Atomic Elements during Differentiation of Pluripotent Stem Cells

PubMed Central

Paulsen, Bruna S.; Rehen, Stevens K.

2011-01-01

The mechanisms underlying pluripotency and differentiation in embryonic and reprogrammed stem cells are unclear. In this work, we characterized the pluripotent state towards neural differentiated state through analysis of trace elements distribution using the Synchrotron Radiation X-ray Fluorescence Spectroscopy. Naive and neural-stimulated embryoid bodies (EB) derived from embryonic and induced pluripotent stem (ES and iPS) cells were irradiated with a spatial resolution of 20 µm to make elemental maps and qualitative chemical analyses. Results show that these embryo-like aggregates exhibit self-organization at the atomic level. Metallic elements content rises and consistent elemental polarization pattern of P and S in both mouse and human pluripotent stem cells were observed, indicating that neural differentiation and elemental polarization are strongly correlated. PMID:22195032

Bioluminescent imaging demonstrates transplanted human embryonic stem cell-derived CD34+ cells preferentially develop into endothelial cells

PubMed Central

Tian, Xinghui; Hexum, Melinda K.; Penchev, Vesselin R.; Taylor, Russell J.; Shultz, Leonard D.; Kaufman, Dan S

2010-01-01

Human embryonic stem cells (hESCs) provide an important resource for novel regenerative medicine therapies and have been used to derive diverse cell populations, including hematopoietic and endothelial cells. However, it remains a challenge to achieve significant engraftment of hESC-derived blood cells when transplanted into animal models. To better understand mechanisms that enhance or limit the in vivo developmental potential of hESC-derived cells, we utilized hESCs that express firefly luciferase (luc) to allow non-invasive, real-time bioluminescent imaging of hESC-derived CD34+ cells transplanted into the liver of neonatal immunodeficient mice. Serial imaging demonstrated stable engraftment and expansion of the luc+ hESC-derived cells in vivo over several months. While we found that these hESC-derived CD34+ cells have bipotential ability to generate both hematopoietic and endothelial lineages in vitro, these studies demonstrate preferential differentiation into endothelial cells in vivo, with only low levels of hematopoietic cell engraftment. Therefore, these studies reveal key differences in the developmental potential of hESC-derived cells using in vitro and in vivo analyses. While transplanted hESC-derived CD34+ cells are well suited for revascularization therapies, additional measures are needed to provide higher levels of long-term hematopoietic engraftment. PMID:19711457

Unsuccessful derivation of human embryonic stem cell lines from pairs of human blastomeres.

PubMed

Fong, Chui-Yee; Richards, Mark; Bongso, Ariff

2006-08-01

Human embryonic stem cells (hESC) that differentiate into all three primordial germ layers have been established. Differentiation of these cells into desirable lineages offers hope for future transplantation therapies. Currently, hESC lines are derived from the inner cell mass (ICM) of blastocysts, leading to destruction of the embryo, and thus the process is ethically controversial. Successful attempts at deriving hESC lines from blastomeres without destruction of the ensuing embryo have not been reported. One or two blastomeres are routinely biopsied from 8-cell embryos for preimplantation genetic diagnosis. In this study it was therefore attempted to derive hESC lines from paired blastomeres. Of 66 pairs of 8-cell stage blastomeres, four pairs produced two morula and two blastocyst-like structures. When plated on mitomycin-C-treated mouse embryonic fibroblasts, one morula and one blastocyst-like structure separately produced small colonies containing hESC-like cells with prominent nucleoli and high nuclear-cytoplasmic ratios. When these colonies were detached and plated onto fresh feeders, there was no further colony formation or ensuing hESC lines. The results showed that it might not be possible to derive hESC lines directly from paired blastomeres. A minimum number of blastomeres in close contact with one another may be required to successfully generate an hESC line as blastomeres, like ICM and hESC cells, may be 'social' cells.

Development and production of good manufacturing practice grade human embryonic stem cell lines as source material for clinical application.

PubMed

De Sousa, P A; Downie, J M; Tye, B J; Bruce, K; Dand, P; Dhanjal, S; Serhal, P; Harper, J; Turner, M; Bateman, M

2016-09-01

From 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources. Copyright © 2016. Published by Elsevier B.V.

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

PubMed

Woll, Petter S; Martin, Colin H; Miller, Jeffrey S; Kaufman, Dan S

2005-10-15

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

Engineering Robust and Functional Vascular Networks in Vivo with Human Adult and Cord Blood-Derived Progenitor Cells

DTIC Science & Technology

2008-12-01

for other sources of ECs such as those derived from embryonic and adult progenitor cells ( Rafii ; Lyden 2003). For example, human ES-derived...functional endothelial precursors. Blood, 95, 952-958. Rafii , S., and D. Lyden, 2003: Therapeutic stem and progenitor cell transplantation for

Histone Deacetylase Inhibitors in Cell Pluripotency, Differentiation, and Reprogramming

PubMed Central

Kretsovali, Androniki; Hadjimichael, Christiana; Charmpilas, Nikolaos

2012-01-01

Histone deacetylase inhibitors (HDACi) are small molecules that have important and pleiotropic effects on cell homeostasis. Under distinct developmental conditions, they can promote either self-renewal or differentiation of embryonic stem cells. In addition, they can promote directed differentiation of embryonic and tissue-specific stem cells along the neuronal, cardiomyocytic, and hepatic lineages. They have been used to facilitate embryo development following somatic cell nuclear transfer and induced pluripotent stem cell derivation by ectopic expression of pluripotency factors. In the latter method, these molecules not only increase effectiveness, but can also render the induction independent of the oncogenes c-Myc and Klf4. Here we review the molecular pathways that are involved in the functions of HDAC inhibitors on stem cell differentiation and reprogramming of somatic cells into pluripotency. Deciphering the mechanisms of HDAC inhibitor actions is very important to enable their exploitation for efficient and simple tissue regeneration therapies. PMID:22550500

[Low expression of activin A in mouse and human embryonic teratocarcinoma cells].

PubMed

Gordeeva, O F

2014-01-01

TGFP3 family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFbeta family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGF@[beta] family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty 1, TGFbeta1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFbeta family (ACTIVINA, NODAL, LEFTY 1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells: Thus, in mouse and human nullipotent teratocarcinoma cells, theexpression of ActivinA is significantly reduced com- pared ivith embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.

Generation of glucose-sensitive insulin-secreting beta-like cells from human embryonic stem cells by incorporating a synthetic lineage-control network.

PubMed

Saxena, Pratik; Bojar, Daniel; Zulewski, Henryk; Fussenegger, Martin

2017-10-10

We previously reported novel technology to differentiate induced pluripotent stem cells (IPSCs) into glucose-sensitive insulin-secreting beta-like cells by engineering a synthetic lineage-control network regulated by the licensed food additive vanillic acid. This genetic network was able to program intricate expression dynamics of the key transcription factors Ngn3 (neurogenin 3, OFF-ON-OFF), Pdx1 (pancreatic and duodenal homeobox 1, ON-OFF-ON) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A, OFF-ON) to guide the differentiation of IPSC-derived pancreatic progenitor cells to beta-like cells. In the present study, we show for the first time that this network can also program the expression dynamics of Ngn3, Pdx1 and MafA in human embryonic stem cell (hESC)-derived pancreatic progenitor cells and drive differentiation of these cells into glucose-sensitive insulin-secreting beta-like cells. Therefore, synthetic lineage-control networks appear to be a robust methodology for differentiating pluripotent stem cells into somatic cell types for basic research and regenerative medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures - a new donor for cell therapy.

PubMed

Wu, Wei; Zeng, Yuxiao; Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

2016-04-19

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution

PubMed Central

Spiewak, Jessica E.

2014-01-01

Summary Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage and mate choice and have played important roles in speciation. Here, we review recent studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns. PMID:25421288

Adult Human Nasal Mesenchymal-Like Stem Cells Restore Cochlear Spiral Ganglion Neurons After Experimental Lesion

PubMed Central

Bas, Esperanza; Van De Water, Thomas R.; Lumbreras, Vicente; Rajguru, Suhrud; Goss, Garrett; Hare, Joshua M.

2014-01-01

A loss of sensory hair cells or spiral ganglion neurons from the inner ear causes deafness, affecting millions of people. Currently, there is no effective therapy to repair the inner ear sensory structures in humans. Cochlear implantation can restore input, but only if auditory neurons remain intact. Efforts to develop stem cell-based treatments for deafness have demonstrated progress, most notably utilizing embryonic-derived cells. In an effort to bypass limitations of embryonic or induced pluripotent stem cells that may impede the translation to clinical applications, we sought to utilize an alternative cell source. Here, we show that adult human mesenchymal-like stem cells (MSCs) obtained from nasal tissue can repair spiral ganglion loss in experimentally lesioned cochlear cultures from neonatal rats. Stem cells engraft into gentamicin-lesioned organotypic cultures and orchestrate the restoration of the spiral ganglion neuronal population, involving both direct neuronal differentiation and secondary effects on endogenous cells. As a physiologic assay, nasal MSC-derived cells engrafted into lesioned spiral ganglia demonstrate responses to infrared laser stimulus that are consistent with those typical of excitable cells. The addition of a pharmacologic activator of the canonical Wnt/β-catenin pathway concurrent with stem cell treatment promoted robust neuronal differentiation. The availability of an effective adult autologous cell source for inner ear tissue repair should contribute to efforts to translate cell-based strategies to the clinic. PMID:24172073

Lower Oncogenic Potential of Human Mesenchymal Stem Cells Derived from Cord Blood Compared to Induced Pluripotent Stem Cells

PubMed Central

Foroutan, T.; Najmi, M.; Kazemi, N.; Hasanlou, M.; Pedram, A.

2015-01-01

Background: In regenerative medicine, use of each of the mesenchymal stem cells derived from bone marrow, cord blood, and adipose tissue, has several cons and pros. Mesenchymal stem cells derived from cord blood have been considered the best source for precursor transplantation. Direct reprogramming of a somatic cell into induced pluripotent stem cells by over-expression of 6 transcription factors Oct4, Sox2, Klf4, lin28, Nanog, and c-Myc has great potential for regenerative medicine, eliminating the ethical issues of embryonic stem cells and the rejection problems of using non-autologous cells. Objective: To compare reprogramming and pluripotent markers OCT4, Sox-2, c-Myc, Klf4, Nanog, and lin28 in mesenchymal stem cells derived from cord blood and induced pluripotent stem cells. Methods: We analyzed the expression level of OCT4, Sox-2, c-Myc, Klf4, Nanog and lin28 genes in human mesenchymal stem cells derived from cord blood and induced pluripotent stem cells by cell culture and RT-PCR. Results: The expression level of pluripotent genes OCT4 and Sox-2, Nanog and lin28 in mesenchymal stem cells derived from cord blood were significantly higher than those in induced pluripotent stem cells. In contrast to OCT-4A and Sox-2, Nanog and lin28, the expression level of oncogenic factors c-Myc and Klf4 were significantly higher in induced pluripotent stem cells than in mesenchymal stem cells derived from cord blood. Conclusion: It could be concluded that mesenchymal stem cells derived from human cord blood have lower oncogenic potential compared to induced pluripotent stem cells. PMID:26306155

Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking.

PubMed

Kallur, Therése; Blomberg, Pontus; Stenfelt, Sonya; Tryggvason, Kristian; Hovatta, Outi

2017-01-01

For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).

Can bone marrow differentiate into renal cells?

PubMed

Imai, Enyu; Ito, Takahito

2002-10-01

A considerable plasticity of adult stem cells has been confirmed in a wide variety of tissues. In particular, the pluripotency of bone marrow-derived stem cells may influence the regeneration of injured tissues and may provide novel avenues in regenerative medicine. Bone marrow contains at least hematopoietic and mesenchymal stem cells, and both can differentiate into a wide range of differentiated cells. Side population (SP) cells, which are originally defined in bone marrow cells by high efflux of DNA-binding dye, seem to be a new class of multipotent stem cells. Irrespective of the approach used to obtain stem cells, the fates of marrow-derived cells following bone marrow transplantation can be traced by labeling donor cells with green fluorescence protein or by identifying donor Y chromosome in female recipients. So far, bone marrow-derived cells have been reported to differentiate into renal cells, including mesangial cells, endothelial cells, podocytes, and tubular cells in the kidney, although controversy exists. Further studies are required to address this issue. Cell therapy will be promising when we learn to control stem cells such as bone marrow-derived stem cells, embryonic stem cells, and resident stem cells in the kidney. Identification of factors that support stem cells or promote their differentiation should provide a relevant step towards cell therapy.

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

PubMed Central

Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

2016-01-01

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

PubMed

Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

2011-11-01

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

Isolation and Characterization of Node/Notochord-Like Cells from Mouse Embryonic Stem Cells

PubMed Central

Winzi, Maria K.; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

2014-01-01

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP+ cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen’s node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures. PMID:21351873

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.

Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress

PubMed Central

Asensi, Karina D; Fortunato, Rodrigo S; dos Santos, Danúbia S; Pacheco, Thaísa S; de Rezende, Danielle F; Rodrigues, Deivid C; Mesquita, Fernanda C P; Kasai-Brunswick, Tais H; de Carvalho, Antonio C Campos; Carvalho, Denise P; Carvalho, Adriana B; Goldenberg, Regina C dos S

2014-01-01

Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H2O2, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS. PMID:24528612

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic Lineage Tracing of Non-Myocyte Population by Dual Recombinases.

PubMed

Li, Yan; He, Lingjuan; Huang, Xiuzhen; Issa Bhaloo, Shirin; Zhao, Huan; Zhang, Shaohua; Pu, Wenjuan; Tian, Xueying; Li, Yi; Liu, Qiaozhen; Yu, Wei; Zhang, Libo; Liu, Xiuxiu; Liu, Kuo; Tang, Juan; Zhang, Hui; Cai, Dongqing; Adams, Ralf H; Xu, Qingbo; Lui, Kathy O; Zhou, Bin

2018-04-26

Background -Whether the adult mammalian heart harbors cardiac stem cells (CSCs) for regeneration of cardiomyocytes is an important yet contentious topic in the field of cardiovascular regeneration. The putative myocyte stem cell populations recognized without specific cell markers such as the cardiosphere-derived cells or with markers such as Sca1 + , Bmi1 + , Isl1 + or Abcg2 + CSCs have been reported. Moreover, it remains unclear whether putative CSCs with unknown or unidentified markers exist and give rise to de novo cardiomyocytes in the adult heart. Methods -To address this question without relying on a particular stem cell marker, we developed a new genetic lineage tracing system to label all non-myocyte populations that contain putative CSCs. Using dual lineage tracing system, we assessed if non-myocytes generated any new myocytes during embryonic development, adult homeostasis and after myocardial infarction. Skeletal muscle was also examined after injury for internal control of new myocytes generation from non-myocytes. Results -By this stem cell marker-free and dual recombinases-mediated cell tracking approach, our fate mapping data show that new myocytes arise from non-myocytes in the embryonic heart, but not in the adult heart during homeostasis or after myocardial infarction. As positive control, our lineage tracing system detected new myocytes derived from non-myocytes in the skeletal muscle after injury. Conclusions -This study provides in vivo genetic evidence for non-myocyte to myocyte conversion in embryonic but not adult heart, arguing again the myogenic potential of putative stem cell populations for cardiac regeneration in the adult stage. This study also provides a new genetic strategy to identify endogenous stem cells, if any, in other organ systems for tissue repair and regeneration.

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.

The miR-290-295 cluster as multi-faceted players in mouse embryonic stem cells.

PubMed

Yuan, Kai; Ai, Wen-Bing; Wan, Lin-Yan; Tan, Xiao; Wu, Jiang-Feng

2017-01-01

Increasing evidence indicates that embryonic stem cell specific microRNAs (miRNAs) play an essential role in the early development of embryo. Among them, the miR-290-295 cluster is the most highly expressed in the mouse embryonic stem cells and involved in various biological processes. In this paper, we reviewed the research progress of the function of the miR-290-295 cluster in embryonic stem cells. The miR-290-295 cluster is involved in regulating embryonic stem cell pluripotency maintenance, self-renewal, and reprogramming somatic cells to an embryonic stem cell-like state. Moreover, the miR-290-295 cluster has a latent pro-survival function in embryonic stem cells and involved in tumourigenesis and senescence with a great significance. Elucidating the interaction between the miR-290-295 cluster and other modes of gene regulation will provide us new ideas on the biology of pluripotent stem cells. In the near future, the broad prospects of the miRNA cluster will be shown in the stem cell field, such as altering cell identities with high efficiency through the transient introduction of tissue-specific miRNA cluster.

Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease.

PubMed

Chumarina, Margarita; Azevedo, Carla; Bigarreau, Julie; Vignon, Clémentine; Kim, Kwang-Soo; Li, Jia-Yi; Roybon, Laurent

2017-03-01

Mouse embryonic stem cell (mESC) lines were derived by crossing heterozygous transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase (TH) promoter, with homozygous alpha-synuclein (aSYN) mice expressing human mutant SNCA A53T under the control of the mouse Prion promoter (MoPrP), or wildtype (WT) mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Transcriptional Differences between Rhesus Embryonic Stem Cells Generated from In Vitro and In Vivo Derived Embryos

PubMed Central

Harvey, Alexandra J.; Mao, Shihong; Lalancette, Claudia; Krawetz, Stephen A.; Brenner, Carol A.

2012-01-01

Numerous studies have focused on the transcriptional signatures that underlie the maintenance of embryonic stem cell (ESC) pluripotency. However, it remains unclear whether ESC retain transcriptional aberrations seen in in vitro cultured embryos. Here we report the first global transcriptional profile comparison between ESC generated from either in vitro cultured or in vivo derived primate embryos by microarray analysis. Genes involved in pluripotency, oxygen regulation and the cell cycle were downregulated in rhesus ESC generated from in vitro cultured embryos (in vitro ESC). Significantly, several gene differences are similarly downregulated in preimplantation embryos cultured in vitro, which have been associated with long term developmental consequences and disease predisposition. This data indicates that prior to derivation, embryo quality may influence the molecular signature of ESC lines, and may differentially impact the physiology of cells prior to or following differentiation. PMID:23028448

Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model.

PubMed

Lotfinia, Majid; Kadivar, Mehdi; Piryaei, Abbas; Pournasr, Behshad; Sardari, Soroush; Sodeifi, Niloofar; Sayahpour, Forugh-Azam; Baharvand, Hossein

2016-12-15

Adult tissue-derived mesenchymal stem cells (MSCs) show tremendous promise for a wide array of therapeutic applications predominantly through paracrine activity. Recent reports showed that human embryonic stem cell (ESC)-derived MSCs are an alternative for regenerative cellular therapy due to manufacturing large quantities of MSCs from a single donor. However, no study has been reported to uncover the secretome of human ESC-MSCs as treatment of an acute liver failure (ALF) mouse model. We demonstrated that human ESC-MSCs showed similar morphology and cell surface markers compared with bone marrow-derived MSCs. ESC-MSCs exhibited a higher growth rate during early in vitro expansion, along with adipogenic and osteogenic differentiation potential. Treatment with ESC-MSC-conditioned medium (CM) led to statistically significant enhancement of primary hepatocyte viability and increased immunomodulatory interleukin-10 secretion from lipopolysaccharide-induced human blood mononuclear cells. Analysis of the MSCs secretome by a protein array screen showed an association between higher frequencies of secretory proteins such as vascular endothelial growth factor (VEGF) and regulation of cell proliferation, cell migration, the development process, immune system process, and apoptosis. In this thioacetamide-induced mouse model of acute liver injury, we observed that systemic infusion of VEGF led to significant survival. These data have provided the first experimental evidence of the therapeutic potential of human ESC-MSC-derived molecules. These molecules show trophic support to hepatocytes, which potentially creates new avenues for the treatment of ALF, as an inflammatory condition.

Derivation of porcine pluripotent stem cells for biomedical research.

PubMed

Shiue, Yow-Ling; Yang, Jenn-Rong; Liao, Yu-Jing; Kuo, Ting-Yung; Liao, Chia-Hsin; Kang, Ching-Hsun; Tai, Chein; Anderson, Gary B; Chen, Lih-Ren

2016-07-01

Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

PubMed

Yoshie, Sachiko; Ogasawara, Yuki; Ikehata, Masateru; Ishii, Kazuyuki; Suzuki, Yukihisa; Wada, Keiji; Wake, Kanako; Nakasono, Satoshi; Taki, Masao; Ohkubo, Chiyoji

2016-01-01

The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF-MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF-MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF-MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2 , and an early developmental gene, Hba-x , in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc . in our daily lives, these results suggested that IF-MF in which the public is exposed to in general living environment would not have embryotoxic effect.

[Embryos and embryo-like entities: problem of definition in the draft of the Swiss embryonic research law].

PubMed

Bürgin, M T; Bürkli, P

2002-11-01

At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.

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.

Differential developmental ability of embryos cloned from tissue-specific stem cells.

PubMed

Inoue, Kimiko; Noda, Shinichi; Ogonuki, Narumi; Miki, Hiromi; Inoue, Shinichi; Katayama, Kazufumi; Mekada, Kazuyuki; Miyoshi, Hiroyuki; Ogura, Atsuo

2007-05-01

Although cloning animals by somatic cell nuclear transfer is generally inefficient, the use of certain nuclear donor cell types may significantly improve or deteriorate outcomes. We evaluated whether two multipotent stem cell lines produced in vitro--neural stem cells (NSCs) and mesenchymal stem cells (MSCs)--could serve as nuclear donors for nuclear transfer cloning. Most (76%) NSC-derived embryos survived the two-cell-to-four-cell transition, the stage when the major zygotic gene activation occurs. Consistent with this observation, the expression patterns of zygotically active genes were better in NSC-derived embryos than in fibroblast clone embryos, which arrested at the two-cell stage more frequently. Embryo transfer experiments demonstrated that at least some of these NSC embryos had the ability to develop to term fetuses (1.6%, 3/189). In contrast, embryos reconstructed using MSCs showed a low rate of in vitro development and never underwent implantation in vivo. Chromosomal analysis of the donor MSCs revealed very frequent aneuploidy, which probably impaired the potential for development of their derived clones. This is the first demonstration that tissue-specific multipotent stem cells produced in vitro can serve as donors of nuclei for cloning mice; however, these cells may be prone to chromosomal aberrations, leading to high embryonic death rates. We found previously that hematopoietic stem cells (HSCs) are very inefficient donor cells because of their failure to activate the genes essential for embryonic development. Taken together, our data led us to conclude that tissue-specific stem cells in mice, namely NSCs, MSCs, and HSCs, exhibited marked variations in the ability to produce cloned offspring and that this ability varies according to both the epigenetic and genetic status of the original genomes. Disclosure of potential conflicts of interest is found at the end of this article.

Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

PubMed

Zhao, Dongxin; Chen, Song; Cai, Jun; Guo, Yushan; Song, Zhihua; Che, Jie; Liu, Chun; Wu, Chen; Ding, Mingxiao; Deng, Hongkui

2009-07-31

The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

PubMed

Thyagarajan, Bhaskar; Scheyhing, Kelly; Xue, Haipeng; Fontes, Andrew; Chesnut, Jon; Rao, Mahendra; Lakshmipathy, Uma

2009-03-01

Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells.

PubMed

Barbet, Romain; Peiffer, Isabelle; Hatzfeld, Antoinette; Charbord, Pierre; Hatzfeld, Jacques A

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

Large Scale Production of Stem Cells and Their Derivatives

NASA Astrophysics Data System (ADS)

Zweigerdt, Robert

Stem cells have been envisioned to become an unlimited cell source for regenerative medicine. Notably, the interest in stem cells lies beyond direct therapeutic applications. They might also provide a previously unavailable source of valuable human cell types for screening platforms, which might facilitate the development of more efficient and safer drugs. The heterogeneity of stem cell types as well as the numerous areas of application suggests that differential processes are mandatory for their in vitro culture. Many of the envisioned applications would require the production of a high number of stem cells and their derivatives in scalable, well-defined and potentially clinical compliant manner under current good manufacturing practice (cGMP). In this review we provide an overview on recent strategies to develop bioprocesses for the expansion, differentiation and enrichment of stem cells and their progenies, presenting examples for adult and embryonic stem cells alike.

In Vitro Differentiation and Propagation of Urothelium from Pluripotent Stem Cell Lines.

PubMed

Osborn, Stephanie L; Kurzrock, Eric A

2018-01-01

Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

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.

Doxycycline Enhances Survival and Self-Renewal of Human Pluripotent Stem Cells

PubMed Central

Chang, Mi-Yoon; Rhee, Yong-Hee; Yi, Sang-Hoon; Lee, Su-Jae; Kim, Rae-Kwon; Kim, Hyongbum; Park, Chang-Hwan; Lee, Sang-Hun

2014-01-01

Summary We here report that doxycycline, an antibacterial agent, exerts dramatic effects on human embryonic stem and induced pluripotent stem cells (hESC/iPSCs) survival and self-renewal. The survival-promoting effect was also manifest in cultures of neural stem cells (NSCs) derived from hESC/iPSCs. These doxycycline effects are not associated with its antibacterial action, but mediated by direct activation of a PI3K-AKT intracellular signal. These findings indicate doxycycline as a useful supplement for stem cell cultures, facilitating their growth and maintenance. PMID:25254347

Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast.

PubMed

Acampora, Dario; Omodei, Daniela; Petrosino, Giuseppe; Garofalo, Arcomaria; Savarese, Marco; Nigro, Vincenzo; Di Giovannantonio, Luca Giovanni; Mercadante, Vincenzo; Simeone, Antonio

2016-06-21

Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Treating Diet-Induced Diabetes and Obesity with Human Embryonic Stem Cell-Derived Pancreatic Progenitor Cells and Antidiabetic Drugs

PubMed Central

Bruin, Jennifer E.; Saber, Nelly; Braun, Natalie; Fox, Jessica K.; Mojibian, Majid; Asadi, Ali; Drohan, Campbell; O’Dwyer, Shannon; Rosman-Balzer, Diana S.; Swiss, Victoria A.; Rezania, Alireza; Kieffer, Timothy J.

2015-01-01

Summary Human embryonic stem cell (hESC)-derived pancreatic progenitor cells effectively reverse hyperglycemia in rodent models of type 1 diabetes, but their capacity to treat type 2 diabetes has not been reported. An immunodeficient model of type 2 diabetes was generated by high-fat diet (HFD) feeding in SCID-beige mice. Exposure to HFDs did not impact the maturation of macroencapsulated pancreatic progenitor cells into glucose-responsive insulin-secreting cells following transplantation, and the cell therapy improved glucose tolerance in HFD-fed transplant recipients after 24 weeks. However, since diet-induced hyperglycemia and obesity were not fully ameliorated by transplantation alone, a second cohort of HFD-fed mice was treated with pancreatic progenitor cells combined with one of three antidiabetic drugs. All combination therapies rapidly improved body weight and co-treatment with either sitagliptin or metformin improved hyperglycemia after only 12 weeks. Therefore, a stem cell-based therapy may be effective for treating type 2 diabetes, particularly in combination with antidiabetic drugs. PMID:25801507

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

PubMed Central

Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

2012-01-01

Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-29

...; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH... Embryonic Stem Cell Line to be Approved for Use in NIH-Funded Research, 0925-0601, Expiration Date 04/30... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be...

Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences

PubMed Central

Yu, Junying; Hu, Kejin; Smuga-Otto, Kim; Tian, Shulan; Stewart, Ron; Slukvin, Igor I.; Thomson, James A.

2009-01-01

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. Here we describe the derivation of human iPS cells using non-integrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells. PMID:19325077

Derivation of the King's College London human embryonic stem cell lines.

PubMed

Stephenson, Emma L; Braude, Peter R

2010-04-01

Since the derivation of the first human embryonic stem cell (hESC) line in 1998, there has been substantial interest in the potential of these cells for regenerative medicine and cell therapy and in the use of hESCs carrying clinically relevant genetic mutations as models for disease research and therapeutic target identification. There is still a need to improve derivation efficiency and further the understanding of the basic biology of these cells and to develop clinical grade culture systems with the aim of producing cell lines suitable for subsequent manipulation for therapy. The derivation of initial hESC lines at King's College London is discussed here, with focus on derivation methodology. Each of the derivations was distinctive. Although the stage and morphology of each blastocyst were generally similar in each attempt, the behaviour of the colonies was unpredictable; colony morphology and development was different with each attempt. Days 5, 6 and 7 blastocysts were used successfully, and the number of days until appearance of stem-like cells varied from 4 to 14 d. Routine characterisation analyses were performed on three lines, all of which displayed appropriate marker expression and survived cryopreservation-thaw cycles. From the lines discussed, four are at various stages of the deposition process with the UKSCB, one is pending submission and two are unsuitable for banking. Continued open and transparent reporting of results and collaborations will maximise the efficiency of derivation and facilitate the development of standardised protocols for the derivation and early culture of hESC lines.

Novel developmental biology-based protocol of embryonic stem cell differentiation to morphologically sound and functional yet immature hepatocytes.

PubMed

Bukong, Terence N; Lo, Tracie; Szabo, Gyongyi; Dolganiuc, Angela

2012-05-01

Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites. Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays. Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase. We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation. © 2012 John Wiley & Sons A/S.

Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

PubMed

Fu, Shangfeng; Ding, Jianwu; Liu, Dewu; Huang, Heping; Li, Min; Liu, Yang; Tu, Longxiang; Liu, Deming

2018-01-01

Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell‑based skin tissue engineering in the future.

Embryonic stem cells: testing the germ-cell theory.

PubMed

Hochedlinger, Konrad

2011-10-25

The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state. Copyright © 2011 Elsevier Ltd. All rights reserved.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

PubMed

Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

2006-11-01

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles.

PubMed

Oh, Jung-Hwa; Son, Mi-Young; Choi, Mi-Sun; Kim, Soojin; Choi, A-Young; Lee, Hyang-Ae; Kim, Ki-Suk; Kim, Janghwan; Song, Chang Woo; Yoon, Seokjoo

2016-05-15

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10-200μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24h. The results showed that Ag NPs evoked significant toxicity in hESC-derived NPCs at 24h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

Safety paradigm: genetic evaluation of therapeutic grade human embryonic stem cells.

PubMed

Stephenson, Emma; Ogilvie, Caroline Mackie; Patel, Heema; Cornwell, Glenda; Jacquet, Laureen; Kadeva, Neli; Braude, Peter; Ilic, Dusko

2010-12-06

The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

PubMed

Pandolfini, Luca; Luzi, Ettore; Bressan, Dario; Ucciferri, Nadia; Bertacchi, Michele; Brandi, Rossella; Rocchiccioli, Silvia; D'Onofrio, Mara; Cremisi, Federico

2016-05-06

Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.

PubMed

Keirstead, Hans S; Nistor, Gabriel; Bernal, Giovanna; Totoiu, Minodora; Cloutier, Frank; Sharp, Kelly; Steward, Oswald

2005-05-11

Demyelination contributes to loss of function after spinal cord injury, and thus a potential therapeutic strategy involves replacing myelin-forming cells. Here, we show that transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into adult rat spinal cord injuries enhances remyelination and promotes improvement of motor function. OPCs were injected 7 d or 10 months after injury. In both cases, transplanted cells survived, redistributed over short distances, and differentiated into oligodendrocytes. Animals that received OPCs 7 d after injury exhibited enhanced remyelination and substantially improved locomotor ability. In contrast, when OPCs were transplanted 10 months after injury, there was no enhanced remyelination or locomotor recovery. These studies document the feasibility of predifferentiating hESCs into functional OPCs and demonstrate their therapeutic potential at early time points after spinal cord injury.

Generating gene knockout rats by homologous recombination in embryonic stem cells

PubMed Central

Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

2013-01-01

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

PubMed

Tan, Xiaobing; Dai, Qingli; Guo, Tao; Xu, Jingshu; Dai, Qingyuan

2018-01-22

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy. Copyright © 2017. Published by Elsevier Inc.

Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches

PubMed Central

Toyoshima, Koh-ei; Asakawa, Kyosuke; Ishibashi, Naoko; Toki, Hiroshi; Ogawa, Miho; Hasegawa, Tomoko; Irié, Tarou; Tachikawa, Tetsuhiko; Sato, Akio; Takeda, Akira; Tsuji, Takashi

2012-01-01

Organ replacement regenerative therapy is purported to enable the replacement of organs damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully functional hair organ regeneration via the intracutaneous transplantation of a bioengineered pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skin-derived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered hair follicle develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered follicles also show restored hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. This study thus reveals the potential applications of adult tissue-derived follicular stem cells as a bioengineered organ replacement therapy. PMID:22510689

Morphology and vasoactive hormone profiles from endothelial cells derived from stem cells of different sources.

PubMed

Reed, Daniel M; Foldes, Gabor; Kirkby, Nicholas S; Ahmetaj-Shala, Blerina; Mataragka, Stefania; Mohamed, Nura A; Francis, Catherine; Gara, Edit; Harding, Sian E; Mitchell, Jane A

2014-12-12

Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation. Copyright © 2014 Elsevier Inc. All rights reserved.

A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

PubMed

Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

2008-10-01

The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

PubMed

Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

PubMed Central

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant. PMID:20587750

The Role of H1 Linker Histone Subtypes in Preserving the Fidelity of Elaboration of Mesendodermal and Neuroectodermal Lineages during Embryonic Development

PubMed Central

Nguyen, Giang D.; Gokhan, Solen; Molero, Aldrin E.; Yang, Seung-Min; Kim, Byung-Ju; Skoultchi, Arthur I.; Mehler, Mark F.

2014-01-01

H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs) depleted of H1c, H1d and H1e subtypes (H1-KO ESCs) by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers. PMID:24802750

Can mammalian cloning combined with embryonic stem cell technologies be used to treat human diseases?

PubMed Central

Hadjantonakis, Anna-Katerina; Papaioannou, Virginia E

2002-01-01

Cloning is commonly perceived as a means of generating genetically identical individuals, but it can also be used to obtain genetically matched embryo-derived stem cells, which could potentially be used in the treatment of patients. A recent report offers the first 'proof of principle' of such cloning for therapeutic purposes, referred to as nuclear transplantation to produce stem cells for autologous transplantation. PMID:12186652

Production of urothelium from pluripotent stem cells for regenerative applications.

PubMed

Osborn, Stephanie L; Kurzrock, Eric A

2015-01-01

As bladder reconstruction strategies evolve, a feasible and safe source of transplantable urothelium becomes a major consideration for patients with advanced bladder disease, particularly cancer. Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are attractive candidates from which to derive urothelium as they renew and proliferate indefinitely in vitro and fulfill the non-autologous and/or non-urologic criteria, respectively, that is required for many patients. This review presents the latest advancements in differentiating urothelium from pluripotent stem cells in vitro in the context of current bladder tissue engineering strategies.

Tracing the destiny of mesenchymal stem cells from embryo to adult bone marrow and white adipose tissue via Pdgfrα expression.

PubMed

Miwa, Hiroyuki; Era, Takumi

2018-01-29

Mesenchymal stem cells (MSCs) are somatic stem cells that can be derived from adult bone marrow (BM) and white adipose tissue (WAT), and that display multipotency and self-renewal capacity. Although MSCs are essential for tissue formation and have already been used in clinical therapy, the origins and markers of these cells remain unknown. In this study, we first investigated the developmental process of MSCs in mouse embryos using the gene encoding platelet-derived growth factor receptor α ( Pdgfra ) as a marker. We then traced cells expressing Pdgfra and other genes (brachyury, Sox1 and Pmx1 ) in various mutant mouse embryos until the adult stage. This tracing of MSC origins and destinies indicates that embryonic MSCs emerge in waves and that almost all adult BM MSCs and WAT MSCs originate from mesoderm and embryonic Pdgfrα-positive cells. Furthermore, we demonstrate that adult Pdgfrα-positive cells are involved in some pathological conditions. © 2018. Published by The Company of Biologists Ltd.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells.

PubMed

Liu, Ying; Giannopoulou, Eugenia G; Wen, Duancheng; Falciatori, Ilaria; Elemento, Olivier; Allis, C David; Rafii, Shahin; Seandel, Marco

2016-04-27

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells

PubMed Central

Liu, Ying; Giannopoulou, Eugenia G.; Wen, Duancheng; Falciatori, Ilaria; Elemento, Olivier; Allis, C. David; Rafii, Shahin; Seandel, Marco

2016-01-01

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming. PMID:27117588

In vitro differentiation of primordial germ cells and oocyte-like cells from stem cells.

PubMed

Costa, José J N; Souza, Glaucinete B; Soares, Maria A A; Ribeiro, Regislane P; van den Hurk, Robert; Silva, José R V

2018-02-01

Infertility is the result of failure due to an organic disorder of the reproductive organs, especially their gametes. Recently, much progress has been made on generating germ cells, including oocytes, from various types of stem cells. This review focuses on advances in female germ cell differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the derivation of female germ cells from several types of stem cells are also highlighted, as well as the ability of stem cells to generate mature and functional female gametes. This review shows that stem cell therapies have opened new frontiers in medicine, especially in the reproductive area, with the possibility of regenerating fertility.

Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

PubMed

Maya-Espinosa, Guadalupe; Collazo-Navarrete, Omar; Millán-Aldaco, Diana; Palomero-Rivero, Marcela; Guerrero-Flores, Gilda; Drucker-Colín, René; Covarrubias, Luis; Guerra-Crespo, Magdalena

2015-02-01

A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies. © 2014 AlphaMed Press.

Concise Review: One Stone for Multiple Birds: Generating Universally Compatible Human Embryonic Stem Cells.

PubMed

Zheng, Dejin; Wang, Xiaofang; Xu, Ren-He

2016-09-01

With ongoing clinical trials, human embryonic stem cells (hESCs) have shown substantial potential for regenerative medicine. However, due to the mismatch of human leukocyte antigens (HLAs) between hESC-derived allografts and recipients, immunosuppressant regimens must be used to prevent immune rejection of the grafts. Considerable efforts have been devoted to overcoming this hurdle via the derivation and banking of human nuclear transfer ESCs, parthenogenetic ESCs, and induced pluripotent stem cells. However, ethical and safety concerns remain, hindering the application of these types of pluripotent cells. Other approaches have recently been explored to generate universally compatible hESCs through the silencing or deletion of HLAs or genes essential for HLA expression, including β-2-microglobulin and class-II MHC transactivator, as well as the induction of immunosuppression via the ectopic expression of non-classical HLAs (e.g., HLA-E and -G), cytotoxic T lymphocyte antigen 4 fused with immunoglobulin, and programmed death ligand-1. In this review, we introduce developments in this line of research and discuss strategies to reduce the tumorigenic concerns regarding hESCs, especially after they acquire the capability to escape immune surveillance. Stem Cells 2016;34:2269-2275. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Chemotherapy-Induced Depletion of OCT4-Positive Cancer Stem Cells in a Mouse Model of Malignant Testicular Cancer.

PubMed

Pierpont, Timothy M; Lyndaker, Amy M; Anderson, Claire M; Jin, Qiming; Moore, Elizabeth S; Roden, Jamie L; Braxton, Alicia; Bagepalli, Lina; Kataria, Nandita; Hu, Hilary Zhaoxu; Garness, Jason; Cook, Matthew S; Capel, Blanche; Schlafer, Donald H; Southard, Teresa; Weiss, Robert S

2017-11-14

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Three-dimensional epithelial tissues generated from human embryonic stem cells.

PubMed

Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

2009-11-01

The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

21st Nantes Actualités Transplantation: "When Stem Cells Meet Immunology".

PubMed

Anegon, Ignacio; Nguyen, Tuan Huy

2017-01-01

"When Stem Cells Meet Immunology" has been the topic of the 21st annual "Nantes Actualités en Transplantation" meeting (June 9-10, 2016, Nantes, France). This meeting brought together pioneers and leading experts in the fields of stem cells, biomaterials and immunoregulation. Presentations covered multipotent (mesenchymal and hematopoietic) and pluripotent stem cells (embryonic and induced) for regenerative medicine of incurable diseases, immunotherapy and blood transfusions. An additional focus had been immune rejections and responses of allogeneic or autologous stem cells. Conversely, stem cells are also able to directly modulate the immune response through the production of immunoregulatory molecules. Moreover, stem cells may also provide an unlimited source of immune cells (DCs, NK cells, B cells, and T cells) that can operate as "super" immune cells, for example, through genetic engineering with chimeric antigen receptors.This meeting report puts presentations into an overall context highlighting new potential biomarkers for potency prediction of mesenchymal stem cell-derived and pluripotent stem cell-derived multicellular organoids. Finally, we propose future directions arising from the flourishing encounter of stem cell and immune biology.

The New Federalism: State Policies Regarding Embryonic Stem Cell Research.

PubMed

Acosta, Nefi D; Golub, Sidney H

2016-09-01

Stem cell policy in the United States is an amalgam of federal and state policies. The scientific development of human pluripotent embryonic stem cells (ESCs) triggered a contentious national stem cell policy debate during the administration of President George W. Bush. The Bush "compromise" that allowed federal funding to study only a very limited number of ESC derived cell lines did not satisfy either the researchers or the patient advocates who saw great medical potential being stifled. Neither more restrictive legislation nor expansion of federal funding proved politically possible and the federal impasse opened the door for a variety of state-based experiments. In 2004, California became the largest and most influential state venture into stem cell research by passing "Prop 71," a voter initiative that created a new stem cell agency and funded it with $3 billion. Several states followed suit with similar programs to protect the right of investigators to do stem cell research and in some cases to invest state funding in such projects. Other states devised legislation to restrict stem cell research and in five states, criminal penalties were included. Thus, the US stem cell policy is a patchwork of multiple, often conflicting, state and federal policies. © 2016 American Society of Law, Medicine & Ethics.

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

PubMed Central

Gori, Jennifer L.; Tian, Xinghui; Swanson, Debra; Gunther, Roland; Shultz, Leonard D.; McIvor, R. Scott; Kaufman, Dan S.

2009-01-01

SUMMARY Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase engraftment of gene-modified hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR – GFP expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR-cells in vivo, and provides a novel approach for combined human cell and gene therapy. PMID:19829316

Quantitative analysis of serotonin secreted by human embryonic stem cells-derived serotonergic neurons via pH-mediated online stacking-CE-ESI-MRM.

PubMed

Zhong, Xuefei; Hao, Ling; Lu, Jianfeng; Ye, Hui; Zhang, Su-Chun; Li, Lingjun

2016-04-01

A CE-ESI-MRM-based assay was developed for targeted analysis of serotonin released by human embryonic stem cells-derived serotonergic neurons in a chemically defined environment. A discontinuous electrolyte system was optimized for pH-mediated online stacking of serotonin. Combining with a liquid-liquid extraction procedure, LOD of serotonin in the Krebs'-Ringer's solution by CE-ESI-MS/MS on a 3D ion trap MS was0.15 ng/mL. The quantitative results confirmed the serotonergic identity of the in vitro developed neurons and the capacity of these neurons to release serotonin in response to stimulus. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reprogramming of Sheep Fibroblasts into Pluripotency under a Drug-Inducible Expression of Mouse-Derived Defined Factors

PubMed Central

Li, Yang; Cang, Ming; Lee, Andrew Stephen; Zhang, Kehua; Liu, Dongjun

2011-01-01

Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes. PMID:21253598

Phase resolved and coherence gated en face reflection imaging of multilayered embryonal carcinoma cells

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Fukami, Tadashi; Iwai, Hidenao; Yamashita, Yutaka

2012-03-01

Embryonal carcinoma (EC) cells, which are cell lines derived from teratocarcinomas, have characteristics in common with stem cells and differentiate into many kinds of functional cells. Similar to embryonic stem (ES) cells, undifferentiated EC cells form multi-layered spheroids. In order to visualize the three-dimensional structure of multilayered EC cells without labeling, we employed full-field interference microscopy with the aid of a low-coherence quantitative phase microscope, which is a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low-coherency of the light-source (halogen lamp), only the light reflected from reflective surface at a specific sectioning height generates an interference image on the CCD camera. P19CL6 EC cells, derived from mouse teratocarcinomas, formed spheroids that are about 50 to 200 micrometers in diameter. Since the height of each cell is around 10 micrometers, it is assumed that each spheroid has 5 to 20 cell layers. The P19CL6 spheroids were imaged in an upright configuration and the horizontally sectioned reflection images of the sample were obtained by sequentially and vertically scanning the zero-path-length height. Our results show the threedimensional structure of the spheroids, in which plasma and nuclear membranes were distinguishably imaged. The results imply that our technique is further capable of imaging induced pluripotent stem (iPS) cells for the assessment of cell properties including their pluripotency.

Genetic strategies to investigate neuronal circuit properties using stem cell-derived neurons

PubMed Central

Garcia, Isabella; Kim, Cynthia; Arenkiel, Benjamin R.

2012-01-01

The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons, now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs toward investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches. PMID:23264761

Changes in microRNA expression during differentiation of embryonic and induced pluripotent stem cells to definitive endoderm.

PubMed

Francis, Natalie; Moore, Melanie; Asan, Simona G; Rutter, Guy A; Burns, Chris

2015-01-01

Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to treat type 1 diabetes through cell replacement therapy. However, the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification, and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation, and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified, suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR, and the relationship between one of these miRNAs, miR-151a-5p, and its predicted target gene, SOX17, was investigated by luciferase assay, and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion, these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Stem cell regulatory function mediated by expression of a novel mouse Oct4 pseudogene

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

Lin, Huey; Shabbir, Arsalan; Molnar, Merced

2007-03-30

Multiple pseudogenes have been proposed for embryonic stem (ES) cell-specific genes, and their abundance suggests that some of these potential pseudogenes may be functional. ES cell-specific expression of Oct4 regulates stem cell pluripotency and self-renewing state. Although Oct4 expression has been reported in adult tissues during gene reprogramming, the detected Oct4 signal might be contributed by Oct4 pseudogenes. Among the multiple Oct4 transcripts characterized here is a {approx}1 kb clone derived from P19 embryonal carcinoma stem cells, which shares a {approx}87% sequence homology with the parent Oct4 gene, and has the potential of encoding an 80-amino acid product (designated asmore » Oct4P1). Adenoviral expression of Oct4P1 in mesenchymal stem cells promotes their proliferation and inhibits their osteochondral differentiation. These dual effects of Oct4P1 are reminiscent of the stem cell regulatory function of the parent Oct4, and suggest that Oct4P1 may be a functional pseudogene or a novel Oct4-related gene with a unique function in stem cells.« less

Transplantation of embryonic stem cell-derived dopaminergic neurons in MPTP-treated monkeys.

PubMed

Takahashi, Jun; Takagi, Yasushi; Saiki, Hidemoto

2009-01-01

One of the target diseases of cell-replacement therapy is Parkinson's disease. Clinical experiences with fetal dopaminergic cell graft have shown that the therapy is effective, but limited and accompanied by side effects, such as dyskinesia. So, the therapy needs to be further improved and sophisticated. Embryonic stem (ES) cells are expected to be another donor cell for the treatment, because of its proliferative and differentiation capacities. For clinical application, experiments using non-human primates are important, because size, anatomy, and biological characteristics of the brain are different between rodents and primates. Here, we would like to discuss induction of dopaminergic neurons from monkey ES cells and cell transplantation into the brain of monkey Parkinson's disease model.

The Effect of Laser Irradiation on Adipose Derived Stem Cell Proliferation and Differentiation

NASA Astrophysics Data System (ADS)

Abrahamse, H.; de Villiers, J.; Mvula, B.

2009-06-01

There are two fundamental types of stem cells: Embryonic Stem cells and Adult Stem cells. Adult Stem cells have a more restricted potential and can usually differentiate into a few different cell types. In the body these cells facilitate the replacement or repair of damaged or diseased cells in organs. Low intensity laser irradiation was shown to increase stem cell migration and stimulate proliferation and it is thought that treatment of these cells with laser irradiation may increase the stem cell harvest and have a positive effect on the viability and proliferation. Our research is aimed at determining the effect of laser irradiation on differentiation of Adipose Derived Stem Cells (ADSCs) into different cell types using a diode laser with a wavelength of 636 nm and at 5 J/cm2. Confirmation of stem cell characteristics and well as subsequent differentiation were assessed using Western blot analysis and cellular morphology supported by fluorescent live cell imaging. Functionality of subsequent differentiated cells was confirmed by measuring adenosine triphosphate (ATP) production and cell viability.

[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.

Pluripotent Human embryonic stem cell derived neural lineages for in vitro modelling of enterovirus 71 infection and therapy.

PubMed

Yap, May Shin; Tang, Yin Quan; Yeo, Yin; Lim, Wei Ling; Lim, Lee Wei; Tan, Kuan Onn; Richards, Mark; Othman, Iekhsan; Poh, Chit Laa; Heng, Boon Chin

2016-01-06

The incidence of neurological complications and fatalities associated with Hand, Foot & Mouth disease has increased over recent years, due to emergence of newly-evolved strains of Enterovirus 71 (EV71). In the search for new antiviral therapeutics against EV71, accurate and sensitive in vitro cellular models for preliminary studies of EV71 pathogenesis is an essential prerequisite, before progressing to expensive and time-consuming live animal studies and clinical trials. This study thus investigated whether neural lineages derived from pluripotent human embryonic stem cells (hESC) can fulfil this purpose. EV71 infection of hESC-derived neural stem cells (NSC) and mature neurons (MN) was carried out in vitro, in comparison with RD and SH-SY5Y cell lines. Upon assessment of post-infection survivability and EV71 production by the various types, it was observed that NSC were significantly more susceptible to EV71 infection compared to MN, RD (rhabdomyosarcoma) and SH-SY5Y cells, which was consistent with previous studies on mice. The SP81 peptide had significantly greater inhibitory effect on EV71 production by NSC and MN compared to the cancer-derived RD and SH-SY5Y cell lines. Hence, this study demonstrates that hESC-derived neural lineages can be utilized as in vitro models for studying EV71 pathogenesis and for screening of antiviral therapeutics.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

PubMed

Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

2009-08-01

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

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

Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.more » - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.« less

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

EPA Science Inventory

The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

PubMed

Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

2014-08-01

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

PubMed

Li, Dong; Zuo, Qisheng; Lian, Chao; Zhang, Lei; Shi, Qingqing; Zhang, Zhentao; Wang, Yingjie; Ahmed, Mahmoud F; Tang, Beibei; Xiao, Tianrong; Zhang, Yani; Li, Bichun

2015-08-01

We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.

Unique differentiation profile of mouse embryonic stem cells in rotary and stirred tank bioreactors.

PubMed

Fridley, Krista M; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B; Roy, Krishnendu

2010-11-01

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.

ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

PubMed

Pettinato, Giuseppe; Vanden Berg-Foels, Wendy S; Zhang, Ning; Wen, Xuejun

2014-01-01

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application.

PubMed

Saito, Shigeo; Lin, Ying-Chu; Murayama, Yoshinobu; Nakamura, Yukio; Eckner, Richard; Niemann, Heiner; Yokoyama, Kazunari K

2015-12-01

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment.

Functional expression and pharmaceutical efficacy of cardiac-specific ion channels in human embryonic stem cell-derived cardiomyocytes.

PubMed

Kim, Han Sol; Yoon, Jung Won; Li, Hongliang; Jeong, Geun Ok; Park, Jin Ju; Shin, Sung Eun; Jang, Il Ho; Kim, Jae Ho; Park, Won Sun

2017-10-23

Cardiomyocytes differentiated from human pluripotent stem cells provide promising tools for screening of cardiotoxic drugs. For evaluation of human pluripotent stem cell-derived cardiomyocytes for cardiotoxicity test, in the present study, human embryonic stem cells (hESCs) were differentiated to cardiomyocytes, followed by metabolic selection to enrich the differentiated cardiomyocytes. The highly purified hESC-derived cardiomyocytes (hESC-CMs) expressed several cardiomyocyte-specific markers including cTnT, MLC2a, and α-SA, but not pluripotency markers, such as OCT4 and NANOG. Patch clamp technique and RT-PCR revealed the expression of cardiomyocyte-specific Na + , Ca 2+ , and K + channels and cardiac action potential in hESC-CMs. To explore the potential use of hESC-CMs as functional cardiomyocytes for drug discovery and cardiotoxicity screening, we examined the effects of bisindolylmaleimide (BIM) (I), which inhibits native cardiac Ca 2+ channels, on the Ca 2+ channel activity of hESC-CMs. We observed a similar response for the BIM (I)-induced modulation of Ca 2+ channels between hESC-CMs and native cardiomyocytes through L-type Ca 2+ channel current. These results suggest that hESC-CMs can be useful for evaluation of pharmaceutical efficacy and safety of novel drug candidate in cardiac research.

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

Embryonic stem-like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo.

PubMed

Wei, Ruxue; Zhao, Xueming; Hao, Haisheng; Du, Weihua; Zhu, Huabin

2016-11-01

The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10 -7  M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The king is dead, long live the king: entering a new era of stem cell research and clinical development.

PubMed

Ichim, Thomas; Riordan, Neil H; Stroncek, David F

2011-12-20

In mid November the biopharma industry was shocked by the announcement from Geron that they were ending work on embryonic stem cell research and therapy. For more than 10 years the public image of all stem cell research has been equated with embryonic stem cells. Unfortunately, a fundamentally important medical and financial fact was being ignored: embryonic stem cell therapy is extremely immature. In parallel to efforts in embryonic stem cell research and development, scientists and physicians in the field of adult stem cells realized that the natural role of adult stem cells in the body is to promote healing and to act like endogenous "repair cells" and, as a result, numerous companies have entered the field of adult stem cell therapy with the goal of expanding numbers of adult stem cells for administration to patients with various conditions. In contrast to embryonic stem cells, which are extremely expensive and potentially dangerous, adult cell cells are inexpensive and have an excellent safety record when used in humans. Many studies are now showing that adult stem cells are practical, patient-applicable, therapeutics that are very close to being available for incorporation into the practice of medicine. These events signal the entrance of the field of stem cells into a new era: an era where hype and misinformation no longer triumph over economic and medical realities.

Promotion of haematopoietic activity in embryonic stem cells by the aorta-gonad-mesonephros microenvironment

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

Krassowska, Anna; Gordon-Keylock, Sabrina; Samuel, Kay

We investigated whether the in vitro differentiation of ES cells into haematopoietic progenitors could be enhanced by exposure to the aorta-gonadal-mesonephros (AGM) microenvironment that is involved in the generation of haematopoietic stem cells (HSC) during embryonic development. We established a co-culture system that combines the requirements for primary organ culture and differentiating ES cells and showed that exposure of differentiating ES cells to the primary AGM region results in a significant increase in the number of ES-derived haematopoietic progenitors. Co-culture of ES cells on the AM20-1B4 stromal cell line derived from the AGM region also increases haematopoietic activity. We concludemore » that factors promoting the haematopoietic activity of differentiating ES cells present in primary AGM explants are partially retained in the AM20.1B4 stromal cell line and that these factors are likely to be different to those required for adult HSC maintenance.« less

Breaking down pluripotency in the porcine embryo reveals both a premature and reticent stem cell state in the inner cell mass and unique expression profiles of the naive and primed stem cell states.

PubMed

Hall, Vanessa Jane; Hyttel, Poul

2014-09-01

To date, it has been difficult to establish bona fide porcine embryonic stem cells (pESC) and stable induced pluripotent stem cells. Reasons for this remain unclear, but they may depend on inappropriate culture conditions. This study reports the most insights to date on genes expressed in the pluripotent cells of the porcine embryo, namely the inner cell mass (ICM), the trophectoderm-covered epiblast (EPI), and the embryonic disc epiblast (ED). Specifically, we reveal that the early porcine ICM represents a premature state of pluripotency due to lack of translation of key pluripotent proteins, and the late ICM enters a transient, reticent pluripotent state which lacks expression of most genes associated with pluripotency. We describe a unique expression profile of the porcine EPI, reflecting the naive stem cell state, including expression of OCT4, NANOG, CRIPTO, and SSEA-1; weak expression of NrOB1 and REX1; but very limited expression of genes in classical pathways involved in regulating pluripotency. The porcine ED, reflecting the primed stem cell state, can be characterized by the expression of OCT4, NANOG, SOX2, KLF4, cMYC, REX1, CRIPTO, and KLF2. Further cell culture experiments using inhibitors against FGF, JAK/STAT, BMP, WNT, and NODAL pathways on cell cultures derived from day 5 and 10 embryos reveal the importance of FGF, JAK/STAT, and BMP signaling in maintaining cell proliferation of pESCs in vitro. Together, this article provides new insights into the regulation of pluripotency, revealing unique stem cell states in the different porcine stem cell populations derived from the early developing embryo.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

PubMed

Novosadova, E V; Manuilova, E S; Arsen'eva, E L; Khaidarova, N V; Dolotov, O V; Inozemtseva, L S; Kozachenkov, K Yu; Tarantul, V Z; Grivennikov, I A

2005-07-01

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

Perspectives on stem cell therapy for cardiac regeneration. Advances and challenges.

PubMed

Choi, Sung Hyun; Jung, Seok Yun; Kwon, Sang-Mo; Baek, Sang Hong

2012-01-01

Ischemic heart disease (IHD) accelerates cardiomyocyte loss, but the developing stem cell research could be useful for regenerating a variety of tissue cells, including cardiomyocytes. Diverse sources of stem cells for IHD have been reported, including embryonic stem cells, induced pluripotent stem cells, skeletal myoblasts, bone marrow-derived stem cells, mesenchymal stem cells, and cardiac stem cells. However, stem cells have unique advantages and disadvantages for cardiac tissue regeneration, which are important considerations in determining the specific cells for improving cell survival and long-term engraftment after transplantation. Additionally, the dosage and administration method of stem cells need to be standardized to increase stability and efficacy for clinical applications. Accordingly, this review presents a summary of the stem cell therapies that have been studied for cardiac regeneration thus far, and discusses the direction of future cardiac regeneration research for stem cells.

Tumorigenicity studies for human pluripotent stem cell-derived products.

PubMed

Kuroda, Takuya; Yasuda, Satoshi; Sato, Yoji

2013-01-01

Human pluripotent stem cells (hPSCs), i.e. human embryonic stem cells and human induced pluripotent stem cells, are able to self-renew and differentiate into multiple cell types. Because of these abilities, numerous attempts have been made to utilize hPSCs in regenerative medicine/cell therapy. hPSCs are, however, also tumorigenic, that is, they can give rise to the progressive growth of tumor nodules in immunologically unresponsive animals. Therefore, assessing and managing the tumorigenicity of all final products is essential in order to prevent ectopic tissue formation, tumor development, and/or malignant transformation elicited by residual pluripotent stem cells after implantation. No detailed guideline for the tumorigenicity testing of hPSC-derived products has yet been issued for regenerative medicine/cell therapy, despite the urgent necessity. Here, we describe the current situations and issues related to the tumorigenicity testing of hPSC-derived products and we review the advantages and disadvantages of several types of tumorigenicity-associated tests. We also refer to important considerations in the execution and design of specific studies to monitor the tumorigenicity of hPSC-derived products.

An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis.

PubMed

Lipskind, Shane; Lindsey, Jennifer S; Gerami-Naini, Behzad; Eaton, Jennifer L; O'Connell, Daniel; Kiezun, Adam; Ho, Joshua W K; Ng, Nicholas; Parasar, Parveen; Ng, Michelle; Nickerson, Michael; Demirci, Utkan; Maas, Richard; Anchan, Raymond M

2018-05-01

Embryoid bodies (EBs) can serve as a system for evaluating pluripotency, cellular differentiation, and tissue morphogenesis. In this study, we use EBs derived from mouse embryonic stem cells (mESCs) and human amniocyte-derived induced pluripotent stem cells (hAdiPSCs) as a model for ovarian granulosa cell (GC) development and steroidogenic cell commitment. We demonstrated that spontaneously differentiated murine EBs (mEBs) and human EBs (hEBs) displayed ovarian GC markers, such as aromatase (CYP19A1), FOXL2, AMHR2, FSHR, and GJA1. Comparative microarray analysis identified both shared and unique gene expression between mEBs and the maturing mouse ovary. Gene sets related to gonadogenesis, lipid metabolism, and ovarian development were significantly overrepresented in EBs. Of the 29 genes, 15 that were differentially regulated in steroidogenic mEBs displayed temporal expression changes between embryonic, postnatal, and mature ovarian tissues by polymerase chain reaction. Importantly, both mEBs and hEBs were capable of gonadotropin-responsive estradiol (E2) synthesis in vitro (217-759 pg/mL). Live fluorescence-activated cell sorting-sorted AMHR2 + granulosa-like cells from mEBs continued to produce E2 after purification (15.3 pg/mL) and secreted significantly more E2 than AMHR2 - cells (8.6 pg/mL, P < .05). We conclude that spontaneously differentiated EBs of both mESC and hAdiPSC origin can serve as a biologically relevant model for ovarian GC differentiation and steroidogenic cell commitment. These cells should be further investigated for therapeutic uses, such as stem cell-based hormone replacement therapy and in vitro maturation of oocytes.

Characterization of axon formation in the embryonic stem cell-derived motoneuron.

PubMed

Pan, Hung-Chuan; Wu, Ya-Ting; Shen, Shih-Cheng; Wang, Chi-Chung; Tsai, Ming-Shiun; Cheng, Fu-Chou; Lin, Shinn-Zong; Chen, Ching-Wen; Liu, Ching-San; Su, Hong-Lin

2011-01-01

The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP(+) neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.

From "ES-like" cells to induced pluripotent stem cells: a historical perspective in domestic animals.

PubMed

Koh, Sehwon; Piedrahita, Jorge A

2014-01-01

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide great potential as cell sources for gene editing to generate genetically modified animals, as well as in the field of regenerative medicine. Stable, long-term ESCs have been established in laboratory mouse and rat; however, isolation of true pluripotent ESCs in domesticated animals such as pigs and dogs have been less successful. Initially, domesticated animal pluripotent cell lines were referred to as "embryonic stem-like" cells owing to their similar morphologic characteristics to mouse ESCs, but accompanied by a limited ability to proliferate in vitro in an undifferentiated state. That is, they shared some but not all the characteristics of true ESCs. More recently, advances in reprogramming using exogenous transcription factors, combined with the utilization of small chemical inhibitors of key biochemical pathways, have led to the isolation of iPSCs. In this review, we provide a historical perspective of the isolation of various types of pluripotent stem cells in domesticated animals. In addition, we summarize the latest progress and limitations in the derivation and application of iPSCs. Copyright © 2014 Elsevier Inc. All rights reserved.

Genome stability of programmed stem cell products.

PubMed

Martin, Ulrich

2017-10-01

Inherited and acquired genomic abnormalities are known to cause genetic diseases and contribute to cancer formation. Recent studies demonstrated a substantial mutational load in mouse and human embryonic and induced pluripotent stem cells (ESCs and iPSCs). Single nucleotide variants, copy number variations, and larger chromosomal abnormalities may influence the differentiation capacity of pluripotent stem cells and the functionality of their derivatives in disease modeling and drug screening, and are considered a serious risk for cellular therapies based on ESC or iPSC derivatives. This review discusses the types and origins of different genetic abnormalities in pluripotent stem cells, methods for their detection, and the mechanisms of development and enrichment during reprogramming and culture expansion. Copyright © 2017 Elsevier B.V. All rights reserved.

Chondrogenesis of Embryonic Stem Cell-Derived Mesenchymal Stem Cells Induced by TGFβ1 and BMP7 Through Increased TGFβ Receptor Expression and Endogenous TGFβ1 Production.

PubMed

Lee, Patrick T; Li, Wan-Ju

2017-01-01

For decades stem cells have proven to be invaluable to the study of tissue development. More recently, mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) (ESC-MSCs) have emerged as a cell source with great potential for the future of biomedical research due to their enhanced proliferative capability compared to adult tissue-derived MSCs and effectiveness of musculoskeletal lineage-specific cell differentiation compared to ESCs. We have previously compared the properties and differentiation potential of ESC-MSCs to bone marrow-derived MSCs. In this study, we evaluated the potential of TGFβ1 and BMP7 to induce chondrogenic differentiation of ESC-MSCs compared to that of TGFβ1 alone and further investigated the cellular phenotype and intracellular signaling in response to these induction conditions. Our results showed that the expression of cartilage-associated markers in ESC-MSCs induced by the TGFβ1 and BMP7 combination was increased compared to induction with TGFβ1 alone. The TGFβ1 and BMP7 combination upregulated the expression of TGFβ receptor and the production of endogenous TGFβs compared to TGFβ1 induction. The growth factor combination also increasingly activated both of the TGF and BMP signaling pathways, and inhibition of the signaling pathways led to reduced chondrogenesis of ESC-MSCs. Our findings suggest that by adding BMP7 to TGFβ1-supplemented induction medium, ESC-MSC chondrogenesis is upregulated through increased production of endogenous TGFβ and activities of TGFβ and BMP signaling. J. Cell. Biochem. 118: 172-181, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro.

PubMed

Teramura, Takeshi; Onodera, Yuta; Murakami, Hideki; Ito, Syunsuke; Mihara, Toshihiro; Takehara, Toshiyuki; Kato, Hiromi; Mitani, Tasuku; Anzai, Masayuki; Matsumoto, Kazuya; Saeki, Kazuhiro; Fukuda, Kanji; Sagawa, Norimasa; Osoi, Yoshihiko

2009-06-01

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

PubMed

Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

2010-04-01

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Ground-state transcriptional requirements for skin-derived precursors.

PubMed

Suflita, Michael T; Pfaltzgraff, Elise R; Mundell, Nathan A; Pevny, Larysa H; Labosky, Patricia A

2013-06-15

Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell.

Ground-State Transcriptional Requirements for Skin-Derived Precursors

PubMed Central

Suflita, Michael T.; Pfaltzgraff, Elise R.; Mundell, Nathan A.; Pevny, Larysa H.

2013-01-01

Skin-derived precursors (SKPs) are an attractive stem cell model for cell-based therapies. SKPs can be readily generated from embryonic and adult mice and adult humans, exhibit a high degree of multipotency, and have the potential to serve as a patient autologous stem cell. The advancement of these cells toward therapeutic use depends on the ability to control precisely the self-renewal and differentiation of SKPs. Here we show that two well-known stem cell factors, Foxd3 and Sox2, are critical regulators of the stem cell properties of SKPs. Deletion of Foxd3 completely abolishes the sphere-forming potential of these cells. In the absence of Sox2, SKP spheres can be formed, but with reduced size and frequency. Our results provide entry points into the gene regulatory networks dictating SKP behavior, and pave the way for future studies on a therapeutically relevant stem cell. PMID:23316968

Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

PubMed Central

2013-01-01

Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

PubMed Central

Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Zandi, Mohammad; Manik, Radhey Sham; Singla, Suresh Kumar; Palta, Prabhat

2015-01-01

Abstract We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro–fertilized, somatic cell nuclear–transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro–produced blastocysts. Most of the ICMs (45–55%) resulted in formation of primary colonies that were subcultured after 8–10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture–derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture–derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium. PMID:26168169

Challenges of primate embryonic stem cell research.

PubMed

Bavister, Barry D; Wolf, Don P; Brenner, Carol A

2005-01-01

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.

Erythro-Myeloid Progenitors: “definitive” hematopoiesis in the conceptus prior to the emergence of hematopoietic stem cells

PubMed Central

Frame, Jenna M.; McGrath, Kathleen E.; Palis, James

2013-01-01

Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes. PMID:24095199

In vitro developmental model of the gastrointestinal tract from mouse embryonic stem cells.

PubMed

Torihashi, Shigeko; Kuwahara, Masaki; Kurahashi, Masaaki

2007-10-01

Mouse embryonic stem (ES) cells are pluripotent and retain their potential to form cells, tissues and organs originated from three embryonic germ layers. Recently, we developed in vitro organ--gut-like structures--from mouse ES cells. They had basically similar morphological features to a mouse gastrointestinal tract in vivo composed of three distinct layers (i.e., epithelium, connective tissue and musculature). Gut-like structures showed spontaneous contractions derived from pacemaker cells (interstitial cells of Cajal) in the musculature. We also examined their formation process and expression pattern of transcription factors crucial for gut organogenesis such as Id2, Sox17, HNF3beta/Foxa2 and GATA4. We found that they mimic the development of embryonic gut in vivo and showed a similar expression pattern of common transcription factors. They also maintain their developmental potential after transplantation to a renal capsule. Therefore, gut-like structures are suitable for in vitro models of gastrointestinal tracts and their development. In addition, we pointed out several unique features different from gut in vivo that provide useful and advantageous tools to investigate the developmental mechanism of the gastrointestinal tract.

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.

PC1/3 Deficiency Impacts Pro-opiomelanocortin Processing in Human Embryonic Stem Cell-Derived Hypothalamic Neurons.

PubMed

Wang, Liheng; Sui, Lina; Panigrahi, Sunil K; Meece, Kana; Xin, Yurong; Kim, Jinrang; Gromada, Jesper; Doege, Claudia A; Wardlaw, Sharon L; Egli, Dieter; Leibel, Rudolph L

2017-02-14

We recently developed a technique for generating hypothalamic neurons from human pluripotent stem cells. Here, as proof of principle, we examine the use of these cells in modeling of a monogenic form of severe obesity: PCSK1 deficiency. The cognate enzyme, PC1/3, processes many prohormones in neuroendocrine and other tissues. We generated PCSK1 (PC1/3)-deficient human embryonic stem cell (hESC) lines using both short hairpin RNA and CRISPR-Cas9, and investigated pro-opiomelanocortin (POMC) processing using hESC-differentiated hypothalamic neurons. The increased levels of unprocessed POMC and the decreased ratios (relative to POMC) of processed POMC-derived peptides in both PCSK1 knockdown and knockout hESC-derived neurons phenocopied POMC processing reported in PC1/3-null mice and PC1/3-deficient patients. PC1/3 deficiency was associated with increased expression of melanocortin receptors and PRCP (prolylcarboxypeptidase, a catabolic enzyme for α-melanocyte stimulating hormone (αMSH)), and reduced adrenocorticotropic hormone secretion. We conclude that the obesity accompanying PCSK1 deficiency may not be primarily due to αMSH deficiency. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Characterizing Human Stem Cell–derived Sensory Neurons at the Single-cell Level Reveals Their Ion Channel Expression and Utility in Pain Research

PubMed Central

Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

2014-01-01

The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell–derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders. PMID:24832007

The Sox2 promoter-driven CD63-GFP transgenic rat model allows tracking of neural stem cell-derived extracellular vesicles.

PubMed

Yoshimura, Aya; Adachi, Naoki; Matsuno, Hitomi; Kawamata, Masaki; Yoshioka, Yusuke; Kikuchi, Hisae; Odaka, Haruki; Numakawa, Tadahiro; Kunugi, Hiroshi; Ochiya, Takahiro; Tamai, Yoshitaka

2018-01-30

Extracellular vesicles (EVs) can modulate microenvironments by transferring biomolecules, including RNAs and proteins derived from releasing cells, to target cells. To understand the molecular mechanisms maintaining the neural stem cell (NSC) niche through EVs, a new transgenic (Tg) rat strain that can release human CD63-GFP-expressing EVs from the NSCs was established. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/human CD63-GFP), and it was expressed in undifferentiated fetal brains. GFP signals were specifically observed in in vitro cultured NSCs obtained from embryonic brains of the Tg rats. We also demonstrated that embryonic NSC (eNSC)-derived EVs were labelled by human CD63-GFP. Furthermore, when we examined the transfer of EVs, eNSC-derived EVs were found to be incorporated into astrocytes and eNSCs, thus implying an EV-mediated communication between different cell types around NSCs. This new Sox2/human CD63-GFP Tg rat strain should provide resources to analyse the cell-to-cell communication via EVs in NSC microenvironments. © 2018. Published by The Company of Biologists Ltd.

Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells.

PubMed

Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

2016-04-01

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells

PubMed Central

Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

2016-01-01

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology.

PubMed

Shin, Hwa Sung; Kim, Hyung Joon; Min, Seul Ki; Kim, Sung Hoon; Lee, Byung Man; Jeon, Noo Li

2010-08-01

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.

Complete TCRα gene locus control region activity in T cells derived in vitro from embryonic stem cells

PubMed Central

Lahiji, Armin; Kučerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D.

2013-01-01

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering. PMID:23720809

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.

Dental Pulp Stem Cells Model Early Life and Imprinted DNA Methylation Patterns.

PubMed

Dunaway, Keith; Goorha, Sarita; Matelski, Lauren; Urraca, Nora; Lein, Pamela J; Korf, Ian; Reiter, Lawrence T; LaSalle, Janine M

2017-04-01

Early embryonic stages of pluripotency are modeled for epigenomic studies primarily with human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs). For analysis of DNA methylation however, ESCs and iPSCs do not accurately reflect the DNA methylation levels found in preimplantation embryos. Whole genome bisulfite sequencing (WGBS) approaches have revealed the presence of large partially methylated domains (PMDs) covering 30%-40% of the genome in oocytes, preimplantation embryos, and placenta. In contrast, ESCs and iPSCs show abnormally high levels of DNA methylation compared to inner cell mass (ICM) or placenta. Here we show that dental pulp stem cells (DPSCs), derived from baby teeth and cultured in serum-containing media, have PMDs and mimic the ICM and placental methylome more closely than iPSCs and ESCs. By principal component analysis, DPSC methylation patterns were more similar to two other neural stem cell types of human derivation (EPI-NCSC and LUHMES) and placenta than were iPSCs, ESCs or other human cell lines (SH-SY5Y, B lymphoblast, IMR90). To test the suitability of DPSCs in modeling epigenetic differences associated with disease, we compared methylation patterns of DPSCs derived from children with chromosome 15q11.2-q13.3 maternal duplication (Dup15q) to controls. Differential methylation region (DMR) analyses revealed the expected Dup15q hypermethylation at the imprinting control region, as well as hypomethylation over SNORD116, and novel DMRs over 147 genes, including several autism candidate genes. Together these data suggest that DPSCs are a useful model for epigenomic and functional studies of human neurodevelopmental disorders. Stem Cells 2017;35:981-988. © 2016 AlphaMed Press.

Putative embryonic stem cells derived from porcine cloned blastocysts using induced pluripotent stem cells as donors.

PubMed

Kim, Eunhye; Hwang, Seon-Ung; Yoo, Hyunju; Yoon, Junchul David; Jeon, Yubyeol; Kim, Hyunggee; Jeung, Eui-Bae; Lee, Chang-Kyu; Hyun, Sang-Hwan

2016-03-01

The establishment of porcine embryonic stem cells (ESCs) would have great impact in biomedical studies and preclinical trials through their use in genetic engineering. However, authentic porcine ESCs have not been established until now. In this study, a total of seven putative ESC lines were derived from porcine embryos of various origins, including in vitro fertilization, parthenogenetic activation, and, in particular, induced pluripotent stem (iPS) nuclear transfer (NT) from a donor cell with induced pluripotent stem cells (iPSCs). To characterize these cell lines, several assays including an assessment of intensive alkaline phosphatase activity, karyotyping, embryoid body formation, expression analysis of the pluripotency-associated markers, and the three germ layerassociated markers were performed. Based on quantitative polymerase chain reaction, the expression levels of REX1 and FGFR2 in iPS-NT lines were higher than those of cells of other origins. Additionally, only iPS-NT lines showed multiple aberrant patterns of nuclear foci elucidated by immunofluorescence staining of H3K27me3 as a marker of the state of X chromosome inactivation and a less mature form of mitochondria like naive ESCs, by transmission electron microscopy. Together, these data suggested that established putative porcine ESC lines generally exhibited a primed pluripotent state, like human ESCs. However, iPS-NT lines have especially unique characteristics distinct from other origins because they have more epigenetic instability and naive-like mitochondrial morphology than other putative ESC lines. This is the first study to establish and characterize the iPSC-derived putative ESC lines and compare them with other lines derived from different origins in pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice

PubMed Central

Lu, Shi-Jiang; Li, Feng; Yin, Hong; Feng, Qiang; Kimbrel, Erin A; Hahm, Eunsil; Thon, Jonathan N; Wang, Wei; Italiano, Joseph E; Cho, Jaehyung; Lanza, Robert

2011-01-01

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells. PMID:21221130

Establishment of novel detection system for embryonic stem cell-derived hepatocyte-like cells based on nongenetic manipulation with indocyanine green.

PubMed

Yoshie, Susumu; Ito, Jun; Shirasawa, Sakiko; Yokoyama, Tadayuki; Fujimura, Yuu; Takeda, Kazuo; Mizuguchi, Masahiro; Matsumoto, Ken; Tomotsune, Daihachiro; Sasaki, Katsunori

2012-01-01

Hepatocytes derived from embryonic stem cells (ESCs) are expected to be useful for basic research and clinical applications. However, in several studies, genetic methods used to detect and obtain them are difficult and pose major safety problems. Therefore, in this study, we established a novel detection system for hepatocytes by using indocyanine green (ICG), which is selectively taken up by hepatocytes, based on nongenetic manipulation. ICG has maximum light absorption near 780 nm, and it fluoresces between 800 and 900 nm. Making use of these properties, we developed flow cytometry equipped with an excitation lazer of 785 nm and specific bandpass filters and successfully detected ESC-derived ICG-positive cells that were periodic acid-Schiff positive and expressed hepatocyte phenotypic mRNAs. These results demonstrate that this detection system based on nongenetic manipulation with ICG will lead to isolate hepatocytes generated from ESCs and provide the appropriate levels of stability, quality, and safety required for cell source for cell-based therapy and pharmaceutical studies such as toxicology.

Mouse mutants from chemically mutagenized embryonic stem cells

PubMed Central

Munroe, Robert J.; Bergstrom, Rebecca A.; Zheng, Qing Yin; Libby, Brian; Smith, Richard; John, Simon W.M.; Schimenti, Kerry J.; Browning, Victoria L.; Schimenti, John C.

2010-01-01

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain1 and interlocus2 variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chi-maeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives. PMID:10700192

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

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

Shikanai, Mima; Asahina, Kinji; Iseki, Sachiko

2009-04-03

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or {alpha}-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells becamemore » mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.« less

Enhanced neuro-therapeutic potential of Wharton's Jelly-derived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture.

PubMed

Drela, Katarzyna; Lech, Wioletta; Figiel-Dabrowska, Anna; Zychowicz, Marzena; Mikula, Michał; Sarnowska, Anna; Domanska-Janik, Krystyna

2016-04-01

Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type "pre-MSC," where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration-associated potency. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity.

PubMed

Higuchi, Akon; Kao, Shih-Hsuan; Ling, Qing-Dong; Chen, Yen-Ming; Li, Hsing-Fen; Alarfaj, Abdullah A; Munusamy, Murugan A; Murugan, Kadarkarai; Chang, Shih-Chang; Lee, Hsin-Chung; Hsu, Shih-Tien; Kumar, S Suresh; Umezawa, Akihiro

2015-12-14

The tentative clinical application of human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human induced pluripotent stem cells, is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore, we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture, whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture.

Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

NASA Astrophysics Data System (ADS)

Meng, Qing-Yuan; Akaike, Toshihiro

2013-03-01

Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.

PubMed

Dubois, Nicole C; Craft, April M; Sharma, Parveen; Elliott, David A; Stanley, Edouard G; Elefanty, Andrew G; Gramolini, Anthony; Keller, Gordon

2011-10-23

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.

Using the mouse embryonic stem cell test (EST) to evaluate the embryotoxicity of haloacetic acids

EPA Science Inventory

The Embryonic Stem Cell Test (EST) is used to predict the embryotoxic potential of a test compound by combining the data from cytotoxicity assays in undifferentiated mouse embryonic stem (mES) cells and differentiated mouse cells with the data from a differentiation assay in mES ...

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.

Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.

PubMed Central

Wood, S A; Pascoe, W S; Schmidt, C; Kemler, R; Evans, M J; Allen, N D

1993-01-01

A method for the production of embryonic stem (ES) cell-embryo chimeras was developed that involves the simple coculture of eight-cell embryos on a lawn of ES cells. After coculture, the embryos with ES cells attached are transferred to normal embryo culture medium and allowed to develop to the blastocyst stage before reimplantation into foster mothers. Although the ES cells initially attach to the outside of the embryos, they primarily colonize the inner cell mass and its derivatives. This method results in the efficient production of chimeras with high levels of chimerism including the germ line. As embryos are handled en masse and manipulative steps are minimal, this method should greatly reduce the time and effort required to produce chimeric mice. Images Fig. 1 Fig. 2 PMID:8506303

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

Metastable Pluripotent States in NOD Mouse Derived ES Cells

PubMed Central

Hanna, Jacob; Markoulaki, Styliani; Mitalipova, Maisam; Cheng, Albert W.; Cassady, John P.; Staerk, Judith; Carey, Bryce W.; Lengner, Christopher J.; Foreman, Ruth; Love, Jennifer; Gao, Qing; Kim, Jongpil; Jaenisch, Rudolf

2009-01-01

Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the post-implantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the non-obese diabetic (NOD) mouse strain, which prior to this study was considered “non-permissive” for ES cell derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ES and iPS cells appear “metastable”, as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors. PMID:19427283

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor.

PubMed

Wakayama, Sayaka; Wakayama, Teruhiko

2010-01-01

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

Three-dimensional neural differentiation of embryonic stem cells with ACM induction in microfibrous matrices in bioreactors.

PubMed

Liu, Ning; Ouyang, Anli; Li, Yan; Yang, Shang-Tian

2013-01-01

The clinical use of pluripotent stem cell (PSC)-derived neural cells requires an efficient differentiation process for mass production in a bioreactor. Toward this goal, neural differentiation of murine embryonic stem cells (ESCs) in three-dimensional (3D) polyethylene terephthalate microfibrous matrices was investigated in this study. To streamline the process and provide a platform for process integration, the neural differentiation of ESCs was induced with astrocyte-conditioned medium without the formation of embryoid bodies, starting from undifferentiated ESC aggregates expanded in a suspension bioreactor. The 3D neural differentiation was able to generate a complex neural network in the matrices. When compared to 2D differentiation, 3D differentiation in microfibrous matrices resulted in a higher percentage of nestin-positive cells (68% vs. 54%) and upregulated gene expressions of nestin, Nurr1, and tyrosine hydroxylase. High purity of neural differentiation in 3D microfibrous matrix was also demonstrated in a spinner bioreactor with 74% nestin + cells. This study demonstrated the feasibility of a scalable process based on 3D differentiation in microfibrous matrices for the production of ESC-derived neural cells. © 2013 American Institute of Chemical Engineers.

Large-scale production of embryonic red blood cells from human embryonic stem cells.

PubMed

Olivier, Emmanuel N; Qiu, Caihong; Velho, Michelle; Hirsch, Rhoda Elison; Bouhassira, Eric E

2006-12-01

To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

Embryonic Stem Cell Patents and Human Dignity

PubMed Central

Resnik, David B.

2009-01-01

This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells. PMID:17922198

Disease and Stem Cell-Based Analysis of the 2014 ASNTR Meeting

PubMed Central

Eve, David J.

2015-01-01

A wide variety of subjects are presented at the annual American Society of Neural Therapy and Repair meeting every year, as typified by this summary of the 2014 meeting. Parkinson’s disease-related presentations were again the most popular topic, with traumatic brain injury, spinal cord injury, and stroke being close behind. Other disorders included Huntington’s disease, brain cancer, and bipolar disorders. Several studies were related to multiple diseases, and many studies attempted to reveal more about the disease process. The use of scaffolds, drugs, and gene therapy as disease models and/or potential therapies were also featured. An increasing proportion of presentations related to stem cells, with the study of multiple stem cell types being the most common. Induced pluripotent stem cells were increasingly popular, including two presentations each on a muscle-derived dedifferentiated cell type and cells derived from bipolar patients. Other stem cells, including neural stem cells, mesenchymal stem cells, umbilical cord blood cells, and embryonic stem cells, were featured. More than 55% of the stem cell studies involved transplantation, with human-derived cells being the most frequently transplanted, while rats were the most common recipient. Two human autologous studies for spinal cord injury and hypoxia-derived encephalopathy, while a further three allogenic studies for stroke and spinal cord injury, were also featured. This year’s meeting highlights the increasing promise of stem cells and other therapies for the treatment of neurodegenerative disorders. PMID:26858901

Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

PubMed Central

James, Daylon; Nam, Hyung-song; Seandel, Marco; Nolan, Daniel; Janovitz, Tyler; Tomishima, Mark; Studer, Lorenz; Lee, Gabsang; Lyden, David; Benezra, Robert; Zaninovic, Nikica; Rosenwaks, Zev; Rabbany, Sina Y; Rafii, Shahin

2010-01-01

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application. PMID:20081865

GENOMIC ADAPTATION OF THE EMBRYONIC STEM CELL TEST (EST) FOR A TOXICOLOGICAL STUDY OF DRINKING WATER DISINFECTION BY-PRODUCTS

EPA Science Inventory

Among the many promised and potential applications of embryonic stem cells, in vitro toxicology is one area in which ES cells have already proven their utility. In 2003, the Embryonic Stem Cell Test (EST) protocol was validated in Europe as an in vitro alternative to live animal...

Human Neural Cell-Based Biosensor

DTIC Science & Technology

2013-05-28

ionotropic receptors in human embryonic stem cell derived neural progenitors. Neuroscience. 2011 Sep 29;192:793-805. Krishnamoorthy M, Gerwe BA, Scharer...coupled receptors . Pharmacol Ther. 2011 Mar;129(3):290-306. Workshop/conference abstracts, presentations, posters, and papers Powe, A, et al

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

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

Fang, Zhen F.; Gai, Hui; Huang, You Z.

2006-11-01

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ESmore » cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.« less

Transplantation of Human Embryonic Stem Cells in Patients with Multiple Sclerosis and Lyme Disease

PubMed Central

Shroff, Geeta

2016-01-01

Case series Patient: Male, 42 • Female, 30 Final Diagnosis: Human embryonic stem cells showed good therapeutic potential for treatment of multiple sclerosis with lyme disease Symptoms: Fatigue • weakness in limbs Medication: — Clinical Procedure: Human embryonic stem cells transplantation Specialty: Transplantology Objective: Rare disease Background: Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease in which the myelin sheath of nerve cells is damaged. It can cause delayed neurologic symptoms similar to those seen in Lyme disease (LD) patients. Thymus derived T-cells (myelin reactive) migrate to the blood brain barrier and stimulate an inflammatory cascade in the central nervous system. Cell based therapies play an important role in treating neurological diseases such as MS and LD. Case Report: Human embryonic stem cell (hESC) therapy was used to treat two patients with both MS and LD. The hESCs were administered via different routes including intramuscular, intravenous, and supplemental routes (e.g., deep spinal, caudal, intercostal through eye drops) to regenerate the injured cells. Both the patients showed remarkable improvement in their functional skills, overall stamina, cognitive abilities, and muscle strength. Furthermore, the improvement in the patients’ conditions were assessed by magnetic resonance tractography and single photon emission computed tomography (SPECT). Conclusions: Therapy with hESCs might emerge as an effective and safe treatment for patients with both MS and LD. Well-designed clinical trials and follow-up studies are needed to prove the long-term efficacy and safety of hESC therapy in the treatment of patients with MS and LD. PMID:27956736

Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells.

PubMed

Soman, Pranav; Tobe, Brian T D; Lee, Jin Woo; Winquist, Alicia M; Singec, Ilyas; Vecchio, Kenneth S; Snyder, Evan Y; Chen, Shaochen

2012-10-01

Access to unlimited numbers of live human neurons derived from stem cells offers unique opportunities for in vitro modeling of neural development, disease-related cellular phenotypes, and drug testing and discovery. However, to develop informative cellular in vitro assays, it is important to consider the relevant in vivo environment of neural tissues. Biomimetic 3D scaffolds are tools to culture human neurons under defined mechanical and physico-chemical properties providing an interconnected porous structure that may potentially enable a higher or more complex organization than traditional two-dimensional monolayer conditions. It is known that even minor variations in the internal geometry and mechanical properties of 3D scaffolds can impact cell behavior including survival, growth, and cell fate choice. In this report, we describe the design and engineering of 3D synthetic polyethylene glycol (PEG)-based and biodegradable gelatin-based scaffolds generated by a free form fabrication technique with precise internal geometry and elastic stiffnesses. We show that human neurons, derived from human embryonic stem (hESC) cells, are able to adhere to these scaffolds and form organoid structures that extend in three dimensions as demonstrated by confocal and electron microscopy. Future refinements of scaffold structure, size and surface chemistries may facilitate long term experiments and designing clinically applicable bioassays.

Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

PubMed

Artioli, Guilherme Giannini; De Oliveira Silvestre, João Guilherme; Guilherme, João Paulo Limongi França; Baptista, Igor Luchini; Ramos, Gracielle Vieira; Da Silva, Willian José; Miyabara, Elen Haruka; Moriscot, Anselmo Sigari

2015-03-01

We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy. © 2014 Wiley Periodicals, Inc.

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

PubMed Central

van der Meulen, Talitha; Xie, Ruiyu; Kelly, Olivia G.; Vale, Wylie W.; Sander, Maike; Huising, Mark O.

2012-01-01

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3+ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3+ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3+ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies. PMID:23251699

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

PubMed

Mandenius, Carl-Fredrik; Andersson, Tommy B; Alves, Paula M; Batzl-Hartmann, Christine; Björquist, Petter; Carrondo, Manuel J T; Chesne, Christophe; Coecke, Sandra; Edsbagge, Josefina; Fredriksson, J Magnus; Gerlach, Jörg C; Heinzle, Elmar; Ingelman-Sundberg, Magnus; Johansson, Inger; Küppers-Munther, Barbara; Müller-Vieira, Ursula; Noor, Fozia; Zeilinger, Katrin

2011-05-01

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance. 2011 FRAME.

Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells.

PubMed

Nakamura, K; Aizawa, K; Yamauchi, J; Tanoue, A

2013-10-01

Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks. © 2013 John Wiley & Sons Ltd.

Investigating the mincing method for isolation of adipose-derived stem cells from pregnant women fat.

PubMed

Li, Yuan-Sheng; Chen, Pao-Jen; Wu, Li-Wei; Chou, Pei-Wen; Sun, Li-Yi; Chiou, Tzyy-Wen

2018-02-01

The success of stem cell application in regenerative medicine, usually require a stable source of stem or progenitor cells. Fat tissue represents a good source of stem cells because it is rich in stem cells and there are fewer ethical issues related to the use of such stem cells, unlike embryonic stem cells. Therefore, there has been increased interest in adipose-derived stem cells (ADSCs) for tissue engineering applications. Here, we aim to provide an easy processing method for isolating adult stem cells from human adipose tissue harvested from the subcutaneous fat of the abdominal wall during gynecologic surgery. We used a homogenizer to mince fat and compared the results with those obtained from the traditional cut method involving a sterile scalpel and forceps. Our results showed that our method provides another stable and quality source of stem cells that could be used in cases with a large quantity of fat. Furthermore, we found that pregnancy adipose-derived stem cells (P-ADSCs) could be maintained in vitro for extended periods with a stable population doubling and low senescence levels. P-ADSCs could also differentiate in vitro into adipogenic, osteogenic, chondrogenic, and insulin-producing cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirates, adipose tissues obtained from pregnant women contain multipotent cells with better proliferation and showed great promise for use in both stem cell banking studies as well as in stem cell therapy.

Efficient and rapid derivation of primitive neural stem cells and generation of brain subtype neurons from human pluripotent stem cells.

PubMed

Yan, Yiping; Shin, Soojung; Jha, Balendu Shekhar; Liu, Qiuyue; Sheng, Jianting; Li, Fuhai; Zhan, Ming; Davis, Janine; Bharti, Kapil; Zeng, Xianmin; Rao, Mahendra; Malik, Nasir; Vemuri, Mohan C

2013-11-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.

Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

PubMed Central

Wada, Tamaki; Honda, Makoto; Minami, Itsunari; Tooi, Norie; Amagai, Yuji; Nakatsuji, Norio; Aiba, Kazuhiro

2009-01-01

Background There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. Methods/Principal Findings We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. Conclusions and Significance The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved. PMID:19701462

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

PubMed Central

Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

2013-01-01

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

Quantification of Retinogenesis in 3D Cultures Reveals Epigenetic Memory and Higher Efficiency in iPSCs Derived from Rod Photoreceptors.

PubMed

Hiler, Daniel; Chen, Xiang; Hazen, Jennifer; Kupriyanov, Sergey; Carroll, Patrick A; Qu, Chunxu; Xu, Beisi; Johnson, Dianna; Griffiths, Lyra; Frase, Sharon; Rodriguez, Alberto R; Martin, Greg; Zhang, Jiakun; Jeon, Jongrye; Fan, Yiping; Finkelstein, David; Eisenman, Robert N; Baldwin, Kristin; Dyer, Michael A

2015-07-02

Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in three-dimensional (3D) organ cultures. Copyright © 2015 Elsevier Inc. All rights reserved.

Human Embryonic Stem Cell Therapy in Crohn’s Disease: A Case Report

PubMed Central

Shroff, Geeta

2016-01-01

Patient: Male, 21 Final Diagnosis: Crohn’s disease Symptoms: Intolerance to specific foods • abdominal pain and diarrhea Medication: Human embryonic stem cell therapy Clinical Procedure: Human embryonic stem cell transplantation Specialty: Gastroenterology Objective: Unusual or unexpected effect of treatment Background: Crohn’s disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565 000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn’s disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn’s disease. Case Report: A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Conclusions: Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn’s disease. PMID:26923312

Long-term culture and cryopreservation does not affect the stability and functionality of human embryonic stem cell-derived hepatocyte-like cells.

PubMed

Mandal, Arundhati; Raju, Sheena; Viswanathan, Chandra

2016-02-01

Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs, obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology.

Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

PubMed

Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

2012-08-01

The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

PubMed

Ji, Junfeng; Risueño, Ruth M; Hong, Seokho; Allan, David; Rosten, Patty; Humphries, Keith; Bhatia, Mickie

2011-04-01

Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self-renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98-HOXA10hd (NA10) on adult murine bone marrow HSC self-renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45(-) GlycophorinA(+) cells and erythroid progenitors (blast-forming unit-erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells. Copyright © 2011 AlphaMed Press.

The Immunogenicity and Immune Tolerance of Pluripotent Stem Cell Derivatives

PubMed Central

Liu, Xin; Li, Wenjuan; Fu, Xuemei; Xu, Yang

2017-01-01

Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and differentiate into all cell types in human body, and therefore hold great potential for cell therapy of currently incurable diseases including neural degenerative diseases, heart failure, and macular degeneration. This potential is further underscored by the promising safety and efficacy data from the ongoing clinical trials of hESC-based therapy of macular degeneration. However, one main challenge for the clinical application of hESC-based therapy is the allogeneic immune rejection of hESC-derived cells by the recipient. The breakthrough of the technology to generate autologous-induced pluripotent stem cells (iPSCs) by nuclear reprogramming of patient’s somatic cells raised the possibility that autologous iPSC-derived cells can be transplanted into the patients without the concern of immune rejection. However, accumulating data indicate that certain iPSC-derived cells can be immunogenic. In addition, the genomic instability associated with iPSCs raises additional safety concern to use iPSC-derived cells in human cell therapy. In this review, we will discuss the mechanism underlying the immunogenicity of the pluripotent stem cells and recent progress in developing immune tolerance strategies of human pluripotent stem cell (hPSC)-derived allografts. The successful development of safe and effective immune tolerance strategy will greatly facilitate the clinical development of hPSC-based cell therapy. PMID:28626459

Chapter 17 Sterile Plate-Based Vitrification of Adherent Human Pluripotent Stem Cells and Their Derivatives Using the TWIST Method.

PubMed

Neubauer, Julia C; Stracke, Frank; Zimmermann, Heiko

2017-01-01

Due to their high biological complexity, e.g., their close cell-to-cell contacts, cryopreservation of human pluripotent stem cells with standard slow-rate protocols often is inefficient and can hardly be standardized. Vitrification that means ultrafast freezing already showed very good viability and recovery rates for this sensitive cell system, but is only applicable for low cell numbers, bears a high risk of contamination, and can hardly be implemented under GxP regulations. In this chapter, a sterile plate-based vitrification method for adherent pluripotent stem cells and their derivatives is presented based on a procedure and device for human embryonic stem cells developed by Beier et al. (Cryobiology 66:8-16, 2013). This protocol overcomes the limitations of conventional vitrification procedures resulting in the highly efficient preservation of ready-to-use adherent pluripotent stem cells with the possibility of vitrifying cells in multi-well formats for direct application in high-throughput screenings.

Human embryonic stem cell therapies for neurodegenerative diseases.

PubMed

Tomaskovic-Crook, Eva; Crook, Jeremy M

2011-06-01

There is a renewed enthusiasm for the clinical translation of human embryonic stem (hES) cells. This is abetted by putative clinically-compliant strategies for hES cell maintenance and directed differentiation, greater understanding of and accessibility to cells through formal cell registries and centralized cell banking for distribution, the revised US government policy on funding hES cell research, and paradoxically the discovery of induced pluripotent stem (iPS) cells. Additionally, as we consider the constraints (practical and fiscal) of delivering cell therapies for global healthcare, the more efficient and economical application of allogeneic vs autologous treatments will bolster the clinical entry of hES cell derivatives. Neurodegenerative disorders such as Parkinson's disease are primary candidates for hES cell therapy, although there are significant hurdles to be overcome. The present review considers key advances and challenges to translating hES cells into novel therapies for neurodegenerative diseases, with special consideration given to Parkinson's disease and Alzheimer's disease. Importantly, despite the focus on degenerative brain disorders and hES cells, many of the issues canvassed by this review are relevant to systemic application of hES cells and other pluripotent stem cells such as iPS cells.

Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells from Patients with Sickle Cell Disease.

PubMed

Uchida, Naoya; Haro-Mora, Juan J; Fujita, Atsushi; Lee, Duck-Yeon; Winkler, Thomas; Hsieh, Matthew M; Tisdale, John F

2017-03-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent an ideal source for in vitro modeling of erythropoiesis and a potential alternative source for red blood cell transfusions. However, iPS cell-derived erythroid cells predominantly produce ε- and γ-globin without β-globin production. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production. In this study, we generated several iPS cell lines derived from bone marrow stromal cells (MSCs) and peripheral blood erythroid progenitors (EPs) from sickle cell disease patients, and evaluated hematopoietic stem/progenitor cell (HSPC) generation after iPS sac induction as well as subsequent erythroid differentiation. MSC-derived iPS sacs yielded greater amounts of immature hematopoietic progenitors (VEGFR2 + GPA-), definitive HSPCs (CD34 + CD45+), and megakaryoerythroid progenitors (GPA + CD41a+), as compared to EP-derived iPS sacs. Erythroid differentiation from MSC-derived iPS sacs resulted in greater amounts of erythroid cells (GPA+) and higher β-globin (and βS-globin) expression, comparable to ES sac-derived cells. These data demonstrate that human MSC-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, likely due to heightened emergence of immature progenitors. Our findings should be important for iPS cell-derived erythroid cell generation. Stem Cells 2017;35:586-596. © 2016 AlphaMed Press.

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

PubMed

Behboodi, E; Bondareva, A; Begin, I; Rao, K; Neveu, N; Pierson, J T; Wylie, C; Piero, F D; Huang, Y J; Zeng, W; Tanco, V; Baldassarre, H; Karatzas, C N; Dobrinski, I

2011-03-01

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells. Copyright © 2011 Wiley-Liss, Inc.

The fine structure of human germ layers in vivo: clues to the early differentiation of embryonic stem cells in vitro.

PubMed

Sathananthan, Henry; Selvaraj, Kamala; Clark, Joan

2011-08-01

The fine structure of the three germ layers in human ectopic embryos (stage 7) have been documented by digital light and electron microscopy. The formation of ectoderm, endoderm and mesoderm and notochordal cells, and also the extraembryonic membranes, amnion and yolk sac, are imaged. The germ layers give rise to all the cells and tissues of the human body. Possible clues to the early differentiation of embryonic stem cells (ESC) in vitro were obtained, since these events are more or less mimicked in cultures of ESC derived from the inner cell mass of human blastocysts. The findings are discussed with reference to previous studies on the fine structure of ESC using the same technique. Copyright © 2011. Published by Elsevier Ltd.

Maturation of Human Embryonic Stem Cell–Derived Pancreatic Progenitors Into Functional Islets Capable of Treating Pre-existing Diabetes in Mice

PubMed Central

Rezania, Alireza; Bruin, Jennifer E.; Riedel, Michael J.; Mojibian, Majid; Asadi, Ali; Xu, Jean; Gauvin, Rebecca; Narayan, Kavitha; Karanu, Francis; O’Neil, John J.; Ao, Ziliang; Warnock, Garth L.

2012-01-01

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes. PMID:22740171

Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

EPA Science Inventory

The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

Using Human Stem Cells to Study the Role of the Stroma in the Initiation of Prostate Cancer

DTIC Science & Technology

2011-03-01

alterations in the epithelium that drives the pr ogressive transformation of nor mal human cells into highly malignant derivatives. It is evident that...of tumor initiation, we propose to use normal human prostate epithelium generated from human embryonic stem cells (hESCs) in tissue recombination...serum free conditions for 5-8 days into endoderm in vitro. Confirm endoderm phenotype using immunohistochemistry and FACs analysis . We conducted

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation.

PubMed

Zhang, Peng; Wu, Xinglong; Hu, Chunchao; Wang, Pengbo; Li, Xiangyun

2012-01-01

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.

YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

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

Han, Dasol; Byun, Sung-Hyun; Park, Soojeong

Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and sizemore » of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.« less

IGF-1 Signaling Plays an Important Role in the Formation of Three-Dimensional Laminated Neural Retina and Other Ocular Structures From Human Embryonic Stem Cells.

PubMed

Mellough, Carla B; Collin, Joseph; Khazim, Mahmoud; White, Kathryn; Sernagor, Evelyne; Steel, David H W; Lako, Majlinda

2015-08-01

We and others have previously demonstrated that retinal cells can be derived from human embryonic stem cells (hESCs) and induced pluripotent stem cells under defined culture conditions. While both cell types can give rise to retinal derivatives in the absence of inductive cues, this requires extended culture periods and gives lower overall yield. Further understanding of this innate differentiation ability, the identification of key factors that drive the differentiation process, and the development of clinically compatible culture conditions to reproducibly generate functional neural retina is an important goal for clinical cell based therapies. We now report that insulin-like growth factor 1 (IGF-1) can orchestrate the formation of three-dimensional ocular-like structures from hESCs which, in addition to retinal pigmented epithelium and neural retina, also contain primitive lens and corneal-like structures. Inhibition of IGF-1 receptor signaling significantly reduces the formation of optic vesicle and optic cups, while exogenous IGF-1 treatment enhances the formation of correctly laminated retinal tissue composed of multiple retinal phenotypes that is reminiscent of the developing vertebrate retina. Most importantly, hESC-derived photoreceptors exhibit advanced maturation features such as the presence of primitive rod- and cone-like photoreceptor inner and outer segments and phototransduction-related functional responses as early as 6.5 weeks of differentiation, making these derivatives promising candidates for cell replacement studies and in vitro disease modeling. © 2015 AlphaMed Press.

The mechanical coupling of adult marrow stromal stem cells during cardiac regeneration assessed in a 2-D co-culture model

PubMed Central

Valarmathi, Mani T.; Fuseler, John W.; Goodwin, Richard L.; Davis, Jeffrey M.; Potts, Jay D.

2011-01-01

Postnatal cardiomyocytes undergo terminal differentiation and a restricted number of human cardiomyocytes retain the ability to divide and regenerate in response to ischemic injury. However, whether these neo-cardiomyocytes are derived from endogenous population of resident cardiac stem cells or from the exogenous double assurance population of resident bone marrow-derived stem cells that populate the damaged myocardium is unresolved and under intense investigation. The vital challenge is to ameliorate and/or regenerate the damaged myocardium. This can be achieved by stimulating proliferation of native quiescent cardiomyocytes and/or cardiac stem cell, or by recruiting exogenous autologous or allogeneic cells such as fetal or embryonic cardiomyocyte progenitors or bone marrow-derived stromal stem cells. The prerequisites are that these neo-cardiomyocytes must have the ability to integrate well within the native myocardium and must exhibit functional synchronization. Adult bone marrow stromal cells (BMSCs) have been shown to differentiate into cardiomyocyte-like cells both in vitro and in vivo. As a result, BMSCs may potentially play an essential role in cardiac repair and regeneration, but this concept requires further validation. In this report, we have provided compelling evidence that functioning cardiac tissue can be generated by the interaction of multipotent BMSCs with embryonic cardiac myocytes (ECMs) in two-dimensional (2-D) co-cultures. The differentiating BMSCs were induced to undergo cardiomyogenic differentiation pathway and were able to express unequivocal electromechanical coupling and functional synchronization with ECMs. Our 2-D co-culture system provides a useful in vitro model to elucidate various molecular mechanisms underpinning the integration and orderly maturation and differentiation of BMSCs into neo-cardiomyocytes during myocardial repair and regeneration. PMID:21288568

Simultaneous suppression of TGF-β and ERK signaling contributes to the highly efficient and reproducible generation of mouse embryonic stem cells from previously considered refractory and non-permissive strains.

PubMed

Hassani, Seyedeh-Nafiseh; Totonchi, Mehdi; Farrokhi, Ali; Taei, Adeleh; Larijani, Mehran Rezaei; Gourabi, Hamid; Baharvand, Hossein

2012-06-01

Mouse embryonic stem cells (ESCs) are pluripotent stem cell lines derived from pre-implantation embryos. The efficiency of mESC generation is affected by genetic variation in mice; that is, some mouse strains are refractory or non-permissive to ESC establishment. Developing an efficient method to derive mESCs from strains of various genetic backgrounds should be valuable for establishment of ESCs in various mammalian species. In the present study, we identified dual inhibition of TGF-β and ERK1/2, by SB431542 and PD0325901, respectively led to the highly efficient and reproducible generation of mESC lines from NMRI, C57BL/6, BALB/c, DBA/2, and FVB/N strains, which previously considered refractory or non-permissive for ESC establishment. These mESCs expressed pluripotency markers and retained the capacity to differentiate into derivatives of all three germ layers. The evaluated lines exhibited high rates of chimerism when reintroduced into blastocysts. To our knowledge, this is the first report of efficient (100%) mESC lines generation from different genetic backgrounds. The application of these two inhibitors will not only solve the problems of mESC derivation but also clarifies new signaling pathways in pluripotent mESCs.

Glucose responsive insulin production from human embryonic germ (EG) cell derivatives.

PubMed

Clark, Gregory O; Yochem, Robert L; Axelman, Joyce; Sheets, Timothy P; Kaczorowski, David J; Shamblott, Michael J

2007-05-11

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and beta-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.

Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles

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

Oh, Jung-Hwa; Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113; Son, Mi-Young

Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10–200 μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24 h. The results showed that Ag NPs evoked significant toxicity in hESC-derivedmore » NPCs at 24 h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. - Highlights: • This system served as a suitable model for developmental neurotoxicity testing. • Ag NPs induce the apoptosis in human neural cells by ROS generation. • Genes for development of neurons were dysregulated in response to Ag NPs. • Molecular events during early developmental neurotoxicity were proposed.« less

Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future.

PubMed

Luo, Mingyue; Chen, Youxin

2018-01-01

As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

Stem cell treatment of degenerative eye disease.

PubMed

Mead, Ben; Berry, Martin; Logan, Ann; Scott, Robert A H; Leadbeater, Wendy; Scheven, Ben A

2015-05-01

Stem cell therapies are being explored extensively as treatments for degenerative eye disease, either for replacing lost neurons, restoring neural circuits or, based on more recent evidence, as paracrine-mediated therapies in which stem cell-derived trophic factors protect compromised endogenous retinal neurons from death and induce the growth of new connections. Retinal progenitor phenotypes induced from embryonic stem cells/induced pluripotent stem cells (ESCs/iPSCs) and endogenous retinal stem cells may replace lost photoreceptors and retinal pigment epithelial (RPE) cells and restore vision in the diseased eye, whereas treatment of injured retinal ganglion cells (RGCs) has so far been reliant on mesenchymal stem cells (MSC). Here, we review the properties of non-retinal-derived adult stem cells, in particular neural stem cells (NSCs), MSC derived from bone marrow (BMSC), adipose tissues (ADSC) and dental pulp (DPSC), together with ESC/iPSC and discuss and compare their potential advantages as therapies designed to provide trophic support, repair and replacement of retinal neurons, RPE and glia in degenerative retinal diseases. We conclude that ESCs/iPSCs have the potential to replace lost retinal cells, whereas MSC may be a useful source of paracrine factors that protect RGC and stimulate regeneration of their axons in the optic nerve in degenerate eye disease. NSC may have potential as both a source of replacement cells and also as mediators of paracrine treatment. Copyright © 2015. Published by Elsevier B.V.

Sex Differences in Maturation of Human Embryonic Stem Cell-Derived β Cells in Mice.

PubMed

Saber, Nelly; Bruin, Jennifer E; O'Dwyer, Shannon; Schuster, Hellen; Rezania, Alireza; Kieffer, Timothy J

2018-04-01

Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate that pancreatic progenitor cells can mature into a mixed population of endocrine cells, including glucose-responsive β cells several months after implantion. However, it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these hESC-derived cells. Here, we investigated the effects of (1) pregnancy on the maturation of human stage 4 (S4) pancreatic progenitor cells and (2) the impact of host sex on both S4 cells and more mature stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-beige mice. Pregnancy led to increased proliferation of endogenous pancreatic β cells, but did not appear to affect proliferation or maturation of S4 cells at midgestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males, whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells before implantation.

Development of an invitro technique to use mouse embryonic stem cell in evaluating effects of xenobiotics

EPA Science Inventory

Our goal has been to develop a high-throughput, in vitro technique for evaluating the effects of xenobiotics using mouse embryonic stem cells (mESCs). We began with the Embryonic Stem Cell Test (EST), which is used to predict the embryotoxic potential of a test compound by combin...

Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

PubMed

Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

2018-05-01

The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

CD71(high) population represents primitive erythroblasts derived from mouse embryonic stem cells.

PubMed

Chao, Ruihua; Gong, Xueping; Wang, Libo; Wang, Pengxiang; Wang, Yuan

2015-01-01

The CD71/Ter119 combination has been widely used to reflect dynamic maturation of erythrocytes in vivo. However, because CD71 is expressed on all proliferating cells, it is unclear whether it can be utilized as an erythrocyte-specific marker during differentiation of embryonic stem cells (ESCs). In this study, we revealed that a population expressing high level of CD71 (CD71(high)) during mouse ESC differentiation represented an in vitro counterpart of yolk sac-derived primitive erythroblasts (EryPs) isolated at 8.5days post coitum. In addition, these CD71(high) cells went through "maturational globin switching" and enucleated during terminal differentiation in vitro that were similar to the yolk sac-derived EryPs in vivo. We further demonstrated that the formation of CD71(high) population was regulated differentially by key factors including Scl, HoxB4, Eaf1, and Klf1. Taken together, our study provides a technical advance that allows efficient segregation of EryPs from differentiated ESCs in vitro for further understanding molecular regulation during primitive erythropoiesis. Copyright © 2014. Published by Elsevier B.V.

Metabolomic Response of Human Embryonic Stem Cell Derived Germ-like Cells after Exposure to Steroid Hormones

EPA Science Inventory

To assess the potential risks of human exposure to endocrine active compounds (EACs), the mechanisms of toxicity must first be identified and characterized. Currently, there are no robust in vitro models for identifying the mechanisms of toxicity in germ cells resulting from EAC ...

Quantitative assessment of neurite outgrowth in human embryonic stem cell derived hN2 cells using automated high-content image analysis

EPA Science Inventory

Throughout development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxic chemicals that interfere with this process may result in permanent deficits in nervous system function. Traditionally, rodent primary ne...

[The use of embryonic stem cells for medical-therapeutical purposes: a study of attitudes among Icelandic physicians, lawyers and clergymen.].

PubMed

Oskarsson, Trausti; Guðmundsson, Flóki; Sigurðsson, Jóhann Agúst; Getz, Linn; Arnason, Vilhjálmur

2003-06-01

To study the bioethical standpoints among three groups of Icelandic professionals in relation to the use of embryonic stem cells for medical-therapeutical purposes. In June 2002, a questionnaire was sent by mail to a random sample of 284 doctors and 293 lawyers, as well as all 168 practicing clergymen in Iceland. The participants' position in relation to the use of embryonic stem cells for therapeutical purposes was elicited through general questions as well as case examples. 290 questionnaires (39%) were returned. 62% of participants believed the embryo to have an ethical status superior to that of biologically comparable life forms. 20% of respondents considered its status as equal to that of a grown human being, whilst 18% considered it equal to biologically comparable primitive life forms. There was a difference between the respondent groups (p<0,05). A vast majority believed the use of embryonic stem cells for therapeutical purposes to be justifiable, although the origin of the stem cells appeared to make a difference to many respondents. 8% of participants took an unconditional position against the use of embryonic stem cells. Among those who considered the use of embryonic stem cells with a therapeutic aim to be justifiable, 71% believed that embryonic stem cells should only be utilized to treat diseases of a severe nature. 64% of participants defended the idea of therapeutic cloning with the intention to treat a patient with Parkinson's disease, but the case history elicited considerable difference between professional groups. Clergymen and lawyers tended to hold firmer attitudes, clergymen against and lawyers for the use of stem cells, whilst medical doctors as a group positioned themselves more towards the middle. Female respondents generally took a more modest stand whilst males were more likely to take a firmer stand in both directions. A vast majority (87%) of the participants believed there to be a need for public debate in relation to the use of embryonic stem cells for therapeutical purposes. Overall, participants views in relation to the use of embryonic stem cells for medical purposes were rather liberal. There were however significant differences between professional groups. The relatively high tolerance in regard to therapeutic cloning is interesting in view of the considerable controversy over this topic in many countries. There appears to be fertile ground for a public debate about the use of embryonic stem cells for medical purposes in Iceland.

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

PubMed Central

Jean, Christian; Fragnet-Trapp, Laetitia; Rémy, Sylvie; Chabanne-Vautherot, Danièle; Montillet, Guillaume; Fuet, Aurélie; Denesvre, Caroline; Pain, Bertrand

2017-01-01

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies. PMID:28406989

Strand displacement amplification for ultrasensitive detection of human pluripotent stem cells.

PubMed

Wu, Wei; Mao, Yiping; Zhao, Shiming; Lu, Xuewen; Liang, Xingguo; Zeng, Lingwen

2015-06-30

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful model system for studies of cellular identity and early mammalian development, which hold great promise for regenerative medicine. It is necessary to develop a convenient method to discriminate hPSCs from other cells in clinics and basic research. Herein, a simple and reliable biosensor for stem cell detection was established. In this biosensor system, stage-specific embryonic antigen-3 (SSEA-3) and stage-specific embryonic antigen-4 (SSEA-4) were used to mark human pluripotent stem cells (hPSCs). Antibody specific for SSEA-3 was coated onto magnetic beads for hPSCs enrichment, and antibody specific for SSEA-4 was conjugated with carboxyl-modified tDNA sequence which was used as template for strand displacement amplification (SDA). The amplified single strand DNA (ssDNA) was detected with a lateral flow biosensor (LFB). This biosensor is capable of detecting a minimum of 19 human embryonic stem cells by a strip reader and 100 human embryonic stem cells by the naked eye within 80min. This approach has also shown excellent specificity to distinguish hPSCs from other types of cells, showing that it is promising for specific and handy detection of human pluripotent stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-28

... Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research... Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research. OMB No. 0925-0601-- Expiration Date... cell lines be approved for use in NIH funded research. Applicants may submit applications at any time...

Neural stem cell-based treatment for neurodegenerative diseases.

PubMed

Kim, Seung U; Lee, Hong J; Kim, Yun B

2013-10-01

Human neurodegenerative diseases such as Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) are caused by a loss of neurons and glia in the brain or spinal cord. Neurons and glial cells have successfully been generated from stem cells such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs), and stem cell-based cell therapies for neurodegenerative diseases have been developed. A recent advance in generation of a new class of pluripotent stem cells, induced pluripotent stem cells (iPSCs), derived from patients' own skin fibroblasts, opens doors for a totally new field of personalized medicine. Transplantation of NSCs, neurons or glia generated from stem cells in animal models of neurodegenerative diseases, including PD, HD, ALS and AD, demonstrates clinical improvement and also life extension of these animals. Additional therapeutic benefits in these animals can be provided by stem cell-mediated gene transfer of therapeutic genes such as neurotrophic factors and enzymes. Although further research is still needed, cell and gene therapy based on stem cells, particularly using neurons and glia derived from iPSCs, ESCs or NSCs, will become a routine treatment for patients suffering from neurodegenerative diseases and also stroke and spinal cord injury. © 2013 Japanese Society of Neuropathology.

Human embryonic stem cell derived islet progenitors mature inside an encapsulation device without evidence of increased biomass or cell escape.

PubMed

Kirk, Kaitlyn; Hao, Ergeng; Lahmy, Reyhaneh; Itkin-Ansari, Pamela

2014-05-01

There are several challenges to successful implementation of a cell therapy for insulin dependent diabetes derived from human embryonic stem cells (hESC). Among these are development of functional insulin producing cells, a clinical delivery method that eliminates the need for chronic immunosuppression, and assurance that hESC derived tumors do not form in the patient. We and others have shown that encapsulation of cells in a bilaminar device (TheraCyte) provides immunoprotection in rodents and primates. Here we monitored human insulin secretion and employed bioluminescent imaging (BLI) to evaluate the maturation, growth, and containment of encapsulated islet progenitors derived from CyT49 hESC, transplanted into mice. Human insulin was detectable by 7 weeks post-transplant and increased 17-fold over the course of 8 weeks, yet during this period the biomass of encapsulated cells remained constant. Remarkably, by 20 weeks post-transplant encapsulated cells secreted sufficient levels of human insulin to ameliorate alloxan induced diabetes. Further, bioluminescent imaging revealed for the first time that hESCs remained fully contained in encapsulation devices for up to 150 days, the longest period tested. Collectively, the data suggest that encapsulated hESC derived islet progenitors hold great promise as an effective and safe cell replacement therapy for insulin dependent diabetes. Copyright © 2014. Published by Elsevier B.V.

Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells

PubMed Central

Svensson, Bengt; Nagubothu, Srinivasa R.; Nord, Christoffer; Cedervall, Jessica; Hultman, Isabell; Ährlund-Richter, Lars; Tolf, Anna; Hertegård, Stellan

2015-01-01

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives. PMID:26557696

Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells.

PubMed

Svensson, Bengt; Nagubothu, Srinivasa R; Nord, Christoffer; Cedervall, Jessica; Hultman, Isabell; Ährlund-Richter, Lars; Tolf, Anna; Hertegård, Stellan

2015-01-01

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives.

Induction of human embryonic and induced pluripotent stem cells into urothelium.

PubMed

Osborn, Stephanie L; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H; Nolta, Jan; Kurzrock, Eric A

2014-05-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

Induction of Human Embryonic and Induced Pluripotent Stem Cells Into Urothelium

PubMed Central

Osborn, Stephanie L.; Thangappan, Ravikumar; Luria, Ayala; Lee, Justin H.; Nolta, Jan

2014-01-01

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium (“Uromedium”), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation. PMID:24657961

In Vivo Generation of Neural Stem Cells Through Teratoma Formation.

PubMed

Hong, Yean Ju; Kim, Jong Soo; Choi, Hyun Woo; Song, Hyuk; Park, Chankyu; Do, Jeong Tae

2016-09-01

Pluripotent stem cells have the potential to differentiate into all cell types of the body in vitro through embryoid body formation or in vivo through teratoma formation. In this study, we attempted to generate in vivo neural stem cells (NSCs) differentiated through teratoma formation using Olig2-GFP transgenic embryonic stem cells (ESCs). After 4 to 6 weeks of injection with Olig2-GFP transgenic ESCs, Olig2-GFP(+) NSCs were identified in teratomas formed in immunodeficient mice. Interestingly, 4-week-old teratomas contained higher percentage of Olig2-GFP(+) cells (∼11%) than 6-week-old teratomas (∼3%). These in vivo-derived NSCs expressed common NSC markers (Nestin and Sox2) and differentiated into terminal neuronal and glial lineages. These results suggest that pure NSC populations exhibiting properties similar to those of brain-derived NSCs can be established through teratoma formation.

Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion.

PubMed

Konig, Niclas; Trolle, Carl; Kapuralin, Katarina; Adameyko, Igor; Mitrecic, Dinko; Aldskogius, Hakan; Shortland, Peter J; Kozlova, Elena N

2017-01-01

Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second-order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)-derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3-L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

HCN4-Overexpressing Mouse Embryonic Stem Cell-Derived Cardiomyocytes Generate a New Rapid Rhythm in Rats with Bradycardia.

PubMed

Saito, Yukihiro; Nakamura, Kazufumi; Yoshida, Masashi; Sugiyama, Hiroki; Takano, Makoto; Nagase, Satoshi; Morita, Hiroshi; Kusano, Kengo F; Ito, Hiroshi

2018-05-30

A biological pacemaker is expected to solve the persisting problems of an artificial cardiac pacemaker including short battery life, lead breaks, infection, and electromagnetic interference. We previously reported HCN4 overexpression enhances pacemaking ability of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) in vitro. However, the effect of these cells on bradycardia in vivo has remained unclear. Therefore, we transplanted HCN4-overexpressing mESC-CMs into bradycardia model animals and investigated whether they could function as a biological pacemaker. The rabbit Hcn4 gene was transfected into mouse embryonic stem cells and induced HCN4-overexpressing mESC-CMs. Non-cardiomyocytes were removed under serum/glucose-free and lactate-supplemented conditions. Cardiac balls containing 5 × 10 3 mESC-CMs were made by using the hanging drop method. One hundred cardiac balls were injected into the left ventricular free wall of complete atrioventricular block (CAVB) model rats. Heart beats were evaluated using an implantable telemetry system 7 to 30 days after cell transplantation. The result showed that ectopic ventricular beats that were faster than the intrinsic escape rhythm were often observed in CAVB model rats transplanted with HCN4-overexpressing mESC-CMs. On the other hand, the rats transplanted with non-overexpressing mESC-CMs showed sporadic single premature ventricular contraction but not sustained ectopic ventricular rhythms. These results indicated that HCN4-overexpressing mESC-CMs produce rapid ectopic ventricular rhythms as a biological pacemaker.

Identification of Proteins Related to Epigenetic Regulation in the Malignant Transformation of Aberrant Karyotypic Human Embryonic Stem Cells by Quantitative Proteomics

PubMed Central

Sun, Yi; Yang, Yixuan; Zeng, Sicong; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

2014-01-01

Previous reports have demonstrated that human embryonic stem cells (hESCs) tend to develop genomic alterations and progress to a malignant state during long-term in vitro culture. This raises concerns of the clinical safety in using cultured hESCs. However, transformed hESCs might serve as an excellent model to determine the process of embryonic stem cell transition. In this study, ITRAQ-based tandem mass spectrometry was used to quantify normal and aberrant karyotypic hESCs proteins from simple to more complex karyotypic abnormalities. We identified and quantified 2583 proteins, and found that the expression levels of 316 proteins that represented at least 23 functional molecular groups were significantly different in both normal and abnormal hESCs. Dysregulated protein expression in epigenetic regulation was further verified in six pairs of hESC lines in early and late passage. In summary, this study is the first large-scale quantitative proteomic analysis of the malignant transformation of aberrant karyotypic hESCs. The data generated should serve as a useful reference of stem cell-derived tumor progression. Increased expression of both HDAC2 and CTNNB1 are detected as early as the pre-neoplastic stage, and might serve as prognostic markers in the malignant transformation of hESCs. PMID:24465727

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.

Trends in the human embryonic stem cell patent field.

PubMed

Karlsson, Ulrika; Hyllner, Johan; Runeberg, Kristina

2007-01-01

The successful derivation of human embryonic stem (hES) cell lines in late 1990s marks the birth of a new era in biomedical research. In the USA, this landmark invention is protected by granted composition-of-matter patents. In addition to these patents, several others have been granted on further development of hES cell research, such as on differentiated cell types and in vitro and in vivo use aspects. In Europe, there is presently no consensus pertaining to the patentability of hES cells, and all patent applications pending at the European patent office are therefore awaiting a principal decision by the Enlarged Board of Appeal. The authors argue that it will be of importance to the stem cell industry that patents are granted on inventions downstream in the value chain, e.g on specialised cell types derived from hES cells and different drug discovery applications. Patents and patent applications on such inventions for the three germ layers ectoderm/neuro, endoderm/hepato and mesoderm/cardio have been examined. The number of patents increased in the period 2001 to 2006 for all three lineages with ectoderm/neuro as the most patent intensive field. There where 9-13 times more US patent applications filed related to the three lineages compared to in Europe.

Various types of stem cells, including a population of very small embryonic-like stem cells, are mobilized into peripheral blood in patients with Crohn's disease.

PubMed

Marlicz, Wojciech; Zuba-Surma, Ewa; Kucia, Magda; Blogowski, Wojciech; Starzynska, Teresa; Ratajczak, Mariusz Z

2012-09-01

Developmentally early cells, including hematopoietic stem progenitor cells (HSPCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs), are mobilized into peripheral blood (PB) in response to tissue/organ injury. We sought to determine whether these cells are mobilized into PB in patients with Crohn's disease (CD). Twenty-five patients with active CD, 20 patients in clinical remission, and 25 age-matched controls were recruited and PB samples harvested. The circulating CD133+/Lin-/CD45+ and CD34+/Lin-/CD45+ cells enriched for HSPCs, CD105+/STRO-1+/CD45- cells enriched for MSCs, CD34+/KDR+/CD31+/CD45-cells enriched for EPCs, and small CXCR4+CD34+CD133+ subsets of Lin-CD45- cells that correspond to the population of VSELs were counted by fluorescence-activated cell sorting (FACS) and evaluated by direct immunofluorescence staining for pluripotency embryonic markers and by reverse-transcription polymerase chain reaction (RT-PCR) for expression of messenger (m)RNAs for a panel of genes expressed in intestine epithelial stem cells. The serum concentration of factors involved in stem cell trafficking, such as stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) were measured by enzyme-linked immunosorbent assay (ELISA). Our data indicate that cells expressing markers for MSCs, EPCs, and small Oct-4+Nanog+SSEA-4+CXCR4+lin-CD45- VSELs are mobilized into PB in CD. The mobilized cells also expressed at the mRNA level genes playing a role in development and regeneration of gastrointestinal epithelium. All these changes were accompanied by increased serum concentrations of VEGF and HGF. CD triggers the mobilization of MSCs, EPCs, and VSELs, while the significance and precise role of these mobilized cells in repair of damaged intestine requires further study. Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.

Using therapeutic cloning to fight human disease: a conundrum or reality?

PubMed

Hall, Vanessa J; Stojkovic, Petra; Stojkovic, Miodrag

2006-07-01

The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

PubMed

Secher, Jan O; Liu, Ying; Petkov, Stoyan; Luo, Yonglun; Li, Dong; Hall, Vanessa J; Schmidt, Mette; Callesen, Henrik; Bentzon, Jacob F; Sørensen, Charlotte B; Freude, Kristine K; Hyttel, Poul

2017-03-01

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

PubMed

Roche, E; Sepulcre, M P; Enseñat-Waser, R; Maestre, I; Reig, J A; Soria, B

2003-07-01

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.

Stem cell maintenance by manipulating signaling pathways: past, current and future

PubMed Central

Chen, Xi; Ye, Shoudong; Ying, Qi-Long

2015-01-01

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retainedin various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways. [BMB Reports 2015; 48(12): 668-676] PMID:26497581

Revocation of European patent for neural progenitors highlights patent challenges for inventions relating to human embryonic stem cells.

PubMed

Rigby, Barbara

2013-11-01

Cells derived from human embryonic stem cells have great therapeutic potential. Patents are key to allowing companies that develop methods of generating such cells to recuperate their investment. However, in Europe, inventions relating to the use of human embryos for commercial purposes are excluded from patentability on moral grounds. The scope of this morality exclusion was recently tested before Germany's highest court and before the European Patent Office (EPO), with diverging results. The decision by the EPO's Opposition Division to revoke EP1040185 relating to neural precursors and methods for their generation has received a mixed reception. The decision has very recently been appealed, and the outcome of this Appeal should provide more definitive guidance on the scope of the morality exclusion.

Three-Step Method for Proliferation and Differentiation of Human Embryonic Stem Cell (hESC)-Derived Male Germ Cells

PubMed Central

Lim, Jung Jin; Shim, Myung Sun; Lee, Jeoung Eun; Lee, Dong Ryul

2014-01-01

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells. PMID:24690677

Pluripotent stem cell-derived natural killer cells for cancer therapy

PubMed Central

Knorr, David A.; Kaufman, Dan S.

2010-01-01

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an accessible, genetically tractable and homogenous starting cell populations to efficiently study human blood cell development. These cell populations provide platforms to develop new cell-based therapies to treat both malignant and non-malignant hematological diseases. Our group has previously demonstrated the ability of hESC-derived hematopoietic precursors to produce functional natural killer (NK) cells as well as an explanation of the underlying mechanism responsible for inefficient development of T and B cells from hESCs. hESCs and iPSCs, which can be reliably engineered in vitro, provide an important new model system to study human lymphocyte development and produce enhanced cell-based therapies with potential to serve as a “universal” source of anti-tumor lymphocytes for novel clinical therapies. This review will focus on the application of hESC-derived NK cells with currently used and novel therapeutics for clinical trials, current barriers to translation, and future applications through genetic engineering approaches. PMID:20801411

TOPICAL REVIEW: Stem cells engineering for cell-based therapy

NASA Astrophysics Data System (ADS)

Taupin, Philippe

2007-09-01

Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, heart and muscular diseases, to neurological diseases, disorders and injuries. Significant progresses have been made in stem cell research over the past decade; the derivation of embryonic stem cells (ESCs) from human tissues, the development of cloning technology by somatic cell nuclear transfer (SCNT) and the confirmation that neurogenesis occurs in the adult mammalian brain and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), including that of humans. Despite these advances, there may be decades before stem cell research will translate into therapy. Stem cell research is also subject to ethical and political debates, controversies and legislation, which slow its progress. Cell engineering has proven successful in bringing genetic research to therapy. In this review, I will review, in two examples, how investigators are applying cell engineering to stem cell biology to circumvent stem cells' ethical and political constraints and bolster stem cell research and therapy.

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

PubMed

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation

PubMed Central

Forzati, Floriana; Federico, Antonella; Pallante, Pierlorenzo; Colamaio, Marianna; Esposito, Francesco; Sepe, Romina; Gargiulo, Sara; Luciano, Antonio; Arra, Claudio; Palma, Giuseppe; Bon, Giulia; Bucher, Stefania; Falcioni, Rita; Brunetti, Arturo; Battista, Sabrina; Fedele, Monica; Fusco, Alfredo

2014-01-01

ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation. PMID:25190058

3 CFR 13505 - Executive Order 13505 of March 9, 2009. Removing Barriers to Responsible Scientific Research...

Code of Federal Regulations, 2010 CFR

2010-01-01

... Barriers to Responsible Scientific Research Involving Human Stem Cells 13505 Order 13505 Presidential... Scientific Research Involving Human Stem Cells By the authority vested in me as President by the Constitution.... Research involving human embryonic stem cells and human non-embryonic stem cells has the potential to lead...

Immune modulatory mesenchymal stem cells derived from human embryonic stem cells through a trophoblast-like stage.

PubMed

Wang, Xiaofang; Lazorchak, Adam S; Song, Li; Li, Enqin; Zhang, Zhenwu; Jiang, Bin; Xu, Ren-He

2016-02-01

Mesenchymal stem/stromal cells (MSCs) have great clinical potential in modulating inflammation and promoting tissue repair. Human embryonic stem cells (hESCs) have recently emerged as a potentially superior cell source for MSCs. However, the generation methods reported so far vary greatly in quality and efficiency. Here, we describe a novel method to rapidly and efficiently produce MSCs from hESCs via a trophoblast-like intermediate stage in approximately 11-16 days. We term these cells "T-MSCs" and show that T-MSCs express a phenotype and differentiation potential minimally required to define MSCs. T-MSCs exhibit potent immunomodulatory activity in vitro as they can remarkably inhibit proliferation of cocultured T and B lymphocytes. Unlike bone marrow MSCs, T-MSCs do not have increased expression of inflammatory mediators in response to IFNγ. Moreover, T-MSCs constitutively express a high level of the immune inhibitory ligand PD-L1 and elicit strong and durable efficacy in two distinct animal models of autoimmune disease, dextran sulfate sodium induced colitis, and experimental autoimmune encephalomyelitis, at doses near those approved for clinical trials. Together, we present a simple and fast derivation method to generate MSCs from hESCs, which possess potent immunomodulatory properties in vitro and in vivo and may serve as a novel and ideal candidate for MSC-based therapies. © 2015 AlphaMed Press.

Induced pluripotent stem cells (iPSCs) derived from different cell sources and their potential for regenerative and personalized medicine.

PubMed

Shtrichman, R; Germanguz, I; Itskovitz-Eldor, J

2013-06-01

Human induced pluripotent stem cells (hiPSCs) have great potential as a robust source of progenitors for regenerative medicine. The novel technology also enables the derivation of patient-specific cells for applications to personalized medicine, such as for personal drug screening and toxicology. However, the biological characteristics of iPSCs are not yet fully understood and their similarity to human embryonic stem cells (hESCs) is still unresolved. Variations among iPSCs, resulting from their original tissue or cell source, and from the experimental protocols used for their derivation, significantly affect epigenetic properties and differentiation potential. Here we review the potential of iPSCs for regenerative and personalized medicine, and assess their expression pattern, epigenetic memory and differentiation capabilities in relation to their parental tissue source. We also summarize the patient-specific iPSCs that have been derived for applications in biological research and drug discovery; and review risks that must be overcome in order to use iPSC technology for clinical applications.

[Expression of embryonic markers in pterygium derived mesenchymal cells].

PubMed

Pascual, G; Montes, M A; Pérez-Rico, C; Pérez-Kohler, B; Bellón, J M; Buján, J

2010-12-01

Destruction of the limbal epithelium barrier is the most important mechanism of pterygium formation (conjunctiva proliferation, encroaching onto the cornea). It is thought to arise from activated and proliferating limbal epithelial stem cells. The objective of this study is to evaluate the presence of undifferentiated mesenchymal cells (stem cells) in cultured cells extracted from human pterygium. Cells from 6 human pterygium were isolated by explantation and placed in cultures with amniomax medium. Once the monolayer was reached the cells were seeded onto 24 well microplates. The cells were studied in the second sub-culture. The immunohistochemical expression of different embryonic stem cell markers, OCT3/4 and CD9, was analysed. The differentiated phenotypes were characterised with the monoclonal antibodies anti-CD31, α-actin and vimentin. All the cell populations obtained from pterygium showed vimentin expression. Less than 1% of the cells were positive for CD31 and α-actin markers. The majority of the cell population was positive for OCT3/4 and CD9. The cell population obtained from pterygium expressed mesenchymal cell phenotype and embryonic markers, such us OCT3/4 and CD9. This undifferentiated population could be involved in the large recurrence rate of this type of tissue after surgery. Copyright © 2010 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

PubMed

Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

2010-10-01

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens. 2010 Elsevier Inc. All rights reserved.

Role of bioinspired polymers in determination of pluripotent stem cell fate

PubMed Central

Abraham, Sheena; Eroshenko, Nikolai; Rao, Raj R

2009-01-01

Human pluripotent stem cells, including embryonic and induced pluripotent stem cells, hold enormous potential for the treatment of many diseases, owing to their ability to generate cell types useful for therapeutic applications. Currently, many stem cell culture propagation and differentiation systems incorporate animal-derived components for promoting self-renewal and differentiation. However, use of these components is labor intensive, carries the risk of xenogeneic contamination and yields compromised experimental results that are difficult to duplicate. From a biomaterials perspective, the generation of an animal- and cell-free biomimetic microenvironment that provides the appropriate physical and chemical cues for stem cell self-renewal or differentiation into specialized cell types would be ideal. This review presents the use of natural and synthetic polymers that support propagation and differentiation of stem cells, in an attempt to obtain a clear understanding of the factors responsible for the determination of stem cell fate. PMID:19580405

HLA Engineering of Human Pluripotent Stem Cells

PubMed Central

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-01-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I–negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8+ T cell responses were reduced in class I–negative cells that had undergone differentiation in embryoid bodies. These B2M−/− ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines. PMID:23629003

HLA engineering of human pluripotent stem cells.

PubMed

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-06-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

PubMed

Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

2009-06-17

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle

PubMed Central

Xu, Zijian; Wang, Wenjie; Jiang, Kaiju; Yu, Zhou; Huang, Huanwei; Wang, Fengchao; Zhou, Bin; Chen, Ting

2015-01-01

Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation. DOI: http://dx.doi.org/10.7554/eLife.10567.001 PMID:26653852

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.

Activin A Modulates CRIPTO-1/HNF4α+ Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells

PubMed Central

Duelen, Robin; Gilbert, Guillaume; Patel, Abdulsamie; de Schaetzen, Nathalie; De Waele, Liesbeth; Roderick, Llewelyn; Sipido, Karin R.; Verfaillie, Catherine M.; Buyse, Gunnar M.

2017-01-01

The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes. PMID:28163723

A comparative study on efficiency of adult fibroblast, putative embryonic stem cell and lymphocyte as donor cells for production of handmade cloned embryos in goat and characterization of putative ntES cells obtained from these embryos.

PubMed

Dutta, Rahul; Malakar, Dhruba; Khate, Keviletsu; Sahu, Shailendra; Akshey, Yogesh; Mukesh, Manishi

2011-09-15

The main purpose of the experiment was to compare the efficiency of three cell types, namely adult fibroblast, putative embryonic stem (ES) cell, and lymphocyte, as donor cells for somatic cell nuclear transfer by handmade cloning in goats. The outcome clearly shows that putative embryonic stem cells, with a cleavage and blastocyst production rate of 74.69% ± 3.92 and 39.75% ± 3.86, respectively, performs better in comparison to adult fibroblast cell and lymphocyte. Between adult fibroblast cell and lymphocyte no statistically significant difference exists at P < 0.05. An overall cleavage and blastocyst formation rate of 67.41% ± 3.92 and 26.96% ± 3.86 was obtained using adult fibroblast donor cells. The study establishes beyond doubt the reprogrammability of lymphocyte by handmade cloning (HMC) protocol with a cleavage and blastocyst production rate of 56.47% ± 3.92 and 24.70% ± 3.86, respectively. PCR analysis of highly polymorphic 286 bp fragment of MHC II DRB genes of cloned embryos and three donor cells were performed to verify the cloned embryos. The amplified PCR products were subjected to SSCP to confirm their genetic identity. The karyotyping of the cloned embryos showed normal chromosomal status as expected in goat. Significantly, in the second stage of the experiment, the produced cloned embryos were successfully used to derive ntES-like cells. The rate of primary colony formation rate was 62.50% ± 4.62 for fibroblast donor cell derived embryos. The same was 60.60% ± 4.62 for putative ES donor cell derived embryos and 66.66% ± 4.62 for lymphocyte donor cell derived embryos, respectively. The putative ntES colonies were positively characterized for alkaline phosphatase, Oct-4, TRA-1-60, TRA-1-81, Sox-2, and Nanog by Immunocytochemistry and Reverse Transcription PCR. To further validate the stem ness, the produced putative ntES colonies were differentiated to embryoid bodies. Immunocytochemistry revealed that embryoid bodies expressed NESTIN specific for ectodermal lineage; GATA-4 for endodermal lineage and smooth muscle actin-I, and troponin-I specific for mesodermal lineage. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. It could be of substantial significance as patient specific ntES cells have proven therapeutic significance. Copyright © 2011 Elsevier Inc. All rights reserved.

Structural Complexity of Non-acid Glycosphingolipids in Human Embryonic Stem Cells Grown under Feeder-free Conditions*

PubMed Central

Barone, Angela; Benktander, John; Ångström, Jonas; Aspegren, Anders; Björquist, Petter; Teneberg, Susann; Breimer, Michael. E.

2013-01-01

Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 109 cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Lex pentaosylceramide, and the Ley hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated. PMID:23404501

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.

Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed bed/column bioreactor.

PubMed

de Peppo, Giuseppe Maria; Sladkova, Martina; Sjövall, Peter; Palmquist, Anders; Oudina, Karim; Hyllner, Johan; Thomsen, Peter; Petite, Hervé; Karlsson, Camilla

2013-01-01

Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.

Embryonic death and the creation of human embryonic stem cells.

PubMed

Landry, Donald W; Zucker, Howard A

2004-11-01

The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.

The involvement of protein kinase C-ε in isoflurane induced preconditioning of human embryonic stem cell--derived Nkx2.5(+) cardiac progenitor cells.

PubMed

Song, In-Ae; Oh, Ah-Young; Kim, Jin-Hee; Choi, Young-Min; Jeon, Young-Tae; Ryu, Jung-Hee; Hwang, Jung-Won

2016-02-20

Anesthetic preconditioning can improve survival of cardiac progenitor cells exposed to oxidative stress. We investigated the role of protein kinase C and isoform protein kinase C-ε in isoflurane-induced preconditioning of cardiac progenitor cells exposed to oxidative stress. Cardiac progenitor cells were obtained from undifferentiated human embryonic stem cells. Immunostaining with anti-Nkx2.5 was used to confirm the differentiated cardiac progenitor cells. Oxidative stress was induced by H2O2 and FeSO4. For anesthetic preconditioning, cardiac progenitor cells were exposed to 0.25, 0.5, and 1.0 mM of isoflurane. PMA and chelerythrine were used for protein kinase C activation and inhibition, while εψRACK and εV1-2 were used for protein kinase C -ε activation and inhibition, respectively. Isoflurane-preconditioning decreased the death rate of Cardiac progenitor cells exposed to oxidative stress (death rates isoflurane 0.5 mM 12.7 ± 9.3%, 1.0 mM 12.0 ± 7.7% vs. control 31.4 ± 10.2%). Inhibitors of both protein kinase C and protein kinase C -ε abolished the preconditioning effect of isoflurane 0.5 mM (death rates 27.6 ± 13.5% and 25.9 ± 8.7% respectively), and activators of both protein kinase C and protein kinase C - ε had protective effects from oxidative stress (death rates 16.0 ± 3.2% and 10.6 ± 3.8% respectively). Both PKC and PKC-ε are involved in isoflurane-induced preconditioning of human embryonic stem cells -derived Nkx2.5(+) Cardiac progenitor cells under oxidative stress.

Response to: Dittrich et al.: Non-Embryo-Destructive Extraction of Pluripotent Embryonic Stem Cells – Overlooked Legal Prohibitions, Professional Legal Consequences and Inconsistencies in Patent Law

PubMed Central

Faltus, T.; Storz, U.

2016-01-01

The publication of “Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine” by Dittrich et al. in Geburtshilfe und Frauenheilkunde 2015; 75: 1239–1242 1 describes various possibilities which could result from the non-embryo-destructive extraction of embryonic stem cells from human blastocysts. But implementing this method is more problematic, both legally and ethically, than the authors have represented it to be and is illegal in Germany. German patent DE 10 2004 062 184 on the non-embryo-destructive extraction of embryonic stem cells referred to by Dittrich et al. contravenes the higher-ranking case-law of the European Court of Justice. Ultimately, the non-embryo-destructive harvesting of embryonic stem cells with the aim of storing these cells for use in potential therapies as proposed by Dittrich et al. is prohibited in Germany and could lead to criminal prosecution. PMID:28094826

Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

PubMed Central

Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

2015-01-01

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. PMID:25849374

Transfer and detection of freshly isolated or cultured chicken (Gallus gallus) and exotic species' embryonic gonadal germ stem cells in host embryos.

PubMed

Imus, Nastassja; Roe, Mandi; Charter, Suellen; Durrant, Barbara; Jensen, Thomas

2014-06-01

The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ovo sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds.

Embryonic Stem Cell-Derived Exosomes Promote Endogenous Repair Mechanisms and Enhance Cardiac Function Following Myocardial Infarction

PubMed Central

Khan, Mohsin; Nickoloff, Emily; Abramova, Tatiana; Johnson, Jennifer; Verma, Suresh Kumar; Krishnamurthy, Prasanna; Mackie, Alexander Roy; Vaughan, Erin; Garikipati, Venkata Naga Srikanth; Benedict, Cynthia; Ramirez, Veronica; Lambers, Erin; Ito, Aiko; Gao, Erhe; Misener, Sol; Luongo, Timothy; Elrod, John; Qin, Gangjian; Houser, Steven R; Koch, Walter J; Kishore, Raj

2015-01-01

Rationale Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes however their effect in the context of the heart is unknown. Objective Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit+ CPCs function can be enhanced with ESC exosomes Methods and Results This study demonstrates that mouse ESC derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented cardiac progenitor cell (CPC) survival, proliferation and cardiac commitment concurrent with increased c-kit+ CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290–295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression and proliferation. Conclusions mES Ex provide a novel cell free system that utilizes the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC based repair programs in the heart. PMID:25904597

A Human Pluripotent Stem Cell Model of Facioscapulohumeral Muscular Dystrophy-Affected Skeletal Muscles.

PubMed

Caron, Leslie; Kher, Devaki; Lee, Kian Leong; McKernan, Robert; Dumevska, Biljana; Hidalgo, Alejandro; Li, Jia; Yang, Henry; Main, Heather; Ferri, Giulia; Petek, Lisa M; Poellinger, Lorenz; Miller, Daniel G; Gabellini, Davide; Schmidt, Uli

2016-09-01

: Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development. ©AlphaMed Press.

Engraftment and Differentiation of Embryonic Stem Cell–Derived Neural Progenitor Cells in the Cochlear Nerve Trunk: Growth of Processes into the Organ of Corti

PubMed Central

Corrales, C. Eduardo; Pan, Luying; Li, Huawei; Liberman, M. Charles; Heller, Stefan; Edge, Albert S.B.

2007-01-01

Hearing loss in mammals is irreversible because cochlear neurons and hair cells do not regenerate. To determine whether we could replace neurons lost to primary neuronal degeneration, we injected EYFP-expressing embryonic stem cell–derived mouse neural progenitor cells into the cochlear nerve trunk in immunosuppressed animals 1 week after destroying the cochlear nerve (spiral ganglion) cells while leaving hair cells intact by ouabain application to the round window at the base of the cochlea in gerbils. At 3 days post transplantation, small grafts were seen that expressed endogenous EYFP and could be immunolabeled for neuron-specific markers. Twelve days after transplantation, the grafts had neurons that extended processes from the nerve core toward the denervated organ of Corti. By 64–98 days, the grafts had sent out abundant processes that occupied a significant portion of the space formerly occupied by the cochlear nerve. The neurites grew in fasciculating bundles projecting through Rosenthal’s canal, the former site of spiral ganglion cells, into the osseous spiral lamina and ultimately into the organ of Corti, where they contacted hair cells. Neuronal counts showed a significant increase in neuronal processes near the sensory epithelium, compared to animals that were denervated without subsequent stem cell transplantation. The regeneration of these neurons shows that neurons differentiated from stem cells have the capacity to grow to a specific target in an animal model of neuronal degeneration. PMID:17013931

Novel Method To Differentiate Human Embryonic Stem Cells Into Dopaminergic Nerve Cells | NCI Technology Transfer Center | TTC

Cancer.gov

The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in licensing opportunities to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.

Engraftment of Human Pluripotent Stem Cell-derived Progenitors in the Inner Ear of Prenatal Mice.

PubMed

Takeda, Hiroki; Hosoya, Makoto; Fujioka, Masato; Saegusa, Chika; Saeki, Tsubasa; Miwa, Toru; Okano, Hideyuki; Minoda, Ryosei

2018-01-31

There is, at present, no curative treatment for genetic hearing loss. We have previously reported that transuterine gene transfer of wild type CONNEXIN30 (CX30) genes into otocysts in CX30-deleted mice could restore hearing. Cell transplantation therapy might be another therapeutic option, although it is still unknown whether stem cell-derived progenitor cells could migrate into mouse otocysts. Here, we show successful cell transplantation of progenitors of outer sulcus cell-like cells derived from human-derived induced pluripotent stem cells into mouse otocysts on embryonic day 11.5. The delivered cells engrafted more frequently in the non-sensory region in the inner ear of CX30-deleted mice than in wild type mice and survived for up to 1 week after transplantation. Some of the engrafted cells expressed CX30 proteins in the non-sensory region. This is the first report that demonstrates successful engraftment of exogenous cells in prenatal developing otocysts in mice. Future studies using this mouse otocystic injection model in vivo will provide further clues for developing treatment modalities for congenital hearing loss in humans.

Efficient Generation of Human Embryonic Stem Cell-Derived Cardiac Progenitors Based on Tissue-Specific Enhanced Green Fluorescence Protein Expression

PubMed Central

Szebényi, Kornélia; Péntek, Adrienn; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I.; Sarkadi, Balázs

2015-01-01

Cardiac progenitor cells (CPCs) are committed to the cardiac lineage but retain their proliferative capacity before becoming quiescent mature cardiomyocytes (CMs). In medical therapy and research, the use of human pluripotent stem cell-derived CPCs would have several advantages compared with mature CMs, as the progenitors show better engraftment into existing heart tissues, and provide unique potential for cardiovascular developmental as well as for pharmacological studies. Here, we demonstrate that the CAG promoter-driven enhanced green fluorescence protein (EGFP) reporter system enables the identification and isolation of embryonic stem cell-derived CPCs. Tracing of CPCs during differentiation confirmed up-regulation of surface markers, previously described to identify cardiac precursors and early CMs. Isolated CPCs express cardiac lineage-specific transcripts, still have proliferating capacity, and can be re-aggregated into embryoid body-like structures (CAG-EGFPhigh rEBs). Expression of troponin T and NKX2.5 mRNA is up-regulated in long-term cultured CAG-EGFPhigh rEBs, in which more than 90% of the cells become Troponin I positive mature CMs. Moreover, about one third of the CAG-EGFPhigh rEBs show spontaneous contractions. The method described here provides a powerful tool to generate expandable cultures of pure human CPCs that can be used for exploring early markers of the cardiac lineage, as well as for drug screening or tissue engineering applications. PMID:24734786

Concomitant differentiation of a population of mouse embryonic stem cells into neuron-like cells and Schwann cell-like cells in a slow-flow microfluidic device

PubMed Central

Ramamurthy, Poornapriya; White, Joshua B.; Park, Joong Yull; Hume, Richard I.; Ebisu, Fumi; Mendez, Flor; Takayama, Shuichi; Barald, Kate F

2016-01-01

Background To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining Spiral Ganglion Neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF-induced mouse embryonic stem cell (mESC)-derived “neurons” could potentially substitute for lost or damaged SGN. mESC-derived “Schwann cells” produce MIF as do all Schwann cells (Huang et al., 2002; Roth et al., 2007, 2008) and could attract SGN to “ cell coated” implant. Results Neuron- and Schwann cell-like cells were produced from a common population of mESC in an ultra-slow flow microfluidic device. As the populations interacted; “neurons” grew over the “Schwann cell” lawn and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF-expressing “Schwann cells” were used to “coat” a CI: mouse SGN and MIF-induced “neurons” grew directionally to the CI and to a wild type but not MIF-knock out Organ of Corti explant. Conclusions Two novel stem cell-based approaches for treating the problem of sensorineural hearing loss are described. PMID:27761977

Derivation of vascular endothelial cells from human embryonic stem cells under GMP-compliant conditions: towards clinical studies in ischaemic disease.

PubMed

Kaupisch, A; Kennedy, L; Stelmanis, V; Tye, B; Kane, N M; Mountford, J C; Courtney, A; Baker, A H

2012-10-01

Revascularisation of ischaemic tissue remains an area of substantial unmet clinical need in cardiovascular disease. Strategies to induce therapeutic angiogenesis are therefore attractive. Our recent focus has been on human embryonic stem cell (hESC) strategies since hESC can be maintained in a pluripotent state or differentiated into any desired cell type, including endothelial cells (EC), under defined differentiation culture conditions. We recently published a protocol for non-good manufacturing practice (GMP) feeder- and serum-free hESC-EC-directed monolayer differentiation to vascular EC demonstrating the potential to generate hESC-derived EC in a GMP-compliant manner suitable for use in clinical trials. In this study we modified that laboratory protocol to GMP compliance. EC production was confirmed by flow cytometry, qRT-PCR and production of vascular structures in Matrigel®, yielding approximately 30 % mature VE-cadherin(+)/PECAM-1(+) cells using the GMP-compliant hESC line RC13. In conclusion, we have successfully demonstrated the production of vascular EC under GMP-compliant conditions suitable for clinical evaluation.

Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.

PubMed

Lund, Raymond D; Wang, Shaomei; Klimanskaya, Irina; Holmes, Toby; Ramos-Kelsey, Rebeca; Lu, Bin; Girman, Sergej; Bischoff, N; Sauvé, Yves; Lanza, Robert

2006-01-01

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.

[Ethical aspects of human embryonic stem cell use and commercial umbilical cord blood stem cell banking. Ethical reflections on the occasion of the regulation of the European Council and Parliament on advanced therapy medicinal products].

PubMed

Virt, G

2010-01-01

The regulation of the European Council and Parliament on advanced therapy medicinal products also includes therapies with human embryonic stem cells. The use of these stem cells is controversially and heavily discussed. Contrary to the use of adult stem cells, medical and ethical problems concerning the use of human embryonic stem cells persists, because this use is based on the destruction of human life at the very beginning. The regulation foresees, therefore, subsidiarity within the European Member States. Although there are no ethical problems in principle with the use of stem cells from the umbilical cord blood, there are social ethical doubts with the banking of these stem cells for autologous use without any currently foreseeable medical advantage by commercial blood banks. Also in this case subsidiarity is valid.

Stem Cell Therapy to Treat Diabetes Mellitus

PubMed Central

Liew, Chee Gee; Andrews, Peter W.

2008-01-01

Transplantation of pancreatic islets offers a direct treatment for type 1 diabetes and in some cases, insulin-dependent type 2 diabetes. However, its widespread use is hampered by a shortage of donor organs. Many extant studies have focused on deriving β-cell progenitors from pancreas and pluripotent stem cells. Efforts to generate β-cells in vitro will help elucidate the mechanisms of β-cell formation and thus provide a versatile in vivo system to evaluate the therapeutic potential of these cells to treat diabetes. Various successful experiments using β-cells in animal models have generated extensive interest in using human embryonic stem cells to restore normoglycemia in diabetic patients. While new techniques are continually unveiled, the success of β-cell generation rests upon successful manipulation of culture conditions and the induction of key regulatory genes implicated in pancreas development. In this review, we compare successfully conducted protocols, highlight essential steps and identify some of the remarkable shortfalls common to these methods. In addition, we discuss recent advancements in the derivation of patient-specific pluripotent stem cells that may facilitate the use of autologous β-cells in stem cell therapy. PMID:19290381

Spectroscopic signature of mouse embryonic stem cell-derived hepatocytes using synchrotron Fourier transform infrared microspectroscopy

NASA Astrophysics Data System (ADS)

Thumanu, Kanjana; Tanthanuch, Waraporn; Ye, Danna; Sangmalee, Anawat; Lorthongpanich, Chanchao; Parnpai, Rangsun; Heraud, Philip

2011-05-01

Stem cell-based therapy for liver regeneration has been proposed to overcome the persistent shortage in the supply of suitable donor organs. A requirement for this to succeed is to find a rapid method to detect functional hepatocytes, differentiated from embryonic stem cells. We propose Fourier transform infrared (FTIR) microspectroscopy as a versatile method to identify the early and last stages of the differentiation process leading to the formation of hepatocytes. Using synchrotron-FTIR microspectroscopy, the means of identifying hepatocytes at the single-cell level is possible and explored. Principal component analysis and subsequent partial least-squares (PLS) discriminant analysis is applied to distinguish endoderm induction from hepatic progenitor cells and matured hepatocyte-like cells. The data are well modeled by PLS with endoderm induction, hepatic progenitor cells, and mature hepatocyte-like cells able to be discriminated with very high sensitivity and specificity. This method provides a practical tool to monitor endoderm induction and has the potential to be applied for quality control of cell differentiation leading to hepatocyte formation.

Asynchronous Replication and Autosome-Pair Non-Equivalence in Human Embryonic Stem Cells

PubMed Central

Dutta, Devkanya; Ensminger, Alexander W.; Zucker, Jacob P.; Chess, Andrew

2009-01-01

A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s) that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells. PMID:19325893

Neuromuscular junction in a microfluidic device.

PubMed

Park, Hyun Sung; Liu, Su; McDonald, John; Thakor, Nitish; Yang, In Hong

2013-01-01

Malfunctions at the site of neuromuscular junction (NMJ) of post-injuries or diseases are major barriers to recovery of function. The ability to efficiently derive motor neurons (MN) from embryonic stem cells has indicated promise toward the development of new therapies in increasing functional outcomes post injury. Recent advances in micro-technologies have provided advanced culture platforms allowing compartmentalization of sub-cellular components of neurons. In this study, we combined these advances in science and technology to develop a compartmentalized in vitro NMJ model. The developed NMJ system is between mouse embryonic stem cell (mESC)-derived MNs and c2c12 myotubes cultured in a compartmentalized polydimethylsiloxane (PDMS) microfluidic device. While some functional in vitro NMJ systems have been reported, this system would further contribute to research in NMJ-related diseases by providing a system to study the site of action of NMJ aimed at improving promoting better functional recovery.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

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

Kawaguchi, Manami; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510; Kitajima, Kenji

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstratedmore » that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.« less

The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

PubMed Central

Kim, Eun Young; Lee, Kyung-Bon; Kim, Min Kyu

2014-01-01

The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140] PMID:24499672

Wnt inhibition promotes vascular specification of embryonic cardiac progenitors

PubMed Central

Reichman, David E.; Park, Laura; Man, Limor; Redmond, David; Chao, Kenny; Harvey, Richard P.; Taketo, Makoto M.; Rosenwaks, Zev

2018-01-01

ABSTRACT Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs. PMID:29217753

Quantitative assessment of neurite outgrowth in human embryonic stem-cell derived neurons using automated high-content image analysis

EPA Science Inventory

During development neurons undergo a number of morphological changes including neurite outgrowth from the cell body. Exposure to neurotoxicants that interfere with this process may cause in permanent deficits in nervous system function. While many studies have used rodent primary...

Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage.

PubMed

Yang, Yu; Bolnick, Alan; Shamir, Alexandra; Abdulhasan, Mohammed; Li, Quanwen; Parker, G C; Puscheck, Elizabeth E; Rappolee, D A

2017-08-01

Data from in vitro and in vivo models suggest that malnutrition and stress trigger adaptive responses, leading to small for gestational age (SGA) blastocysts with fewer cell numbers. These stress responses are initially adaptive, but become maladaptive with increasing stress exposures. The common stress responses of the blastocyst-derived stem cells, pluripotent embryonic and multipotent placental trophoblast stem cells (ESCs and TSCs), are decreased growth and potency, and increased, imbalanced and irreversible differentiation. SGA embryos may fail to produce sufficient antiluteolytic placental hormone to maintain corpus luteum progesterone secretion that provides nutrition at the implantation site. Myriad stress inputs for the stem cells in the embryo can occur in vitro during in vitro fertilization/assisted reproductive technology (IVF/ART) or in vivo. Paradoxically, stresses that diminish stem cell growth lead to a higher level of differentiation simultaneously which further decreases ESC or TSC numbers in an attempt to functionally compensate for fewer cells. In addition, prolonged or strong stress can cause irreversible differentiation. Resultant stem cell depletion is proposed as a cause of miscarriage via a "quiet" death of an ostensibly adaptive response of stem cells instead of a reactive, violent loss of stem cells or their differentiated progenies.

Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay

EPA Science Inventory

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing development...

Pluripotent cells in farm animals: state of the art and future perspectives.

PubMed

Nowak-Imialek, Monika; Niemann, Heiner

2012-01-01

Pluripotent cells, such as embryonic stem (ES) cells, embryonic germ cells and embryonic carcinoma cells are a unique type of cell because they remain undifferentiated indefinitely in in vitro culture, show self-renewal and possess the ability to differentiate into derivatives of the three germ layers. These capabilities make them a unique in vitro model for studying development, differentiation and for targeted modification of the genome. True pluripotent ESCs have only been described in the laboratory mouse and rat. However, rodent physiology and anatomy differ substantially from that of humans, detracting from the value of the rodent model for studies of human diseases and the development of cellular therapies in regenerative medicine. Recently, progress in the isolation of pluripotent cells in farm animals has been made and new technologies for reprogramming of somatic cells into a pluripotent state have been developed. Prior to clinical application of therapeutic cells differentiated from pluripotent stem cells in human patients, their survival and the absence of tumourigenic potential must be assessed in suitable preclinical large animal models. The establishment of pluripotent cell lines in farm animals may provide new opportunities for the production of transgenic animals, would facilitate development and validation of large animal models for evaluating ESC-based therapies and would thus contribute to the improvement of human and animal health. This review summarises the recent progress in the derivation of pluripotent and reprogrammed cells from farm animals. We refer to our recent review on this area, to which this article is complementary.

[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.

Current state of the opportunities for derivation of germ-like cells from pluripotent stem cells: are you a man, or a mouse?

PubMed Central

Petkova, Rumena; Arabadjiev, Borislav; Chakarov, Stoyan; Pankov, Roumen

2014-01-01

The concept of pluripotency as a prerogative of cells of early mammal embryos and cultured embryonic stem cells (ESC) has been invalidated with the advent of induced pluripotent stem cells. Later, it became clear that the ability to generate all cell types of the adult organism is also a questionable aspect of pluripotency, as there are cell types, such as germ cells, which are difficult to produce from pluripotent stem cells. Recently it has been proposed that there are at least two different states of pluripotency; namely, the naïve, or ground state, and the primed state, which may differ radically in terms of timeline of existence, signalling mechanisms, cell properties, capacity for differentiation into different cell types, etc. Germ-like male and female rodent cells have been successfully produced in vitro from ESC and induced pluripotent stem cells. The attempts to derive primate primordial germ cells (PGC) and germ cells in vitro from pluripotent stem cells, however, still have a low success rate, especially with the female germline. The paper reviews the properties of rodent and primate ESC with regard to their capacity for differentiation in vitro to germ-like cells, outlining the possible caveats to derivation of PGC and germ cells from primate and human pluripotent cells. PMID:26019504

MicroRNA-195 targets ADP-ribosylation factor-like protein 2 to induce apoptosis in human embryonic stem cell-derived neural progenitor cells

PubMed Central

Zhou, Y; Jiang, H; Gu, J; Tang, Y; Shen, N; Jin, Y

2013-01-01

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy, drug screening and toxicity testing of neural degenerative diseases. However, the molecular regulation of their proliferation and apoptosis, which needs to be revealed before clinical application, is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here, we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells, hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently, global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically, a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression, revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover, forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly, we found that paraquat, a neurotoxin, not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs, indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably, inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively, miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would facilitate our manipulation of the neurological pathogenesis. PMID:23807224

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs

PubMed Central

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A.

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial–mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues. PMID:22539926

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs.

PubMed

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial-mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues.

Human Embryonic Stem Cell-Derived Mesenchymal Stromal Cells Decrease the Development of Severe Experimental Autoimmune Uveitis in B10.RIII Mice.

PubMed

Qin, Yu; Chan, Ann M; Chang, Yu-Ling; Matynia, Anna; Kouris, Nicholas A; Kimbrel, Erin A; Ashki, Negin; Parikh, Sachin; Gorin, Michael B; Lanza, Robert; Levinson, Ralph D; Gordon, Lynn K

2017-09-15

We investigated the effect of exogenously administered human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) in experimental autoimmune uveitis (EAU) in B10.RIII mice, a murine model of severe uveitis. B10.RIII mice were immunized with an uveitogenic peptide, and intraperitoneal injections of 5 million hESC-MSCs per animal were given on the same day. Behavioral light sensitivity assays, histological evaluation, cytokine production, and regulatory T cells were analyzed at the peak of the disease. Histological and behavioral evidence demonstrated that early systemic treatment with hESC-MSCs decreases the development of severe EAU in B10.RIII mice. hESC-MSCs suppress Th17 and upregulate Th1 and Th2 responses as well as IL-2 and GM-CSF in splenocytes from hESC-MSC-treated mice. MSCs that originate from hESC decrease the development of severe EAU in B10.RIII mice, likely through systemic immune modulation. Further investigation is needed to determine any potential effect on active EAU.

BDNF improves the efficacy ERG amplitude maintenance by transplantation of retinal stem cells in RCS rats.

PubMed

Tian, Chunyu; Weng, Chuan Chuang; Yin, Zheng Qin

2010-01-01

The aim of this study was to evaluate the efficacy of subretinal transplantation of rat retinal stem cell when combined with Brain-derived neurotrophic factor (BDNF) in a rat model of retinal degeneration - Royal College of Surgeons (RCS) rats. Retinal stem cells were derived from embryonic day 17 Long-Evans rats and pre-labeled with fluorescence pigment-DiI prior to transplant procedures. RCS rats received injections of retinal stem cells, stem cells+BDNF, phosphate buffered saline or BNDF alone (n = 3 eyes for each procedure). At 1, 2 and 3 months after transplantation, the electroretinogram (ERG) was assessed and the outer nuclear layer thickness measured. The eyes receiving retinal stem cell and stem cell+BDNF transplants showed better photoreceptor maintenance than the other groups (P < 0.01) at all time points. One month after retina transplantation, the amplitudes of rod-ERG and Max-ERG b waves were significantly higher the eyes with stem cells+BDNF (P < 0.01), however, this difference was not seen at two and three months post transplantation. BDNF treatment alone group (without transplanted cells) had no effect when compared to buffer injections. The present results indicate that BDNF can enhance the short-term efficacy of the retinal stem cell transplantation in treating retinal degenerative disease.

Characterization of Epicardial-Derived Cardiac Interstitial Cells: Differentiation and Mobilization of Heart Fibroblast Progenitors

PubMed Central

Ehrbar, Martin; Pérez-Pomares, José M.

2013-01-01

The non-muscular cells that populate the space found between cardiomyocyte fibers are known as ‘cardiac interstitial cells’ (CICs). CICs are heterogeneous in nature and include different cardiac progenitor/stem cells, cardiac fibroblasts and other cell types. Upon heart damage CICs soon respond by initiating a reparative response that transforms with time into extensive fibrosis and heart failure. Despite the biomedical relevance of CICs, controversy remains on the ontogenetic relationship existing between the different cell kinds homing at the cardiac interstitium, as well as on the molecular signals that regulate their differentiation, maturation, mutual interaction and role in adult cardiac homeostasis and disease. Our work focuses on the analysis of epicardial-derived cells, the first cell type that colonizes the cardiac interstitium. We present here a characterization and an experimental analysis of the differentiation potential and mobilization properties of a new cell line derived from mouse embryonic epicardium (EPIC). Our results indicate that these cells express some markers associated with cardiovascular stemness and retain part of the multipotent properties of embryonic epicardial derivatives, spontaneously differentiating into smooth muscle, and fibroblast/myofibroblast-like cells. Epicardium-derived cells are also shown to initiate a characteristic response to different growth factors, to display a characteristic proteolytic expression profile and to degrade biological matrices in 3D in vitro assays. Taken together, these data indicate that EPICs are relevant to the analysis of epicardial-derived CICs, and are a god model for the research on cardiac fibroblasts and the role these cells play in ventricular remodeling in both ischemic or non/ischemic myocardial disease. PMID:23349729

Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

PubMed Central

Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S.; Chang, Alfred E.; Ito, Fumito

2016-01-01

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

Electrophysiological properties of neurons derived from human stem cells and iNeurons in vitro.

PubMed

Halliwell, Robert F

2017-06-01

Functional studies of neurons have traditionally used nervous system tissues from a variety of non-human vertebrate and invertebrate species, even when the focus of much of this research has been directed at understanding human brain function. Over the last decade, the identification and isolation of human stem cells from embryonic, tissue (or adult) and induced pluripotent stem cells (iPSCs) has revolutionized the availability of human neurons for experimental studies in vitro. In addition, the direct conversion of terminally differentiated fibroblasts into Induced neurons (iN) has generated great excitement because of the likely value of such human stem cell derived neurons (hSCNs) and iN cells in drug discovery, neuropharmacology, neurotoxicology and regenerative medicine. This review addresses the current state of our knowledge of functional receptors and ion channels expressed in neurons derived from human stem cells and iNeurons and identifies gaps and questions that might be investigated in future studies; it focusses almost exclusively on what is known about the electrophysiological properties of neurons derived from human stem cells and iN cells in vitro with an emphasis on voltage and ligand gated ion channels, since these mediate synaptic signalling in the nervous system and they are at the heart of neuropharmacology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Therapeutic Effects of Human Amniotic Fluid-Derived Stem Cells on Renal Interstitial Fibrosis in a Murine Model of Unilateral Ureteral Obstruction

PubMed Central

Yin, Zhongcheng; Zhou, Xudong; Li, Xiaoju; Xiao, Aiguo

2013-01-01

Interstitial fibrosis is regarded as the main pathway for the progression of chronic kidney disease (CKD) and is often associated with severe renal dysfunction. Stem cell-based therapies may provide alternative approaches for the treatment of CKD. Human amniotic fluid-derived stem cells (hAFSCs) are a novel stem cell population, which exhibit both embryonic and mesenchymal stem cell characteristics. Herein, the present study investigated whether the transplantation of hAFSCs into renal tissues could improve renal interstitial fibrosis in a murine model of unilateral ureteral obstruction (UUO). We showed that hAFSCs provided a protective effect and alleviated interstitial fibrosis as reflected by an increase in microvascular density; additionally, hAFSCs treatment beneficially modulated protein levels of vascular endothelial growth factor (VEGF), hypoxia inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1). Therefore, we hypothesize that hAFSCs could represent an alternative, readily available source of stem cells that can be applied for the treatment of renal interstitial fibrosis. PMID:23724119

Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain.

PubMed

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.

Nuclear Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain

PubMed Central

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain. PMID:17901127

Screening of bioactive peptides using an embryonic stem cell-based neurodifferentiation assay.

PubMed

Xu, Ruodan; Feyeux, Maxime; Julien, Stéphanie; Nemes, Csilla; Albrechtsen, Morten; Dinnyés, Andras; Krause, Karl-Heinz

2014-05-01

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.

Stem cell function during plant vascular development

PubMed Central

Miyashima, Shunsuke; Sebastian, Jose; Lee, Ji-Young; Helariutta, Yka

2013-01-01

The plant vascular system, composed of xylem and phloem, evolved to connect plant organs and transport various molecules between them. During the post-embryonic growth, these conductive tissues constitutively form from cells that are derived from a lateral meristem, commonly called procambium and cambium. Procambium/cambium contains pluripotent stem cells and provides a microenvironment that maintains the stem cell population. Because vascular plants continue to form new tissues and organs throughout their life cycle, the formation and maintenance of stem cells are crucial for plant growth and development. In this decade, there has been considerable progress in understanding the molecular control of the organization and maintenance of stem cells in vascular plants. Noticeable advance has been made in elucidating the role of transcription factors and major plant hormones in stem cell maintenance and vascular tissue differentiation. These studies suggest the shared regulatory mechanisms among various types of plant stem cell pools. In this review, we focus on two aspects of stem cell function in the vascular cambium, cell proliferation and cell differentiation. PMID:23169537

Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

NASA Astrophysics Data System (ADS)

Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z.; Gimzewski, James K.; Nakano, Atsushi

2013-04-01

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

Fetal Membranes-Derived Stem Cells Microenvironment.

PubMed

Favaron, Phelipe Oliveira; Miglino, Maria Angelica

2017-01-01

Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

PubMed Central

Ou, Xuan; Chae, Hee-Don; Wang, Rui-Hong; Shelley, William C.; Cooper, Scott; Taylor, Tammi; Kim, Young-June; Deng, Chu-Xia; Yoder, Mervin C.

2011-01-01

SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1−/− mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1−/− mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/++/−, and −/− mice. SIRT1−/− ESCs formed fewer mature blast cell colonies. Replated SIRT1−/− blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1−/−-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1−/− ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1−/− ESC differentiation deficiencies. SIRT1−/− yolk sacs manifested fewer primitive erythroid precursors. SIRT1−/− and SIRT1+/− adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. PMID:20966168

The influence of early embryo traits on human embryonic stem cell derivation efficiency.

PubMed

O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

2011-05-01

Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

Concise Review: Parthenote Stem Cells for Regenerative Medicine: Genetic, Epigenetic, and Developmental Features

PubMed Central

Daughtry, Brittany

2014-01-01

Embryonic stem cells (ESCs) have the potential to provide unlimited cells and tissues for regenerative medicine. ESCs derived from fertilized embryos, however, will most likely be rejected by a patient’s immune system unless appropriately immunomatched. Pluripotent stem cells (PSCs) genetically identical to a patient can now be established by reprogramming of somatic cells. However, practical applications of PSCs for personalized therapies are projected to be unfeasible because of the enormous cost and time required to produce clinical-grade cells for each patient. ESCs derived from parthenogenetic embryos (pESCs) that are homozygous for human leukocyte antigens may serve as an attractive alternative for immunomatched therapies for a large population of patients. In this study, we describe the biology and genetic nature of mammalian parthenogenesis and review potential advantages and limitations of pESCs for cell-based therapies. PMID:24443005

Comparative study of P19 EC stem cell differentiation in between conventional hanging drop and the zebrafish chorion as a bio-derived material.

PubMed

Dae Seok Na; Lee, Hwang; Sun Uk Kim; Chang Nam Hwang; Sang Ho Lee; Ji Yoon Kang; Jai Kyeong Kim; James Jungho Pak

2008-07-01

Various materials including glass and polymers have been widely used for stem cell culture due to their biocompatibility. However, the roles of these materials are fundamentally limited because they cannot realize or imitate the complex biological functions of living tissues, except in very simple cases. Here, the development of a bio-derived material suitable for stem cell culture and improvement of differentiation efficiency to specific cell lineages with no stimulating agents by using a chorion obtained from a fertilized zebrafish egg through the removal of the yolk and embryonic cell mass from the egg is reported. Mouse P19 EC stem cells introduced into the empty chorion form a uniform embryoid body (EB) without addition of any inducing agent. It is demonstrated that the zebrafish chorion with nanopores improves efficiencies greatly in the EB formation, cell proliferation, and lineage-specific differentiations compared to those of the conventional hanging drop culture method.

An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.

PubMed

Zhou, Jun-Mei; Chu, Jian-Xin; Chen, Xue-Jin

2008-01-01

Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.

Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow

PubMed Central

Zhu, Jie; Wang, Hong; Yang, Shuo; Guo, Liqiao; Li, Zhen; Wang, Wei; Wang, Suhan; Huang, Wenting; Wang, Liping; Yang, Tan; Ma, Qiang; Bi, Yongyi

2013-01-01

Benzene is an occupational toxicant and an environmental pollutant that potentially causes hematotoxicity and leukemia in exposed populations. Epidemiological studies suggest an association between an increased incidence of childhood leukemia and benzene exposure during the early stages of pregnancy. However, experimental evidence supporting the association is lacking at the present time. It is believed that benzene and its metabolites target hematopoietic stem cells (HSCs) to cause toxicity and cancer in the hematopoietic system. In the current study, we compared the effects of hydroquinone (HQ), a major metabolite of benzene in humans and animals, on mouse embryonic yolk sac hematopoietic stem cells (YS-HSCs) and adult bone marrow hematopoietic stem cells (BM-HSCs). YS-HSCs and BM-HSCs were isolated and enriched, and were exposed to HQ at increasing concentrations. HQ reduced the proliferation and the differentiation and colony formation, but increased the apoptosis of both YS-HSCs and BM-HSCs. However, the cytotoxic and apoptotic effects of HQ were more apparent and reduction of colony formation by HQ was more severe in YS-HSCs than in BM-HSCs. Differences in gene expression profiles were observed in HQ-treated YS-HSCs and BM-HSCs. Cyp4f18 was induced by HQ both in YS-HSCs and BM-HSCs, whereas DNA-PKcs was induced in BM-HSCs only. The results revealed differential effects of benzene metabolites on embryonic and adult HSCs. The study established an experimental system for comparison of the hematopoietic toxicity and leukemogenicity of benzene and metabolites during mouse embryonic development and adulthood. PMID:23940708

Human Embryonic Stem Cell Therapy in Crohn's Disease: A Case Report.

PubMed

Shroff, Geeta

2016-02-29

Crohn's disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565,000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn's disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn's disease. A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn's disease.

Stem cells: science, policy, and ethics

PubMed Central

Fischbach, Gerald D.; Fischbach, Ruth L.

2004-01-01

Human embryonic stem cells offer the promise of a new regenerative medicine in which damaged adult cells can be replaced with new cells. Research is needed to determine the most viable stem cell lines and reliable ways to promote the differentiation of pluripotent stem cells into specific cell types (neurons, muscle cells, etc.). To create new cell lines, it is necessary to destroy preimplantation blastocysts. This has led to an intense debate that threatens to limit embryonic stem cell research. The profound ethical issues raised call for informed, dispassionate debate. PMID:15545983

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

Glucose responsive insulin production from human embryonic germ (EG) cell derivatives

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

Clark, Gregory O.; Yochem, Robert L.; Axelman, Joyce

2007-05-11

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and {beta}-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation ofmore » preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.« less

Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells

PubMed Central

Scottoni, Federico; Crowley, Claire; Fiadeiro, Rebeca; Maghsoudlou, Panagiotis; Pellegata, Alessandro Filippo; Mazzacuva, Francesca; Gjinovci, Asllan; Lyne, Anne-Marie; Zulini, Justine; Little, Daniel; Mosaku, Olukunbi; Kelly, Deirdre; De Coppi, Paolo; Gissen, Paul

2017-01-01

Liver transplantation is the definitive treatment of liver failure but donor organ shortage limits its availability. Stem cells are highly expandable and have the potential to differentiate into any specialist cell. Use of patient-derived induced Pluripotent Stem Cells (hiPSCs) has the additional advantage for organ regeneration therapies by removing the need for immunosuppression. We compared hepatocyte differentiation of human embryonic stem cells (hESCs) and hiPSCs in a mouse decellularised liver scaffold (3D) with standard in vitro protocol (2D). Mouse livers were decellularised preserving micro-architecture, blood vessel network and extracellular matrix. hESCs and hiPSCs were primed towards the definitive endoderm. Cells were then seeded either in 3D or 2D cultures and the hepatocyte differentiation was continued. Both hESCs and hiPSCs differentiated more efficiently in 3D than in 2D, with higher and earlier expression of mature hepatocyte marker albumin, lipid and glycogen synthesis associated with a decrease in expression of fetal hepatocyte marker alpha-fetoprotein. Thus we conclude that stem cell hepatocyte differentiation in 3D culture promotes faster cell maturation. This finding suggests that optimised 3D protocols could allow generation of mature liver cells not achieved so far in standard 2D conditions and lead to improvement in cell models of liver disease and regenerative medicine applications. PMID:29261712

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells.

PubMed

Pellett, Sabine; Du, Zhong-wei; Pier, Christina L; Tepp, William H; Zhang, Su-chun; Johnson, Eric A

2011-01-07

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA. Published by Elsevier Inc.

Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.

PubMed

Ramalho-Santos, João; Varum, Sandra; Amaral, Sandra; Mota, Paula C; Sousa, Ana Paula; Amaral, Alexandra

2009-01-01

Mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species (ROS) production, calcium signalling and apoptosis; and mitochondrial defects are known to cause physiological dysfunction, including infertility. The goal of this review was to identify and discuss common themes in mitochondrial function related to mammalian reproduction. The scientific literature was searched for studies reporting on the several aspects of mitochondrial activity in mammalian testis, sperm, oocytes, early embryos and embryonic stem cells. ATP synthesis and ROS production are the most discussed aspects of mitochondrial function. Metabolic shifts from mitochondria-produced ATP to glycolysis occur at several stages, notably during gametogenesis and early embryo development, either reflecting developmental switches or substrate availability. The exact role of sperm mitochondria is especially controversial. Mitochondria-generated ROS function in signalling but are mostly described when produced under pathological conditions. Mitochondria-based calcium signalling is primarily important in embryo activation and embryonic stem cell differentiation. Besides pathologically triggered apoptosis, mitochondria participate in apoptotic events related to the regulation of spermatogonial cell number, as well as gamete, embryo and embryonic stem cell quality. Interestingly, data from knock-out (KO) mice is not always straightforward in terms of expected phenotypes. Finally, recent data suggests that mitochondrial activity can modulate embryonic stem cell pluripotency as well as differentiation into distinct cellular fates. Mitochondria-based events regulate different aspects of reproductive function, but these are not uniform throughout the several systems reviewed. Low mitochondrial activity seems a feature of 'stemness', being described in spermatogonia, early embryo, inner cell mass cells and embryonic stem cells.

Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages.

PubMed

Jiao, Fei; Wang, Juan; Dong, Zhao-Lun; Wu, Min-Juan; Zhao, Ting-Bao; Li, Dan-Dan; Wang, Xin

2012-08-01

Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA)

PubMed Central

Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

2015-01-01

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA. PMID:26258776

A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA).

PubMed

Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

2015-08-06

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

PubMed

Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

2012-05-01

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. Copyright © 2012 AlphaMed Press.

Improving and accelerating the differentiation and functional maturation of human stem cell-derived neurons: role of extracellular calcium and GABA.

PubMed

Kemp, Paul J; Rushton, David J; Yarova, Polina L; Schnell, Christian; Geater, Charlene; Hancock, Jane M; Wieland, Annalena; Hughes, Alis; Badder, Luned; Cope, Emma; Riccardi, Daniela; Randall, Andrew D; Brown, Jonathan T; Allen, Nicholas D; Telezhkin, Vsevolod

2016-11-15

Neurons differentiated from pluripotent stem cells using established neural culture conditions often exhibit functional deficits. Recently, we have developed enhanced media which both synchronize the neurogenesis of pluripotent stem cell-derived neural progenitors and accelerate their functional maturation; together these media are termed SynaptoJuice. This pair of media are pro-synaptogenic and generate authentic, mature synaptic networks of connected forebrain neurons from a variety of induced pluripotent and embryonic stem cell lines. Such enhanced rate and extent of synchronized maturation of pluripotent stem cell-derived neural progenitor cells generates neurons which are characterized by a relatively hyperpolarized resting membrane potential, higher spontaneous and induced action potential activity, enhanced synaptic activity, more complete development of a mature inhibitory GABA A receptor phenotype and faster production of electrical network activity when compared to standard differentiation media. This entire process - from pre-patterned neural progenitor to active neuron - takes 3 weeks or less, making it an ideal platform for drug discovery and disease modelling in the fields of human neurodegenerative and neuropsychiatric disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease and Schizophrenia. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Establishment of stem cell lines from nuclear transferred and parthenogenetically activated mouse oocytes for therapeutic cloning.

PubMed

Ju, Jin Young; Park, Chun Young; Gupta, Mukesh Kumar; Uhm, Sang Jun; Paik, Eun Chan; Ryoo, Zae Young; Cho, Youl Hee; Chung, Kil Saeng; Lee, Hoon Taek

2008-05-01

To establish embryonic stem cell lines from nuclear transfer of somatic cell nuclei isolated from the same oocyte donor and from parthenogenetic activation. The study also evaluated the effect of the micromanipulation procedure on the outcome of somatic cell nuclear transfer in mice. Randomized, prospective study. Hospital-based assisted reproductive technology laboratory. F(1) (C57BL/6 x 129P3/J) mice. Metaphase II-stage oocytes were either parthenogenetically activated or nuclear transferred with cumulus cell nuclei or parthenogenetically activated after a sham-manipulation procedure. Embryogenesis and embryonic stem cell establishment. The development rate to morula/blastocyst of nuclear transferred oocytes (27.9% +/- 5.9%) was significantly lower than that of the sham-manipulated (84.1% +/- 5.6%) or parthenogenetic (98.6% +/- 1.4%) groups. A sharp decrease in cleavage potential was obvious in the two- to four-cell transition for the nuclear transferred embryos (79.0% +/- 4.6% and 43.3% +/- 5.0%), implying incomplete nuclear reprogramming in arrested oocytes. However, the cleavage, as well as the development rate, of parthenogenetic and sham-manipulated groups did not differ significantly. The embryonic stem cell line establishment rate was higher from parthenogenetically activated oocytes (15.7%) than nuclear transferred (4.3%) or sham-manipulated oocytes (12.5%). Cell colonies from all groups displayed typical morphology of mice embryonic stem cells and could be maintained successfully with undifferentiated morphology after continuous proliferation for more than 120 passages still maintaining normal karyotype. All these cells were positive for mice embryonic stem cell markers such as Oct-4 and SSEA-1 based on immunocytochemistry and reverse transcriptase-polymerase chain reaction. The clonal origin of the ntES cell line and the parthenogenetic embryonic stem cell lines were confirmed by polymerase chain reaction analysis of the polymorphic markers. Blastocyst injection experiments demonstrated that these lines contributed to resulting chimeras and are germ-line competent. We report the establishment of ntES cell lines from somatic cells isolated from same individual. Our data also suggest that embryo micromanipulation procedure during the nuclear transfer procedure influences the developmental ability and embryonic stem cell establishment rate of nuclear transferred embryos.

Human embryonic and induced pluripotent stem cell-derived cardiomyocytes exhibit beat rate variability and power-law behavior.

PubMed

Mandel, Yael; Weissman, Amir; Schick, Revital; Barad, Lili; Novak, Atara; Meiry, Gideon; Goldberg, Stanislav; Lorber, Avraham; Rosen, Michael R; Itskovitz-Eldor, Joseph; Binah, Ofer

2012-02-21

The sinoatrial node is the main impulse-generating tissue in the heart. Atrioventricular conduction block and arrhythmias caused by sinoatrial node dysfunction are clinically important and generally treated with electronic pacemakers. Although an excellent solution, electronic pacemakers incorporate limitations that have stimulated research on biological pacing. To assess the suitability of potential biological pacemakers, we tested the hypothesis that the spontaneous electric activity of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exhibit beat rate variability and power-law behavior comparable to those of human sinoatrial node. We recorded extracellular electrograms from hESC-CMs and iPSC-CMs under stable conditions for up to 15 days. The beat rate time series of the spontaneous activity were examined in terms of their power spectral density and additional methods derived from nonlinear dynamics. The major findings were that the mean beat rate of hESC-CMs and iPSC-CMs was stable throughout the 15-day follow-up period and was similar in both cell types, that hESC-CMs and iPSC-CMs exhibited intrinsic beat rate variability and fractal behavior, and that isoproterenol increased and carbamylcholine decreased the beating rate in both hESC-CMs and iPSC-CMs. This is the first study demonstrating that hESC-CMs and iPSC-CMs exhibit beat rate variability and power-law behavior as in humans, thus supporting the potential capability of these cell sources to serve as biological pacemakers. Our ability to generate sinoatrial-compatible spontaneous cardiomyocytes from the patient's own hair (via keratinocyte-derived iPSCs), thus eliminating the critical need for immunosuppression, renders these myocytes an attractive cell source as biological pacemakers.

Are You Teaching Your Students about Stem Cells?

ERIC Educational Resources Information Center

Concannon, James; Brown, Patrick L.; Brandt, Trisha

2009-01-01

This activity targets students' misconceptions about embryonic and adult stem cells while also addressing an important grades 9-12 science content standard. The authors designed the activity to provide students an opportunity to explore differences between embryonic and adult stem cells prior to formal explanation. The overarching goal of this…

MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

PubMed Central

Virant-Klun, Irma; Ståhlberg, Anders; Kubista, Mikael; Skutella, Thomas

2016-01-01

MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells. PMID:26664407

Effects of hawthorn ( Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells.

PubMed

Pahlavan, Sara; Tousi, Marziyeh Shalchi; Ayyari, Mahdi; Alirezalu, Abolfazl; Ansari, Hassan; Saric, Tomo; Baharvand, Hossein

2018-03-01

Cardiac arrhythmias are major life-threatening conditions. The landmark discovery of induced pluripotent stem cells has provided a promising in vitro system for modeling hereditary cardiac arrhythmias as well as drug development and toxicity testing. Nowadays, nutraceuticals are frequently used as supplements for cardiovascular therapy. Here we studied the cardiac effects of hawthorn ( Crataegus pentagyna) leaf extract using cardiomyocytes (CMs) differentiated from healthy human embryonic stem cells, long QT syndrome type 2 (LQTS2), and catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) patient-specific induced pluripotent stem cells. The hydroalcoholic extract resulted in a dose-dependent negative chronotropic effect in all CM preparations leading to a significant reduction at 1000 µg/ml. This was accompanied by prolongation of field potential durations, although with different magnitudes in CMs from different human embryonic stem cell and iPSC lines. Hawthorn further prolonged field potential durations in LQTS2 CMs but reduced the beating frequencies and occurrence of immature field potentials triggered by β 1 -adrenergic stimulation in CPVT1 CMs at 300 and 1000 µg/ml. Furthermore, isoquercetin and vitexin flavonoids significantly slowed down isoproterenol (5 µM)-induced beating frequencies at 3 and 10 µg/ml. Therefore, C. pentagyna leaf extract and its isoquercetin and vitexin flavonoids may be introduced as a novel nutraceutical with antiarrhythmic potential for CPVT1 patients.-Pahlavan, S., Tousi, M. S., Ayyari, M., Alirezalu, A., Ansari, H., Saric, T., Baharvand, H. Effects of hawthorn ( Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells.

Mechanisms of cellular therapy in respiratory diseases.

PubMed

Abreu, Soraia C; Antunes, Mariana A; Pelosi, Paolo; Morales, Marcelo M; Rocco, Patricia R M

2011-09-01

Stem cells present a variety of clinical implications in the lungs. According to their origin, these cells can be divided into embryonic and adult stem cells; however, due to the important ethical and safety limitations that are involved in the embryonic stem cell use, most studies have chosen to focus on adult stem cell therapy. This article aims to present and clarify the recent advances in the field of stem cell biology, as well as to highlight the effects of mesenchymal stem cell (MSC) therapy in the context of acute lung injury/acute respiratory distress syndrome and chronic disorders such as lung fibrosis and chronic obstructive pulmonary disease. For this purpose, we performed a critical review of adult stem cell therapies, covering the main clinical and experimental studies published in Pubmed databases in the past 11 years. Different characteristics were extracted from these articles, such as: the experimental model, strain, cellular type and administration route used as well as the positive or negative effects obtained. There is evidence for beneficial effects of MSC on lung development, repair, and remodeling. The engraftment in the injured lung does not occur easily, but several studies report that paracrine factors can be effective in reducing inflammation and promoting tissue repair. MSC releases several growth factors and anti-inflammatory cytokines that regulate endothelial and epithelial permeability and reduce the severity of inflammation. A better understanding of the mechanisms that control cell division and differentiation, as well as of their paracrine effects, is required to enable the optimal use of bone marrow-derived stem cell therapy to treat human respiratory diseases.

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.

Self-organization phenomena in embryonic stem cell-derived embryoid bodies: axis formation and breaking of symmetry during cardiomyogenesis.

PubMed

Fuchs, Christiane; Scheinast, Matthias; Pasteiner, Waltraud; Lagger, Sabine; Hofner, Manuela; Hoellrigl, Alexandra; Schultheis, Martina; Weitzer, Georg

2012-01-01

Aggregation of embryonic stem cells gives rise to embryoid bodies (EBs) which undergo developmental processes reminiscent of early eutherian embryonic development. Development of the three germ layers suggests that gastrulation takes place. In vivo, gastrulation is a highly ordered process but in EBs only few data support the hypothesis that self-organization of differentiating cells leads to morphology, reminiscent of the early gastrula. Here we demonstrate that a timely implantation-like process is a prerequisite for the breaking of the radial symmetry of suspended EBs. Attached to a surface, EBs develop a bilateral symmetry and presumptive mesodermal cells emerge between the center of the EBs and a horseshoe-shaped ridge of cells. The development of an epithelial sheet of cells on one side of the EBs allows us to define an 'anterior' and a 'posterior' end of the EBs. In the mesodermal area, first cardiomyocytes (CMCs) develop mainly next to this epithelial sheet of cells. Development of twice as many CMCs at the 'left' side of the EBs breaks the bilateral symmetry and suggests that cardiomyogenesis reflects a local or temporal asymmetry in EBs. The asymmetric appearance of CMCs but not the development of mesoderm can be disturbed by ectopic expression of the muscle-specific protein Desmin. Later, the bilateral morphology becomes blurred by an apparently chaotic differentiation of many cell types. The absence of comparable structures in aggregates of cardiovascular progenitor cells isolated from the heart demonstrates that the self-organization of cells during a gastrulation-like process is a unique feature of embryonic stem cells. Copyright © 2011 S. Karger AG, Basel.

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.

Investigation for the differentiation process of mouse ES cells by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Yamaguchi, Yoshinori; El-Hagrasy, Maha A.; Shimizu, Eiichi; Saito, Masato; Tamiya, Eiichi

2012-03-01

The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine, guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified. The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process. Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with different degrees of maturation.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity.

PubMed

Brevini, T A L; Pennarossa, G; Vanelli, A; Maffei, S; Gandolfi, F

2012-03-01

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenote's ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocyte's ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause. Copyright © 2012 Elsevier Inc. All rights reserved.