Differentiation and characterization of rhesus monkey atrial and ventricular cardiomyocytes from induced pluripotent stem cells.

PubMed

Zhang, Xiaoqian; Cao, Henghua; Bai, Shuyun; Huo, Weibang; Ma, Yue

2017-04-01

The combination of non-human primate animals and their induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provides not only transplantation models for cell-based therapy of heart diseases, but also opportunities for heart-related drug research on both cellular and animal levels. However, the subtypes and electrophysiology properties of non-human primate iPSC-CMs hadn't been detailed characterized. In this study, we generated rhesus monkey induced pluripotent stem cells (riPSCs), and efficiently differentiated them into ventricular or atrial cardiomyocytes by modulating retinoic acid (RA) pathways. Our results revealed that the electrophysiological characteristics and response to canonical drugs of riPSC-CMs were similar with those of human pluripotent stem cell derived CMs. Therefore, rhesus monkeys and their iPSC-CMs provide a powerful and practicable system for heart related biomedical research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways.

PubMed

Li, Xiang; Michaeloudes, Charalambos; Zhang, Yuelin; Wiegman, Coen H; Adcock, Ian M; Lian, Qizhou; Mak, Judith C W; Bhavsar, Pankaj K; Chung, Kian Fan

2018-05-01

Oxidative stress-induced mitochondrial dysfunction can contribute to inflammation and remodeling in patients with chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. We sought to examine the effect of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) on oxidative stress-induce mitochondrial dysfunction in human airway smooth muscle cells (ASMCs) in vitro and in mouse lungs in vivo. ASMCs were cocultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis were measured. Conditioned medium from iPSC-MSCs and transwell cocultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyperresponsiveness in ozone-exposed mice was also investigated. Coculture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis, and ΔΨm loss in ASMCs. iPSC-MSC-conditioned medium or transwell cocultures with iPSC-MSCs reduced CSM-induced mitochondrial ROS but not ΔΨm or apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs was observed after direct coculture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyperresponsiveness, and inflammation in mouse lungs. iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs while reducing airway inflammation and hyperresponsiveness. These effects are, at least in part, dependent on cell-cell contact, which allows for mitochondrial transfer, and paracrine regulation. Therefore iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases, such as COPD. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

PubMed

Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

2016-12-01

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na +  current (I Na ), nifedipine, a blocker of L-type Ca 2+  current (I CaL ), and E4031, a blocker of the rapid component of delayed rectifier K +  current (I Kr ). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K +  current (I Ks ). In hiPSC-derived cardiomyocytes of cardiac origin, I Na , I CaL , I Kr , and I Ks were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic cell origins. Further studies are warranted to characterize electrophysiological properties of hiPSC-derived cardiomyocytes generated from CPCs. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For Permissions, please email: journals.permissions@oup.com.

Metformin Preconditioning of Human induced Pluripotent Stem Cell-derived Neural Stem Cells Promotes Their Engraftment and Improves Post-Stroke Regeneration and Recovery.

PubMed

Ould-Brahim, Fares; Sarma, Sailendra Nath; Syal, Charvi; Lu, Kevin Jiaqi; Seegobin, Matthew; Carter, Anthony; Jeffers, Matthew S; Doré, Carole; Stanford, William; Corbett, Dale; Wang, Jing

2018-06-12

While transplantation of hiPSC-derived neural stem cells (hiPSC-NSCs) shows therapeutic potential in animal stroke models, major concerns for translating hiPSC therapy to the clinic are efficacy and safety. Therefore, there is a demand to develop an optimal strategy to enhance the engraftment and regenerative capacity of transplanted hiPSC-NSCs in order to produce fully differentiated neural cells to replace lost brain tissues. Metformin, an FDA approved drug, is an optimal neuroregenerative agent that not only promotes NSC proliferation but also drives NSC towards differentiation. In this regard, we hypothesize that preconditioning of hiPSC-NSCs with metformin before transplantation into the stroke-damaged brain will improve engraftment and regenerative capabilities of hiPSC-NSCs, ultimately enhancing functional recovery. Here we show that pretreatment of hiPSC-NSCs with metformin enhances the proliferation and differentiation of hiPSC-NSCs in culture. Furthermore, metformin-preconditioned hiPSC-NSCs show increased engraftment 1-week post-transplant in a rat endothelin-1 focal ischemic stroke model. In addition, metformin preconditioned cell grafts exhibit increased survival compared to naïve cell grafts at 7-week post-transplant. Analysis of the grafts demonstrates that metformin preconditioning enhances the differentiation of hiPSC-NSCs. As an outcome, rats receiving metformin preconditioned cells display accelerated gross motor recovery and reduced infarct volume. These studies represent a vital step forward in the optimization of hiPSC-NSC based transplantation to promote post-stroke recovery.

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System.

PubMed

Hunsberger, Joshua G; Efthymiou, Anastasia G; Malik, Nasir; Behl, Mamta; Mead, Ivy L; Zeng, Xianmin; Simeonov, Anton; Rao, Mahendra

2015-08-15

There is great need to develop more predictive drug discovery tools to identify new therapies to treat diseases of the central nervous system (CNS). Current nonpluripotent stem cell-based models often utilize non-CNS immortalized cell lines and do not enable the development of personalized models of disease. In this review, we discuss why in vitro models are necessary for translational research and outline the unique advantages of induced pluripotent stem cell (iPSC)-based models over those of current systems. We suggest that iPSC-based models can be patient specific and isogenic lines can be differentiated into many neural cell types for detailed comparisons. iPSC-derived cells can be combined to form small organoids, or large panels of lines can be developed that enable new forms of analysis. iPSC and embryonic stem cell-derived cells can be readily engineered to develop reporters for lineage studies or mechanism of action experiments further extending the utility of iPSC-based systems. We conclude by describing novel technologies that include strategies for the development of diversity panels, novel genomic engineering tools, new three-dimensional organoid systems, and modified high-content screens that may bring toxicology into the 21st century. The strategic integration of these technologies with the advantages of iPSC-derived cell technology, we believe, will be a paradigm shift for toxicology and drug discovery efforts.

Co-Seeding Human Endothelial Cells with Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells on Calcium Phosphate Scaffold Enhances Osteogenesis and Vascularization in Rats.

PubMed

Liu, Xian; Chen, Wenchuan; Zhang, Chi; Thein-Han, Wahwah; Hu, Kevin; Reynolds, Mark A; Bao, Chongyun; Wang, Ping; Zhao, Liang; Xu, Hockin H K

2017-06-01

A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CPC-HUVEC); (3) hiPSC-MSC-seeded CPC (CPC-hiPSC-MSC); and (4) HUVECs co-cultured with hiPSC-MSCs on CPC scaffolds (co-culture group). After 12 weeks, the co-culture group achieved the greatest new bone area percentage of 46.38% ± 3.8% among all groups (p < 0.05), which was more than four folds of the 10.61% ± 1.43% of CPC control. In conclusion, HUVECs co-cultured with hiPSC-MSCs substantially promoted bone regeneration. The novel construct of HUVECs co-cultured with hiPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.

Control of dental-derived induced pluripotent stem cells through modified surfaces for dental application.

PubMed

Choi, Hyunmin; Park, Kyu-Hyung; Lee, Ah-Reum; Mun, Chin Hee; Shin, Yong Dae; Park, Yong-Beom; Park, Young-Bum

2017-07-01

The aim of this study is to investigate the behaviour of iPSc derived from dental stem cells in terms of initial adhesion, differentiation potential on differently surface-treated titanium disc. iPSc derived from human gingival fibroblasts (hGFs) were established using 4-reprogramming factors transduction with Sendai virus. The hGF-iPSc established in this study exhibited the morphology and growth properties similar to human embryonic stem (ES) cells and expressed pluripotency makers. Alkaline Phosphatase (AP) staining, Embryoid Body (EB) formation and in vitro differentiation and karyotyping further confirmed pluripotency of hGF-iPSc. Then, hGF-iPSc were cultured on machined- and Sandblasted and acid etched (SLA)-treated titanium discs with osteogenic induction medium and their morphological as well as quantitative changes according to different surface types were investigated using Alizrin Red S staining, Scanning electron microscopy (SEM), Flow cytometry and RT-PCR. Time-dependent and surface-dependent morphological changes as well as quantitative change in osteogenic differentiation of hGF-iPSc were identified and osteogenic gene expression of hGF-iPSc cultured on SLA-treated titanium disc found to be greater than machined titanium disc, suggesting the fate of hGF-iPSc may be determined by the characteristics of surface to which hGF-iPSc first adhere. iPSc derived from dental stem cell can be one of the most promising and practical cell sources for personalized regenerative dentistry and their morphological change as well as quantitative change in osteogenic differentiation according to different surface types may be further utilized for future clinical application incorporated with dental implant.

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

PubMed Central

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A.; Alves, Paula M.

2016-01-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. Significance The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. PMID:27025693

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing.

PubMed

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A; Serra, Margarida; Alves, Paula M

2016-05-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. ©AlphaMed Press.

Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells

NASA Astrophysics Data System (ADS)

Bared, Kalia

The discovery of induced pluripotent stem cells (iPSC) provided a novel technology for the study of development and pharmacology and complement embryonic stem cells (ES) for cell therapy applications. Though iPSC are derived from adult tissue they are comparable to ES cells in their behavior; multi-lineage differentiation and self-renewal. This makes iPSC research appealing because they can be studied in great detail and expanded in culture broadly. Fibroblasts were the first cell type reprogrammed to an iPSC using a retrovirus vector, since then alternative cell types including lymphocytes have been used to generate iPSC. Different types of vectors have also been developed to enhance iPSC formation and quality. However, specific T lymphocyte subsets have not been shown to reprogram to a pluripotent state to date. Here, we proposed to derive iPSC from peripheral blood effector and central memory T cells, reasoning that the resultant iPSC will maintain the epigenetic memory of a T lymphocyte, including the T cell receptor (TCR) gene rearrangement. This epigenetic memory will enable the differentiation and expansion of T cell iPSC into professional T cells containing a specific TCR. These could then be used for cell therapy to target specific antigens, as well as to improve culture techniques to expand T cells in vitro. We studied different gene delivery methods to derive iPSC from different types of T lymphocytes. We assessed the viability of viral transduction using flow cytometry to detect green fluorescent marker contained in the viral construct and quantitative real time polymerase chain reaction (qRT-PCR) to detect Oct4, Klf4, Sox2, and c-Myc gene expression. Our results demonstrate that the Sendai virus construct is the most feasible platform to reprogram T lymphocytes. We anticipate that this platform will provide an efficient and safe approach to derive iPSC from different T cell subsets, including memory T cells.

Generation of a human induced pluripotent stem cell line from urinary cells of a healthy donor using integration free Sendai virus technology.

PubMed

Rossbach, Bella; Hildebrand, Laura; El-Ahmad, Linda; Stachelscheid, Harald; Reinke, Petra; Kurtz, Andreas

2017-05-01

We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 28year old healthy female donor. The cells were reprogrammed using a non-integrating viral vector and have shown full differentiation potential. Together with the iPSC line, the donor provided blood cells for the study of immunological effects of the iPSC line and its derivatives in autologous and allogeneic settings. The line is available and registered in the human pluripotent stem cell registry as BCRTi005-A. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Generation of a human induced pluripotent stem cell line from urinary cells of a healthy donor using an integration free vector.

PubMed

Rossbach, Bella; Hildebrand, Laura; El-Ahmad, Linda; Stachelscheid, Harald; Reinke, Petra; Kurtz, Andreas

2016-03-01

We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 30 year old healthy female donor. The cells were reprogrammed using a non-integrating viral vector and have shown full differentiation potential. Together with the iPSC-line, the donor provided blood cells for the study of immunological effects of the iPSC line and its derivatives in autologous and allogeneic settings. The line is available and registered in the human pluripotent stem cell registry as BCRTi004-A. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

Recent technological updates and clinical applications of induced pluripotent stem cells.

PubMed

Diecke, Sebastian; Jung, Seung Min; Lee, Jaecheol; Ju, Ji Hyeon

2014-09-01

Induced pluripotent stem cells (iPSCs) were first described in 2006 and have since emerged as a promising cell source for clinical applications. The rapid progression in iPSC technology is still ongoing and directed toward increasing the efficacy of iPSC production and reducing the immunogenic and tumorigenic potential of these cells. Enormous efforts have been made to apply iPSC-based technology in the clinic, for drug screening approaches and cell replacement therapy. Moreover, disease modeling using patient-specific iPSCs continues to expand our knowledge regarding the pathophysiology and prospective treatment of rare disorders. Furthermore, autologous stem cell therapy with patient-specific iPSCs shows great propensity for the minimization of immune reactions and the provision of a limitless supply of cells for transplantation. In this review, we discuss the recent updates in iPSC technology and the use of iPSCs in disease modeling and regenerative medicine.

Induced Accelerated Aging in Induced Pluripotent Stem Cell Lines from Patients with Parkinson’s Disease

DTIC Science & Technology

2013-11-01

clones . Western blot analysis will be used to detect the protein expression after selection. 2. Differentiation into oligoprecursor cells (OPCs... monkey and mouse which will be tested in iPSC derived neurons aged with progerin. 13 Key Research Accomplishments: • Milestone 1 (month 1-2...iPSC clones with drug-inducible progerin construct we established the plasmid transfection for iPSC induced neural stem cells, the retroviral

High-Content Electrophysiological Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes (hPSC-CMs).

PubMed

Kong, Chi-Wing; Geng, Lin; Li, Ronald A

2018-01-01

Considerable interest has been raised to develop human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as a model for drug discovery and cardiotoxicity screening. High-content electrophysiological analysis of currents generated by transmembrane cell surface ion channels has been pursued to complement such emerging applications. Here we describe practical procedures and considerations for accomplishing successful assays of hPSC-CMs using an automated planar patch-clamp system.

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.

Immunomodulatory Properties of Induced Pluripotent Stem Cell-Derived Mesenchymal Cells.

PubMed

Ng, Jia; Hynes, Kim; White, Gregory; Sivanathan, Kisha Nandini; Vandyke, Kate; Bartold, Peter Mark; Gronthos, Stan

2016-12-01

MSC-like populations derived from induced pluripotent stem cells (iPSC-MSC) serve as an alternative stem cell source due to their high proliferative capacity. In this study, we assessed the immunomodulatory potential of iPSC-MSC generated from periodontal ligament (PDL) and gingival (GF) tissue. The iPSC-MSC lines exhibited a similar level of suppression of mitogen-stimulated peripheral blood mononuclear cells (PBMNC) proliferation compared to their respective parental fibroblast populations in vitro. Moreover, iPSC-MSC demonstrated the ability to suppress T-cells effector cells, Th1/Th2/Th17 populations, and increase levels of Treg cells. In order to investigate the mechanisms involved, expression of common MSC-derived soluble factors known to supress lymphocyte proliferation were assessed in iPSC-MSC cultured with PBMNC with direct cell-cell contact or separated in transwells. Real-time PCR analysis of factors known to be involved in MSC mediated immune regulation, found a general trend of elevated IDO1 and IL6 transcript levels in iPSC-MSC lines and their respective primary cells co-cultured with activated PBMNC, with a wide range of gene expression levels between the different mesenchymal cell types. The results suggest that different iPSC-MSC may be useful as a potential alternative source of cells for future clinical use in therapeutic applications because of their potent immunosuppressive properties. J. Cell. Biochem. 117: 2844-2853, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Induced Pluripotent Stem Cells 10 Years Later: For Cardiac Applications.

PubMed

Yoshida, Yoshinori; Yamanaka, Shinya

2017-06-09

Induced pluripotent stem cells (iPSCs) are reprogrammed cells that have features similar to embryonic stem cells, such as the capacity of self-renewal and differentiation into many types of cells, including cardiac myocytes. Although initially the reprogramming efficiency was low, several improvements in reprogramming methods have achieved robust and efficient generation of iPSCs without genomic insertion of transgenes. iPSCs display clonal variations in epigenetic and genomic profiles and cellular behavior in differentiation. iPSC-derived cardiac myocytes (iPSC cardiac myocytes) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models, and are useful for drug discovery and toxicology testing. In addition, iPSC cardiac myocytes can help with patient stratification in regard to drug responsiveness. Furthermore, they can be used as source cells for cardiac regeneration in animal models. Here, we review recent progress in iPSC technology and its applications to cardiac diseases. © 2017 American Heart Association, Inc.

Fail-Safe System against Potential Tumorigenicity after Transplantation of iPSC Derivatives.

PubMed

Itakura, Go; Kawabata, Soya; Ando, Miki; Nishiyama, Yuichiro; Sugai, Keiko; Ozaki, Masahiro; Iida, Tsuyoshi; Ookubo, Toshiki; Kojima, Kota; Kashiwagi, Rei; Yasutake, Kaori; Nakauchi, Hiromitsu; Miyoshi, Hiroyuki; Nagoshi, Narihito; Kohyama, Jun; Iwanami, Akio; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

2017-03-14

Human induced pluripotent stem cells (iPSCs) are promising in regenerative medicine. However, the risks of teratoma formation and the overgrowth of the transplanted cells continue to be major hurdles that must be overcome. Here, we examined the efficacy of the inducible caspase-9 (iCaspase9) gene as a fail-safe against undesired tumorigenic transformation of iPSC-derived somatic cells. We used a lentiviral vector to transduce iCaspase9 into two iPSC lines and assessed its efficacy in vitro and in vivo. In vitro, the iCaspase9 system induced apoptosis in approximately 95% of both iPSCs and iPSC-derived neural stem/progenitor cells (iPSC-NS/PCs). To determine in vivo function, we transplanted iPSC-NS/PCs into the injured spinal cord of NOD/SCID mice. All transplanted cells whose mass effect was hindering motor function recovery were ablated upon transduction of iCaspase9. Our results suggest that the iCaspase9 system may serve as an important countermeasure against post-transplantation adverse events in stem cell transplant therapies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Human Finger-Prick Induced Pluripotent Stem Cells Facilitate the Development of Stem Cell Banking

PubMed Central

Tan, Hong-Kee; Toh, Cheng-Xu Delon; Ma, Dongrui; Yang, Binxia; Liu, Tong Ming; Lu, Jun; Wong, Chee-Wai; Tan, Tze-Kai; Li, Hu; Syn, Christopher; Tan, Eng-Lee; Lim, Bing; Lim, Yoon-Pin; Cook, Stuart A.

2014-01-01

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients can be a good model for studying human diseases and for future therapeutic regenerative medicine. Current initiatives to establish human iPSC (hiPSC) banking face challenges in recruiting large numbers of donors with diverse diseased, genetic, and phenotypic representations. In this study, we describe the efficient derivation of transgene-free hiPSCs from human finger-prick blood. Finger-prick sample collection can be performed on a “do-it-yourself” basis by donors and sent to the hiPSC facility for reprogramming. We show that single-drop volumes of finger-prick samples are sufficient for performing cellular reprogramming, DNA sequencing, and blood serotyping in parallel. Our novel strategy has the potential to facilitate the development of large-scale hiPSC banking worldwide. PMID:24646489

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

PubMed Central

Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

2015-01-01

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

Mitophagy-driven mitochondrial rejuvenation regulates stem cell fate

PubMed Central

Vazquez-Martin, Alejandro; Van den Haute, Chris; Cufí, Sílvia; Corominas-Faja, Bruna; Cuyàs, Elisabet; Lopez-Bonet, Eugeni; Rodriguez-Gallego, Esther; Fernández-Arroyo, Salvador; Joven, Jorge; Baekelandt, Veerle; Menendez, Javier A.

2016-01-01

Our understanding on how selective mitochondrial autophagy, or mitophagy, can sustain the archetypal properties of stem cells is incomplete. PTEN-induced putative kinase 1 (PINK1) plays a key role in the maintenance of mitochondrial morphology and function and in the selective degradation of damaged mitochondria by mitophagy. Here, using embryonic fibroblasts from PINK1 gene-knockout (KO) mice, we evaluated whether mitophagy is a causal mechanism for the control of cell-fate plasticity and maintenance of pluripotency. Loss of PINK1-dependent mitophagy was sufficient to dramatically decrease the speed and efficiency of induced pluripotent stem cell (iPSC) reprogramming. Mitophagy-deficient iPSC colonies, which were characterized by a mixture of mature and immature mitochondria, seemed unstable, with a strong tendency to spontaneously differentiate and form heterogeneous populations of cells. Although mitophagy-deficient iPSC colonies normally expressed pluripotent markers, functional monitoring of cellular bioenergetics revealed an attenuated glycolysis in mitophagy-deficient iPSC cells. Targeted metabolomics showed a notable alteration in numerous glycolysis- and TCA-related metabolites in mitophagy-deficient iPSC cells, including a significant decrease in the intracellular levels of α-ketoglutarate -a key suppressor of the differentiation path in stem cells. Mitophagy-deficient iPSC colonies exhibited a notably reduced teratoma-initiating capacity, but fully retained their pluripotency and multi-germ layer differentiation capacity in vivo. PINK1-dependent mitophagy pathway is an important mitochondrial switch that determines the efficiency and quality of somatic reprogramming. Mitophagy-driven mitochondrial rejuvenation might contribute to the ability of iPSCs to suppress differentiation by directing bioenergetic transition and metabolome remodeling traits. These findings provide new insights into how mitophagy might influence the stem cell decisions to retain pluripotency or differentiate in tissue regeneration and aging, tumor growth, and regenerative medicine. PMID:27295498

Induced pluripotent stem cell technology: Toward the future of cardiac arrhythmias.

PubMed

Gnecchi, Massimiliano; Stefanello, Manuela; Mura, Manuela

2017-06-15

The development of human induced pluripotent stem cell (iPSC) technology has revitalized the efforts made in the last decade to exploit the potential of human embryonic stem cells (ESCs) for scientific research. In the field of cardiac arrhythmias, the possibility of generating an unlimited amount of patient-specific cardiomyocyte-like cells (iPSC-CMs) has clear advantages compared with the use of ESC-derived cardiac cells. In particular, with the introduction and implementation of the large-scale precision medicine initiative, we anticipate that the iPSC technology will play an important role in the advancement of cardiovascular research and medicine. This platform is not free from technical limitations that must be carefully taken into account; however, the utility of iPSC-CMs in disease modeling and drug testing studies is hardly questionable. Here, we summarize some of the progresses made in the field of iPSC technology applied to inherited cardiac arrhythmias, with particular emphasis on the use of iPSC-CMs for modelling the long QT syndrome and for the development of personalized drug and molecular therapies. The growing role of iPSC technology in the practice of precision medicine will also be discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Systematic optimization of human pluripotent stem cells media using Design of Experiments

NASA Astrophysics Data System (ADS)

Marinho, Paulo A.; Chailangkarn, Thanathom; Muotri, Alysson R.

2015-05-01

Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media.

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration.

PubMed

El Khatib, Moustafa M; Ohmine, Seiga; Jacobus, Egon J; Tonne, Jason M; Morsy, Salma G; Holditch, Sara J; Schreiber, Claire A; Uetsuka, Koji; Fusaki, Noemi; Wigle, Dennis A; Terzic, Andre; Kudva, Yogish C; Ikeda, Yasuhiro

2016-05-01

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature β cell phenotype would lead to safe islet replacement therapy for diabetes. ©AlphaMed Press.

Efficient generation of induced pluripotent stem cells from human bone marrow mesenchymal stem cells.

PubMed

Yulin, X; Lizhen, L; Lifei, Z; Shan, F; Ru, L; Kaimin, H; Huang, H

2012-01-01

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

Temporally coordinated spiking activity of human induced pluripotent stem cell-derived neurons co-cultured with astrocytes.

PubMed

Kayama, Tasuku; Suzuki, Ikuro; Odawara, Aoi; Sasaki, Takuya; Ikegaya, Yuji

2018-01-01

In culture conditions, human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks, which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons, the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study, we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Hydrogel fibers encapsulating hiPSC-MSCs, hESC-MSCs and hUCMSCs in injectable calcium phosphate scaffold for bone tissue engineering

PubMed Central

Wang, Lin; Wang, Ping; Weir, Michael D.; Reynolds, Mark A.; Zhao, Liang; Xu, Hockin H. K.

2016-01-01

Human induced pluripotent stem cells (hiPSCs), human embryonic stem cells (hESCs) and human umbilical cord MSCs (hUCMSCs) are exciting cell sources for use in regenerative medicine. There has been no report on long hydrogel fibers encapsulating stem cells inside injectable calcium phosphate cement (CPC) scaffold for bone tissue engineering. The objectives of this study were to: (1) develop a novel injectable CPC construct containing hydrogel fibers encapsulating cells for bone engineering, and (2) investigate and compare cell viability, proliferation and osteogenic differentiation of hiPSC-MSCs, hESC-MSCs and hUCMSCs in injectable CPC. The stem cell-encapsulating pastes were fully injectable under a small injection force, and the injection did not harm the cells, compared to cells without injection (p > 0.1). Mechanical properties of stem cell-CPC construct were much higher than previous injectable polymers and hydrogels for cell delivery. hiPSC-MSCs, hESC-MSCs and hUCMSCs in hydrogel fibers in CPC had excellent proliferation and osteogenic differentiation. All three cells yielded high alkaline phosphatase, runt-related transcription factor, collagen I, and osteocalcin expressions (mean ± sd; n = 6). Cell-synthesized minerals increased substantially with time (p < 0.05), with no significant difference among the three types of cells (p > 0.1). Mineralization by hiPSC-MSCs, hESC-MSCs and hUCMSCs in CPC at 14 d was 13-fold that at 1 d. In conclusion, all three types of cells (hiPSC-MSCs, hESC-MSCs and hUCMSCs) in CPC scaffold showed high potential for bone tissue engineering, and the novel injectable CPC construct with cell-encapsulating hydrogel fibers is promising to enhance bone regeneration in dental, craniofacial and orthopedic applications. PMID:27811389

Disease modeling and cell based therapy with iPSC: future therapeutic option with fast and safe application.

PubMed

Kim, Changsung

2014-03-01

Induced pluripotent stem cell (iPSC) technology has shown us great hope to treat various human diseases which have been known as untreatable and further endows personalized medicine for future therapy without ethical issues and immunological rejection which embryonic stem cell (hES) treatment has faced. It has been agreed that iPSCs knowledge can be harnessed from disease modeling which mimics human pathological development rather than trials utilizing conventional rodent and cell lines. Now, we can routinely generate iPSC from patient specific cell sources, such as skin fibroblast, hair follicle cells, patient blood samples and even urine containing small amount of epithelial cells. iPSC has both similarity and dissimilarity to hES. iPSC is similar enough to regenerate tissue and even full organism as ES does, however what we want for therapeutic advantage is limited to regenerated tissue and lineage specific differentiation. Depending on the lineage and type of cells, both tissue memory containing (DNA rearrangement/epigenetics) and non-containing iPSC can be generated. This makes iPSC even better choice to perform disease modeling as well as cell based therapy. Tissue memory containing iPSC from mature leukocytes would be beneficial for curing cancer and infectious disease. In this review, the benefit of iPSC for translational approaches will be presented.

3D printing human induced pluripotent stem cells with novel hydroxypropyl chitin bioink: scalable expansion and uniform aggregation.

PubMed

Li, Yang; Jiang, Xulin; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

2018-06-28

Human induced pluripotent stem cells (hiPSCs) are more likely to successfully avoid the immunological rejection and ethical problems that are often encountered by human embryonic stem cells in various stem cell studies and applications. To transfer hiPSCs from the laboratory to clinical applications, researchers must obtain sufficient cell numbers. In this study, 3D cell printing was used as a novel method for iPSC scalable expansion. Hydroxypropyl chitin (HPCH), utilized as a new type of bioink, and a set of optimized printing parameters were shown to achieve high cell survival (> 90%) after the printing process and high proliferation efficiency (~ 32.3 folds) during subsequent 10-day culture. After the culture, high levels of pluripotency maintenance were recognized by both qualitative and quantitative detections. Compared with static suspension (SS) culture, hiPSC aggregates formed in 3D printed constructs showed a higher uniformity in size. Using novel dual-fluorescent labelling method, hiPSC aggregates in the constructs were found more inclined to form by <i>in situ</i> proliferation rather than multicellular aggregation. This study revealed unique advantages of non-ionic crosslinking bioink material HPCH, including high gel strength and rapid temperature response in hiPSC printing, and achieved primed state hiPSC printing for the first time. Features achieved in this study, such as high cell yield, high pluripotency maintenance and uniform aggregation provide good foundations for further hiPSC studies on 3D micro-tissue differentiation and drug screening. © 2018 IOP Publishing Ltd.

Bioprocessing of Cryopreservation for Large-Scale Banking of Human Pluripotent Stem Cells

PubMed Central

Ma, Teng

2012-01-01

Abstract Human pluripotent stem cell (hPSC)-derived cell therapy requires production of therapeutic cells in large quantity, which starts from thawing the cryopreserved cells from a working cell bank or a master cell bank. An optimal cryopreservation and thaw process determines the efficiency of hPSC expansion and plays a significant role in the subsequent lineage-specific differentiation. However, cryopreservation in hPSC bioprocessing has been a challenge due to the unique growth requirements of hPSC, the sensitivity to cryoinjury, and the unscalable cryopreservation procedures commonly used in the laboratory. Tremendous progress has been made to identify the regulatory pathways regulating hPSC responses during cryopreservation and the development of small molecule interventions that effectively improves the efficiency of cryopreservation. The adaption of these methods in current good manufacturing practices (cGMP)-compliant cryopreservation processes not only improves cell survival, but also their therapeutic potency. This review summarizes the advances in these areas and discusses the technical requirements in the development of cGMP-compliant hPSC cryopreservation process. PMID:23515461

Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

PubMed

Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

2017-11-01

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

PubMed

Folmes, Clifford D L; Martinez-Fernandez, Almudena; Perales-Clemente, Ester; Li, Xing; McDonald, Amber; Oglesbee, Devin; Hrstka, Sybil C; Perez-Terzic, Carmen; Terzic, Andre; Nelson, Timothy J

2013-07-01

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. Copyright © 2013 AlphaMed Press.

Isolation and characterization of human CXCR4-positive pancreatic cells.

PubMed

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

2007-01-01

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

Modeling Neuropsychiatric and Neurodegenerative Diseases With Induced Pluripotent Stem Cells.

PubMed

LaMarca, Elizabeth A; Powell, Samuel K; Akbarian, Schahram; Brennand, Kristen J

2018-01-01

Human-induced pluripotent stem cells (hiPSCs) have revolutionized our ability to model neuropsychiatric and neurodegenerative diseases, and recent progress in the field is paving the way for improved therapeutics. In this review, we discuss major advances in generating hiPSC-derived neural cells and cutting-edge techniques that are transforming hiPSC technology, such as three-dimensional "mini-brains" and clustered, regularly interspersed short palindromic repeats (CRISPR)-Cas systems. We examine specific examples of how hiPSC-derived neural cells are being used to uncover the pathophysiology of schizophrenia and Parkinson's disease, and consider the future of this groundbreaking research.

Functional Comparison of Induced Pluripotent Stem Cell- and Blood-Derived GPIIbIIIa Deficient Platelets

PubMed Central

Haas, Jessica; Sandrock-Lang, Kirstin; Gärtner, Florian; Jung, Christian Billy; Zieger, Barbara; Parrotta, Elvira; Kurnik, Karin; Sinnecker, Daniel; Wanner, Gerhard; Laugwitz, Karl-Ludwig; Massberg, Steffen; Moretti, Alessandra

2015-01-01

Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application. PMID:25607928

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.

Stem Cells and Calcium Phosphate Cement Scaffolds for Bone Regeneration

PubMed Central

Wang, P.; Zhao, L.; Chen, W.; Liu, X.; Weir, M.D.; Xu, H.H.K.

2014-01-01

Calcium phosphate cements (CPCs) have excellent biocompatibility and osteoconductivity for dental, craniofacial, and orthopedic applications. This article reviews recent developments in stem cell delivery via CPC for bone regeneration. This includes: (1) biofunctionalization of the CPC scaffold, (2) co-culturing of osteoblasts/endothelial cells and prevascularization of CPC, (3) seeding of CPC with different stem cell species, (4) human umbilical cord mesenchymal stem cell (hUCMSC) and bone marrow MSC (hBMSC) seeding on CPC for bone regeneration, and (5) human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) seeding with CPC for bone regeneration. Cells exhibited good attachment/proliferation in CPC scaffolds. Stem-cell-CPC constructs generated more new bone and blood vessels in vivo than did the CPC control without cells. hUCMSCs, hESC-MSCs, and hiPSC-MSCs in CPC generated new bone and blood vessels similar to those of hBMSCs; hence, they were viable cell sources for bone engineering. CPC with hESC-MSCs and hiPSC-MSCs generated new bone two- to three-fold that of the CPC control. Therefore, this article demonstrates that: (1) CPC scaffolds are suitable for delivering cells; (2) hUCMSCs, hESCs, and hiPSCs are promising alternatives to hBMSCs, which require invasive procedures to harvest with limited cell quantity; and (3) stem-cell-CPC constructs are highly promising for bone regeneration in dental, craniofacial, and orthopedic applications. PMID:24799422

Evaluation of the immunogenicity of human iPS cell-derived neural stem/progenitor cells in vitro.

PubMed

Ozaki, Masahiro; Iwanami, Akio; Nagoshi, Narihito; Kohyama, Jun; Itakura, Go; Iwai, Hiroki; Nishimura, Soraya; Nishiyama, Yuichiro; Kawabata, Soya; Sugai, Keiko; Iida, Tsuyoshi; Matsubayashi, Kohei; Isoda, Miho; Kashiwagi, Rei; Toyama, Yoshiaki; Matsumoto, Morio; Okano, Hideyuki; Nakamura, Masaya

2017-03-01

To achieve the goal of a first-in-human trial for human induced pluripotent stem cell (hiPSC)-based transplantation for the treatment of various diseases, allogeneic human leukocyte antigen (HLA)-matched hiPSC cell banks represent a realistic tool from the perspective of quality control and cost performance. Furthermore, considering the limited therapeutic time-window for acute injuries, including neurotraumatic injuries, an iPS cell bank is of potential interest. However, due to the relatively immunoprivileged environment of the central nervous system, it is unclear whether HLA matching is required in hiPSC-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation for the treatment of neurodegenerative diseases and neurotraumatic injuries. In this study, we evaluated the significance of HLA matching in hiPSC-NS/PC transplantation by performing modified mixed lymphocyte reaction (MLR) assays with hiPSC-NS/PCs. Compared to fetus-derived NS/PCs, the expression levels of human leukocyte antigen-antigen D related (HLA-DR) and co-stimulatory molecules on hiPSC-NS/PCs were significantly low, even with the addition of tumor necrosis factor-α (TNFα) and/or interferon-γ (IFNγ) to mimic the inflammatory environment surrounding transplanted hiPSC-NS/PCs in injured tissues. Interestingly, both the allogeneic HLA-matched and the HLA-mismatched responses were similarly low in the modified MLR assay. Furthermore, the autologous response was also similar to the allogeneic response. hiPSC-NS/PCs suppressed the proliferative responses of allogeneic HLA-mismatched peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Thus, the low antigen-presenting function and immunosuppressive effects of hiPSC-NS/PCs result in a depressed immune response, even in an allogeneic HLA-mismatched setting. It is crucial to verify whether these in vitro results are reproducible in a clinical setting. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells.

PubMed

Wakui, Takashi; Matsumoto, Tsuyoshi; Matsubara, Kenta; Kawasaki, Tomoyuki; Yamaguchi, Hiroshi; Akutsu, Hidenori

2017-10-01

We propose an image analysis method for quality evaluation of human pluripotent stem cells based on biologically interpretable features. It is important to maintain the undifferentiated state of induced pluripotent stem cells (iPSCs) while culturing the cells during propagation. Cell culture experts visually select good quality cells exhibiting the morphological features characteristic of undifferentiated cells. Experts have empirically determined that these features comprise prominent and abundant nucleoli, less intercellular spacing, and fewer differentiating cellular nuclei. We quantified these features based on experts' visual inspection of phase contrast images of iPSCs and found that these features are effective for evaluating iPSC quality. We then developed an iPSC quality evaluation method using an image analysis technique. The method allowed accurate classification, equivalent to visual inspection by experts, of three iPSC cell lines.

A Standard Nomenclature for Referencing and Authentication of Pluripotent Stem Cells.

PubMed

Kurtz, Andreas; Seltmann, Stefanie; Bairoch, Amos; Bittner, Marie-Sophie; Bruce, Kevin; Capes-Davis, Amanda; Clarke, Laura; Crook, Jeremy M; Daheron, Laurence; Dewender, Johannes; Faulconbridge, Adam; Fujibuchi, Wataru; Gutteridge, Alexander; Hei, Derek J; Kim, Yong-Ou; Kim, Jung-Hyun; Kokocinski, Anja Kolb-; Lekschas, Fritz; Lomax, Geoffrey P; Loring, Jeanne F; Ludwig, Tenneille; Mah, Nancy; Matsui, Tohru; Müller, Robert; Parkinson, Helen; Sheldon, Michael; Smith, Kelly; Stachelscheid, Harald; Stacey, Glyn; Streeter, Ian; Veiga, Anna; Xu, Ren-He

2018-01-09

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Feaster, Tromondae K; Cadar, Adrian G; Wang, Lili; Williams, Charles H; Chun, Young Wook; Hempel, Jonathan E; Bloodworth, Nathaniel; Merryman, W David; Lim, Chee Chew; Wu, Joseph C; Knollmann, Björn C; Hong, Charles C

2015-12-04

The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. © 2015 American Heart Association, Inc.

Nucleolar molecular signature of pluripotent stem cells.

PubMed

Pliss, Artem; Kuzmin, Andrey N; Kachynski, Aliaksandr V; Jiang, Houbo; Hu, Zhixing; Ren, Yong; Feng, Jian; Prasad, Paras N

2013-04-02

Induced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells to the pluripotent state. Identification and quantitative characterization of changes in the molecular organization of the cell during the process of cellular reprogramming is valuable for stem cell research and advancement of its therapeutic applications. Here we employ quantitative Raman microspectroscopy and biomolecular component analysis (BCA) for a comparative analysis of the molecular composition of nucleoli in skin fibroblasts and iPSC derived from them. We report that the cultured fibroblasts obtained from different human subjects, share comparable concentrations of proteins, RNA, DNA, and lipids in the molecular composition of nucleoli. The nucleolar molecular environment is drastically changed in the corresponding iPSC. We measured that the transition from skin fibroblasts to iPSC is accompanied by a statistically significant increase in protein concentrations ~1.3-fold, RNA concentrations ~1.3-fold, and DNA concentrations ~1.4-fold, while no statistically significant difference was found for the lipid concentrations. The analysis of molecular vibrations associated with diverse aminoacids and protein conformations indicates that nucleoli of skin fibroblasts contain similar subsets of proteins, with prevalence of tyrosine. In iPSC, we observed a higher signal from tryptophan with an increase in the random coil and α helix protein conformations, indicating changes in the subset of nucleolar proteins during cell reprogramming. At the same time, the concentrations of major types of macromolecules and protein conformations in the nucleoli of iPSC and human embryonic stem cells (hESC) were found to be similar. We discuss these results in the context of nucleolar function and conclude that the nucleolar molecular content is correlated with the cellular differentiation status. The approach described here shows the potential for spectroscopically monitoring changes in macromolecular organization of the cell at different stages of reprogramming.

From iPSC towards cardiac tissue-a road under construction.

PubMed

Peischard, Stefan; Piccini, Ilaria; Strutz-Seebohm, Nathalie; Greber, Boris; Seebohm, Guiscard

2017-10-01

The possibility to generate induced pluripotent stem cells (iPSC) opens the way to generate virtually all cell types of our human body. In combination with modern gene editing techniques like CRISPR/CAS, a new set of powerful tools becomes available for life science. Scientific fields like genotype and cell type-specific pharmacology, disease modeling, stem cell biology, and developmental biology have been dramatically fostered and their faces have been changed. However, as golden as the age of iPSC-derived cells and their manipulation has started, the shine begins to tarnish. Researchers face more and more practical problems intrinsic to the system. These problems are related to the specific culturing conditions which are not yet sufficient to mimic the natural environment of native stem cells differentiating towards adult cells. However, researchers work hard to uncover these factors. Here, we review a common standard approach to generate iPSCs and transduce these to iPSC cardiomyocytes. Further, we review recent achievements and discuss their current limitations and future perspectives. We are on track, but the road is still under construction.

Regulatory Insight into the European Human Pluripotent Stem Cell Registry

PubMed Central

Kurtz, Andreas; Stacey, Glyn; Kidane, Luam; Seriola, Anna; Stachelscheid, Harald; Veiga, Anna

2014-01-01

Abstract The European pluripotent stem cell registry aims at listing qualified pluripotent stem cell (PSC) lines that are available globally together with relevant information for each cell line. Specific emphasis is being put on documenting ethical procurement of the cells and providing evidence of pluripotency. The report discusses the tasks and challenges for a global PSC registry as an instrument to develop collaboration, to access cells from diverse resources and banks, and to implement standards, and as a means to follow up usage of cells and support adherence to regulatory and scientific standards and transparency for stakeholders. PMID:25457963

Regulatory insight into the European human pluripotent stem cell registry.

PubMed

Kurtz, Andreas; Stacey, Glyn; Kidane, Luam; Seriola, Anna; Stachelscheid, Harald; Veiga, Anna

2014-12-01

The European pluripotent stem cell registry aims at listing qualified pluripotent stem cell (PSC) lines that are available globally together with relevant information for each cell line. Specific emphasis is being put on documenting ethical procurement of the cells and providing evidence of pluripotency. The report discusses the tasks and challenges for a global PSC registry as an instrument to develop collaboration, to access cells from diverse resources and banks, and to implement standards, and as a means to follow up usage of cells and support adherence to regulatory and scientific standards and transparency for stakeholders.

Engineered heart tissues and induced pluripotent stem cells: Macro- and microstructures for disease modeling, drug screening, and translational studies.

PubMed

Tzatzalos, Evangeline; Abilez, Oscar J; Shukla, Praveen; Wu, Joseph C

2016-01-15

Engineered heart tissue has emerged as a personalized platform for drug screening. With the advent of induced pluripotent stem cell (iPSC) technology, patient-specific stem cells can be developed and expanded into an indefinite source of cells. Subsequent developments in cardiovascular biology have led to efficient differentiation of cardiomyocytes, the force-producing cells of the heart. iPSC-derived cardiomyocytes (iPSC-CMs) have provided potentially limitless quantities of well-characterized, healthy, and disease-specific CMs, which in turn has enabled and driven the generation and scale-up of human physiological and disease-relevant engineered heart tissues. The combined technologies of engineered heart tissue and iPSC-CMs are being used to study diseases and to test drugs, and in the process, have advanced the field of cardiovascular tissue engineering into the field of precision medicine. In this review, we will discuss current developments in engineered heart tissue, including iPSC-CMs as a novel cell source. We examine new research directions that have improved the function of engineered heart tissue by using mechanical or electrical conditioning or the incorporation of non-cardiomyocyte stromal cells. Finally, we discuss how engineered heart tissue can evolve into a powerful tool for therapeutic drug testing. Copyright © 2015 Elsevier B.V. All rights reserved.

Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

PubMed

Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

2014-07-04

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Controversies in Cardiovascular Research: Induced pluripotent stem cell-derived cardiomyocytes – boutique science or valuable arrhythmia model?

PubMed Central

Knollmann, Björn C

2013-01-01

As part of the series on Controversies in Cardiovascular Research, the article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize anti-arrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders such as long QT syndrome, Brugada Syndrome or Catecholaminergic Polymorphic Ventricular Tachycardia. PMID:23569106

Pathological classification of human iPSC-derived neural stem/progenitor cells towards safety assessment of transplantation therapy for CNS diseases.

PubMed

Sugai, Keiko; Fukuzawa, Ryuji; Shofuda, Tomoko; Fukusumi, Hayato; Kawabata, Soya; Nishiyama, Yuichiro; Higuchi, Yuichiro; Kawai, Kenji; Isoda, Miho; Kanematsu, Daisuke; Hashimoto-Tamaoki, Tomoko; Kohyama, Jun; Iwanami, Akio; Suemizu, Hiroshi; Ikeda, Eiji; Matsumoto, Morio; Kanemura, Yonehiro; Nakamura, Masaya; Okano, Hideyuki

2016-09-19

The risk of tumorigenicity is a hurdle for regenerative medicine using induced pluripotent stem cells (iPSCs). Although teratoma formation is readily distinguishable, the malignant transformation of iPSC derivatives has not been clearly defined due to insufficient analysis of histology and phenotype. In the present study, we evaluated the histology of neural stem/progenitor cells (NSPCs) generated from integration-free human peripheral blood mononuclear cell (PBMC)-derived iPSCs (iPSC-NSPCs) following transplantation into central nervous system (CNS) of immunodeficient mice. We found that transplanted iPSC-NSPCs produced differentiation patterns resembling those in embryonic CNS development, and that the microenvironment of the final site of migration affected their maturational stage. Genomic instability of iPSCs correlated with increased proliferation of transplants, although no carcinogenesis was evident. The histological classifications presented here may provide cues for addressing potential safety issues confronting regenerative medicine involving iPSCs.

Microcarrier-based platforms for in vitro expansion and differentiation of human pluripotent stem cells in bioreactor culture systems.

PubMed

Badenes, Sara M; Fernandes, Tiago G; Rodrigues, Carlos A V; Diogo, Maria Margarida; Cabral, Joaquim M S

2016-09-20

Human pluripotent stem cells (hPSC) have attracted a great attention as an unlimited source of cells for cell therapies and other in vitro biomedical applications such as drug screening, toxicology assays and disease modeling. The implementation of scalable culture platforms for the large-scale production of hPSC and their derivatives is mandatory to fulfill the requirement of obtaining large numbers of cells for these applications. Microcarrier technology has been emerging as an effective approach for the large scale ex vivo hPSC expansion and differentiation. This review presents recent achievements in hPSC microcarrier-based culture systems and discusses the crucial aspects that influence the performance of these culture platforms. Recent progress includes addressing chemically-defined culture conditions for manufacturing of hPSC and their derivatives, with the development of xeno-free media and microcarrier coatings to meet good manufacturing practice (GMP) quality requirements. Finally, examples of integrated platforms including hPSC expansion and directed differentiation to specific lineages are also presented in this review. Copyright © 2016 Elsevier B.V. All rights reserved.

Differentiation and Characterization of Dopaminergic Neurons From Baboon Induced Pluripotent Stem Cells.

PubMed

Grow, Douglas A; Simmons, DeNard V; Gomez, Jorge A; Wanat, Matthew J; McCarrey, John R; Paladini, Carlos A; Navara, Christopher S

2016-09-01

: The progressive death of dopamine producing neurons in the substantia nigra pars compacta is the principal cause of symptoms of Parkinson's disease (PD). Stem cells have potential therapeutic use in replacing these cells and restoring function. To facilitate development of this approach, we sought to establish a preclinical model based on a large nonhuman primate for testing the efficacy and safety of stem cell-based transplantation. To this end, we differentiated baboon fibroblast-derived induced pluripotent stem cells (biPSCs) into dopaminergic neurons with the application of specific morphogens and growth factors. We confirmed that biPSC-derived dopaminergic neurons resemble those found in the human midbrain based on cell type-specific expression of dopamine markers TH and GIRK2. Using the reverse transcriptase quantitative polymerase chain reaction, we also showed that biPSC-derived dopaminergic neurons express PAX6, FOXA2, LMX1A, NURR1, and TH genes characteristic of this cell type in vivo. We used perforated patch-clamp electrophysiology to demonstrate that biPSC-derived dopaminergic neurons fired spontaneous rhythmic action potentials and high-frequency action potentials with spike frequency adaption upon injection of depolarizing current. Finally, we showed that biPSC-derived neurons released catecholamines in response to electrical stimulation. These results demonstrate the utility of the baboon model for testing and optimizing the efficacy and safety of stem cell-based therapeutic approaches for the treatment of PD. Functional dopamine neurons were produced from baboon induced pluripotent stem cells, and their properties were compared to baboon midbrain cells in vivo. The baboon has advantages as a clinically relevant model in which to optimize the efficacy and safety of stem cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. Baboons possess crucial neuroanatomical and immunological similarities to humans, and baboon pluripotent stem cells can be differentiated into functional neurons that mimic those in the human brain, thus laying the foundation for the utility of the baboon model for evaluating stem cell therapies. ©AlphaMed Press.

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

PubMed

Li, Xiao-Li; Zeng, Di; Chen, Yan; Ding, Lu; Li, Wen-Ju; Wei, Ting; Ou, Dong-Bo; Yan, Song; Wang, Bin; Zheng, Qiang-Sun

2017-02-01

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α 1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α 2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α 1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. These results demonstrate that activation of ARs, particularly of α 1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling. © 2016 John Wiley & Sons Ltd.

Engineering-derived approaches for iPSC preparation, expansion, differentiation and applications.

PubMed

Li, Yang; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

2017-07-31

Remarkable achievements have been made since induced pluripotent stem cells (iPSCs) were first introduced in 2006. Compared with non-pluripotent stem cells, iPSC research faces several additional complexities, such as the choice of extracellular matrix proteins, growth and differentiation factors, as well as technical challenges related to self-renewal and directed differentiation. Overcoming these challenges requires the integration of knowledge and technologies from multiple fields including cell biology, biomaterial science, engineering, physics and medicine. Here, engineering-derived iPSC approaches are reviewed according to three aspects of iPSC studies: preparation, expansion, differentiation and applications. Engineering strategies, such as 3D systems establishment, cell-matrix mechanics and the regulation of biophysical and biochemical cues, together with engineering techniques, such as 3D scaffolds, cell microspheres and bioreactors, have been applied to iPSC studies and have generated insightful results and even mini-organs such as retinas, livers and intestines. Specific results are given to demonstrate how these approaches impact iPSC behavior, and related mechanisms are discussed. In addition, cell printing technologies are presented as an advanced engineering-derived approach since they have been applied in both iPSC studies and the construction of diverse tissues and organs. Further development and possible innovations of cell printing technologies are presented in terms of creating complex and functional iPSC-derived living tissues and organs.

Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

NASA Astrophysics Data System (ADS)

Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

2016-02-01

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

Viral Vector-Based Innovative Approaches to Directly Abolishing Tumorigenic Pluripotent Stem Cells for Safer Regenerative Medicine.

PubMed

Mitsui, Kaoru; Ide, Kanako; Takahashi, Tomoyuki; Kosai, Ken-Ichiro

2017-06-16

Human pluripotent stem cells (hPSCs) are a promising source of regenerative material for clinical applications. However, hPSC transplant therapies pose the risk of teratoma formation and malignant transformation of undifferentiated remnants. These problems underscore the importance of developing technologies that completely prevent tumorigenesis to ensure safe clinical application. Research to date has contributed to establishing safe hPSC lines, improving the efficiency of differentiation induction, and indirectly ensuring the safety of products. Despite such efforts, guaranteeing the clinical safety of regenerative medicine products remains a key challenge. Given the intrinsic genome instability of hPSCs, selective growth advantage of cancer cells, and lessons learned through failures in previous attempts at hematopoietic stem cell gene therapy, conventional strategies are unlikely to completely overcome issues related to hPSC tumorigenesis. Researchers have recently embarked on studies aimed at locating and directly treating hPSC-derived tumorigenic cells. In particular, novel approaches to directly killing tumorigenic cells by transduction of suicide genes and oncolytic viruses are expected to improve the safety of hPSC-based therapy. This article discusses the current status and future perspectives of methods aimed at directly eradicating undifferentiated tumorigenic hPSCs, with a focus on viral vector transduction.

Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening.

PubMed

Sharma, Arun; Wu, Joseph C; Wu, Sean M

2013-12-24

Human induced pluripotent stem cells (hiPSCs) have emerged as a novel tool for drug discovery and therapy in cardiovascular medicine. hiPSCs are functionally similar to human embryonic stem cells (hESCs) and can be derived autologously without the ethical challenges associated with hESCs. Given the limited regenerative capacity of the human heart following myocardial injury, cardiomyocytes derived from hiPSCs (hiPSC-CMs) have garnered significant attention from basic and translational scientists as a promising cell source for replacement therapy. However, ongoing issues such as cell immaturity, scale of production, inter-line variability, and cell purity will need to be resolved before human clinical trials can begin. Meanwhile, the use of hiPSCs to explore cellular mechanisms of cardiovascular diseases in vitro has proven to be extremely valuable. For example, hiPSC-CMs have been shown to recapitulate disease phenotypes from patients with monogenic cardiovascular disorders. Furthermore, patient-derived hiPSC-CMs are now providing new insights regarding drug efficacy and toxicity. This review will highlight recent advances in utilizing hiPSC-CMs for cardiac disease modeling in vitro and as a platform for drug validation. The advantages and disadvantages of using hiPSC-CMs for drug screening purposes will be explored as well.

Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

PubMed Central

Lundy, Scott D.; Zhu, Wei-Zhong

2013-01-01

Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20–40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80–120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including β-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue. PMID:23461462

Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

PubMed

Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

2017-03-15

Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells. Copyright © 2017 The Authors. 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

Effects of the Post-Spinal Cord Injury Microenvironment on the Differentiation Capacity of Human Neural Stem Cells Derived from Induced Pluripotent Stem Cells.

PubMed

López-Serrano, Clara; Torres-Espín, Abel; Hernández, Joaquim; Alvarez-Palomo, Ana B; Requena, Jordi; Gasull, Xavier; Edel, Michael J; Navarro, Xavier

2016-10-01

Spinal cord injury (SCI) causes loss of neural functions below the level of the lesion due to interruption of spinal pathways and secondary neurodegenerative processes. The transplant of neural stem cells (NSCs) is a promising approach for the repair of SCI. Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) is expected to provide an autologous source of iPSC-derived NSCs, avoiding the immune response as well as ethical issues. However, there is still limited information on the behavior and differentiation pattern of transplanted iPSC-derived NSCs within the damaged spinal cord. We transplanted iPSC-derived NSCs, obtained from adult human somatic cells, into rats at 0 or 7 days after SCI, and evaluated motor-evoked potentials and locomotion of the animals. We histologically analyzed engraftment, proliferation, and differentiation of the iPSC-derived NSCs and the spared tissue in the spinal cords at 7, 21, and 63 days posttransplant. Both transplanted groups showed a late decline in functional recovery compared to vehicle-injected groups. Histological analysis showed proliferation of transplanted cells within the tissue and that cells formed a mass. At the final time point, most grafted cells differentiated to neural and astroglial lineages, but not into oligodendrocytes, while some grafted cells remained undifferentiated and proliferative. The proinflammatory tissue microenviroment of the injured spinal cord induced proliferation of the grafted cells and, therefore, there are possible risks associated with iPSC-derived NSC transplantation. New approaches are needed to promote and guide cell differentiation, as well as reduce their tumorigenicity once the cells are transplanted at the lesion site.

Applications of patient-specific induced pluripotent stem cells; focused on disease modeling, drug screening and therapeutic potentials for liver disease.

PubMed

Chun, Yong Soon; Chaudhari, Pooja; Jang, Yoon-Young

2010-12-14

The recent advances in the induced pluripotent stem cell (iPSC) research have significantly changed our perspectives on regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. In this review, we describe the human iPSC generation from developmentally diverse origins (i.e. endoderm-, mesoderm-, and ectoderm- tissue derived human iPSCs) and multistage hepatic differentiation protocols, and discuss both basic and clinical applications of these cells including disease modeling, drug toxicity screening/drug discovery, gene therapy and cell replacement therapy.

Is Human-induced Pluripotent Stem Cell the Best Optimal?

PubMed

Wang, Feng; Kong, Jie; Cui, Yi-Yao; Liu, Peng; Wen, Jian-Yan

2018-04-05

Since the advent of induced pluripotent stem cell (iPSC) technology a decade ago, enormous progress has been made in stem cell biology and regenerative medicine. Human iPSCs have been widely used for disease modeling, drug discovery, and cell therapy development. In this review, we discuss the progress in applications of iPSC technology that are particularly relevant to drug discovery and regenerative medicine, and consider the remaining challenges and the emerging opportunities in the field. Articles in this review were searched from PubMed database from January 2014 to December 2017. Original articles about iPSCs and cardiovascular diseases were included and analyzed. iPSC holds great promises for human disease modeling, drug discovery, and stem cell-based therapy, and this potential is only beginning to be realized. However, several important issues remain to be addressed. The recent availability of human cardiomyocytes derived from iPSCs opens new opportunities to build in vitro models of cardiac disease, screening for new drugs and patient-specific cardiac therapy.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

PubMed

Klawitter, Sabine; Fuchs, Nina V; Upton, Kyle R; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L; Faulkner, Geoffrey J; Schumann, Gerald G

2016-01-08

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells

PubMed Central

Klawitter, Sabine; Fuchs, Nina V.; Upton, Kyle R.; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J.; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J. Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J.; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L.; Faulkner, Geoffrey J.; Schumann, Gerald G.

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs. PMID:26743714

Concise Review: Cardiac Disease Modeling Using Induced Pluripotent Stem Cells.

PubMed

Yang, Chunbo; Al-Aama, Jumana; Stojkovic, Miodrag; Keavney, Bernard; Trafford, Andrew; Lako, Majlinda; Armstrong, Lyle

2015-09-01

Genetic cardiac diseases are major causes of morbidity and mortality. Although animal models have been created to provide some useful insights into the pathogenesis of genetic cardiac diseases, the significant species differences and the lack of genetic information for complex genetic diseases markedly attenuate the application values of such data. Generation of induced pluripotent stem cells (iPSCs) from patient-specific specimens and subsequent derivation of cardiomyocytes offer novel avenues to study the mechanisms underlying cardiac diseases, to identify new causative genes, and to provide insights into the disease aetiology. In recent years, the list of human iPSC-based models for genetic cardiac diseases has been expanding rapidly, although there are still remaining concerns on the level of functionality of iPSC-derived cardiomyocytes and their ability to be used for modeling complex cardiac diseases in adults. This review focuses on the development of cardiomyocyte induction from pluripotent stem cells, the recent progress in heart disease modeling using iPSC-derived cardiomyocytes, and the challenges associated with understanding complex genetic diseases. To address these issues, we examine the similarity between iPSC-derived cardiomyocytes and their ex vivo counterparts and how this relates to the method used to differentiate the pluripotent stem cells into a cardiomyocyte phenotype. We progress to examine categories of congenital cardiac abnormalities that are suitable for iPSC-based disease modeling. © AlphaMed Press.

Using induced pluripotent stem cells derived neurons to model brain diseases.

PubMed

McKinney, Cindy E

2017-07-01

The ability to use induced pluripotent stem cells (iPSC) to model brain diseases is a powerful tool for unraveling mechanistic alterations in these disorders. Rodent models of brain diseases have spurred understanding of pathology but the concern arises that they may not recapitulate the full spectrum of neuron disruptions associated with human neuropathology. iPSC derived neurons, or other neural cell types, provide the ability to access pathology in cells derived directly from a patient's blood sample or skin biopsy where availability of brain tissue is limiting. Thus, utilization of iPSC to study brain diseases provides an unlimited resource for disease modelling but may also be used for drug screening for effective therapies and may potentially be used to regenerate aged or damaged cells in the future. Many brain diseases across the spectrum of neurodevelopment, neurodegenerative and neuropsychiatric are being approached by iPSC models. The goal of an iPSC based disease model is to identify a cellular phenotype that discriminates the disease-bearing cells from the control cells. In this mini-review, the importance of iPSC cell models validated for pluripotency, germline competency and function assessments is discussed. Selected examples for the variety of brain diseases that are being approached by iPSC technology to discover or establish the molecular basis of the neuropathology are discussed.

Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

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

Huang, Xin; Tian, Changhai; Liu, Miao

2012-04-06

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using thismore » platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.« less

Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates.

PubMed

Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

2017-11-15

Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and electrical activity, calcium cycling, and force generation, is therefore of paramount importance. hPSC-CMs cultured on stiff substrates like glass or polystyrene do not have the ability to shorten during contraction, making them less suitable for the study of hPSC-CM contractile function. Other approaches require highly specialized hardware and are difficult to reproduce. Here we describe a protocol for the preparation of hPSC-CMs on soft substrates that enable shortening, and subsequently the simultaneous quantitative analysis of their contractile and electrical activity, calcium cycling, and force generation at single-cell resolution. This protocol requires only affordable and readily available materials and works with standard imaging hardware. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by non-integrating plasmid expression

PubMed Central

Cheng, Linzhao; Hansen, Nancy F.; Zhao, Ling; Du, Yutao; Zou, Chunlin; Donovan, Frank X.; Chou, Bin-Kuan; Zhou, Guangyu; Li, Shijie; Dowey, Sarah N.; Ye, Zhaohui; Chandrasekharappa, Settara C.; Yang, Huanming; Mullikin, James C.; Liu, P. Paul

2012-01-01

Summary The utility of induced pluripotent stem cells (iPSCs) as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. Here we report the whole-genome sequencing of three human iPSC lines derived from two cell types of an adult donor by episomal vectors. The vector sequence was undetectable in the deeply sequenced iPSC lines. We identified 1058–1808 heterozygous single nucleotide variants (SNVs), but no copy number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but ~50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during integration-free iPSC induction. PMID:22385660

Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

PubMed Central

Khan, Mahmood; Xu, Yanyi; Hua, Serena; Johnson, Jed; Belevych, Andriy; Janssen, Paul M. L.; Gyorke, Sandor; Guan, Jianjun; Angelos, Mark G.

2015-01-01

Introduction Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. Methods hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. Results SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Conclusions Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch. PMID:25993466

Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch.

PubMed

Khan, Mahmood; Xu, Yanyi; Hua, Serena; Johnson, Jed; Belevych, Andriy; Janssen, Paul M L; Gyorke, Sandor; Guan, Jianjun; Angelos, Mark G

2015-01-01

Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.

Intrinsically active and pacemaker neurons in pluripotent stem cell-derived neuronal populations.

PubMed

Illes, Sebastian; Jakab, Martin; Beyer, Felix; Gelfert, Renate; Couillard-Despres, Sébastien; Schnitzler, Alfons; Ritter, Markus; Aigner, Ludwig

2014-03-11

Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks.

Intrinsically Active and Pacemaker Neurons in Pluripotent Stem Cell-Derived Neuronal Populations

PubMed Central

Illes, Sebastian; Jakab, Martin; Beyer, Felix; Gelfert, Renate; Couillard-Despres, Sébastien; Schnitzler, Alfons; Ritter, Markus; Aigner, Ludwig

2014-01-01

Summary Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks. PMID:24672755

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

PubMed

Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

2016-01-01

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement.

PubMed

Di Foggia, Valentina; Makwana, Priyanka; Ali, Robin R; Sowden, Jane C

2016-06-01

Stem cell therapies are being explored as potential treatments for retinal disease. How to replace neurons in a degenerated retina presents a continued challenge for the regenerative medicine field that, if achieved, could restore sight. The major issues are: (i) the source and availability of donor cells for transplantation; (ii) the differentiation of stem cells into the required retinal cells; and (iii) the delivery, integration, functionality, and survival of new cells in the host neural network. This review considers the use of induced pluripotent stem cells (iPSC), currently under intense investigation, as a platform for cell transplantation therapy. Moreover, patient-specific iPSC are being developed for autologous cell transplantation and as a tool for modeling specific retinal diseases, testing gene therapies, and drug screening.

Production of Human Pluripotent Stem Cell Therapeutics Under Defined Xeno-free Conditions: Progress and Challenges

PubMed Central

Fan, Yongjia; Wu, Jincheng; Ashok, Preeti; Hsiung, Michael; Tzanakakis, Emmanuel S.

2014-01-01

Recent advances on human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have brought us closer to the realization of their clinical potential. Nonetheless, tissue engineering and regenerative medicine applications will require the generation of hPSC products well beyond the laboratory scale. This also mandates the production of hPSC therapeutics in fully-defined, xeno-free systems and in a reproducible manner. Toward this goal, we summarize current developments in defined media free of animal-derived components for hPSC culture. Bioinspired and synthetic extracellular matrices for the attachment growth and differentiation of hPSCs are also reviewed. Given that most progress in xeno-free medium and substrate development has been demonstrated in two-dimensional rather than three dimensional culture systems, translation from the former to the latter poses unique difficulties. These challenges are discussed in the context of cultivation platforms of hPSCs as aggregates, on microcarriers or after encapsulation in biocompatible scaffolds. PMID:25077810

Production of human pluripotent stem cell therapeutics under defined xeno-free conditions: progress and challenges.

PubMed

Fan, Yongjia; Wu, Jincheng; Ashok, Preeti; Hsiung, Michael; Tzanakakis, Emmanuel S

2015-02-01

Recent advances on human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have brought us closer to the realization of their clinical potential. Nonetheless, tissue engineering and regenerative medicine applications will require the generation of hPSC products well beyond the laboratory scale. This also mandates the production of hPSC therapeutics in fully-defined, xeno-free systems and in a reproducible manner. Toward this goal, we summarize current developments in defined media free of animal-derived components for hPSC culture. Bioinspired and synthetic extracellular matrices for the attachment, growth and differentiation of hPSCs are also reviewed. Given that most progress in xeno-free medium and substrate development has been demonstrated in two-dimensional rather than three dimensional culture systems, translation from the former to the latter poses unique difficulties. These challenges are discussed in the context of cultivation platforms of hPSCs as aggregates, on microcarriers or after encapsulation in biocompatible scaffolds.

Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue.

PubMed

Ishikawa, Tetsuya

2017-05-26

To investigate genotype variation among induced pluripotent stem cell (iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer ( OCT3/4 , SOX2 , and KLF4 ). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using next-generation sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants (SNVs) (missense, nonsense, and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbSNP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2 , TTN , ULK4 , TSSK1B , FLT4 , STK19 , STK31 , TRRAP , WNK1 , PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer (stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the non-cancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer (stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells. The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.

Translational Control in Bone Marrow Failure

DTIC Science & Technology

2015-05-01

HCLS1 associated protein X-1 (HAX1), cause hereditary forms of neutropenia . Previously, competing hypotheses have posited that mutant forms of...derived induced pluripotent stem cell (iPSC) model of ELANE-associated neutropenia . During the second year of this project, in order to facilitate...pathology. 3 2. KEY WORDS neutropenia bone marrow failure neutrophil elastase ELANE HAX1 alternate translation induced pluripotent stem cells (iPSC

Patient-Specific Pluripotent Stem Cells in Neurological Diseases

PubMed Central

Durnaoglu, Serpen; Genc, Sermin; Genc, Kursad

2011-01-01

Many human neurological diseases are not currently curable and result in devastating neurologic sequelae. The increasing availability of induced pluripotent stem cells (iPSCs) derived from adult human somatic cells provides new prospects for cellreplacement strategies and disease-related basic research in a broad spectrum of human neurologic diseases. Patient-specific iPSC-based modeling of neurogenetic and neurodegenerative diseases is an emerging efficient tool for in vitro modeling to understand disease and to screen for genes and drugs that modify the disease process. With the exponential increase in iPSC research in recent years, human iPSCs have been successfully derived with different technologies and from various cell types. Although there remain a great deal to learn about patient-specific iPSC safety, the reprogramming mechanisms, better ways to direct a specific reprogramming, ideal cell source for cellular grafts, and the mechanisms by which transplanted stem cells lead to an enhanced functional recovery and structural reorganization, the discovery of the therapeutic potential of iPSCs offers new opportunities for the treatment of incurable neurologic diseases. However, iPSC-based therapeutic strategies need to be thoroughly evaluated in preclinical animal models of neurological diseases before they can be applied in a clinical setting. PMID:21776279

Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells.

PubMed

Wilson, Hannah K; Canfield, Scott G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V

2015-05-21

Brain microvascular-like endothelial cells (BMECs) derived from human pluripotent stem cells (hPSCs) have significant promise as tools for drug screening and studying the structure and function of the BBB in health and disease. The density of hPSCs is a key factor in regulating cell fate and yield during differentiation. Prior reports of hPSC differentiation to BMECs have seeded hPSCs in aggregates, leading to non-uniform cell densities that may result in differentiation heterogeneity. Here we report a singularized-cell seeding approach compatible with hPSC-derived BMEC differentiation protocols and evaluate the effects of initial hPSC seeding density on the subsequent differentiation, yield, and blood-brain barrier (BBB) phenotype. A range of densities of hPSCs was seeded and differentiated, with the resultant endothelial cell yield quantified via VE-cadherin flow cytometry. Barrier phenotype of purified hPSC-derived BMECs was measured via transendothelial electrical resistance (TEER), and purification protocols were subsequently optimized to maximize TEER. Expression of characteristic vascular markers, tight junction proteins, and transporters was confirmed by immunocytochemistry and quantified by flow cytometry. P-glycoprotein and MRP-family transporter activity was assessed by intracellular accumulation assay. The initial hPSC seeding density of approximately 30,000 cells/cm(2) served to maximize the yield of VE-cadherin+ BMECs per input hPSC. BMECs displayed the highest TEER (>2,000 Ω × cm(2)) within this same range of initial seeding densities, although optimization of the BMEC purification method could minimize the seeding density dependence for some lines. Localization and expression levels of tight junction proteins as well as efflux transporter activity were largely independent of hPSC seeding density. Finally, the utility of the singularized-cell seeding approach was demonstrated by scaling the differentiation and purification process down from 6-well to 96-well culture without impacting BBB phenotype. Given the yield and barrier dependence on initial seeding density, the singularized-cell seeding approach reported here should enhance the reproducibility and scalability of hPSC-derived BBB models, particularly for the application to new pluripotent stem cell lines.

Integrated processes for expansion and differentiation of human pluripotent stem cells in suspended microcarriers cultures

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

Lam, Alan Tin-Lun, E-mail: alan_lam@bti.a-star.edu.sg; Chen, Allen Kuan-Liang; Ting, Sherwin Qi-Peng

Current methods for human pluripotent stem cells (hPSC) expansion and differentiation can be limited in scalability and costly (due to their labor intensive nature). This can limit their use in cell therapy, drug screening and toxicity assays. One of the approaches that can overcome these limitations is microcarrier (MC) based cultures in which cells are expanded as cell/MC aggregates and then directly differentiated as embryoid bodies (EBs) in the same agitated reactor. This integrated process can be scaled up and eliminate the need for some culture manipulation used in common monolayer and EBs cultures. This review describes the principles ofmore » such microcarriers based integrated hPSC expansion and differentiation process, and parameters that can affect its efficiency (such as MC type and extracellular matrix proteins coatings, cell/MC aggregates size, and agitation). Finally examples of integrated process for generation cardiomyocytes (CM) and neural progenitor cells (NPC) as well as challenges to be solved are described. - Highlights: • Expansion of hPSC on microcarriers. • Differentiation of hPSC on microcarriers. • Parameters that can affect the expansion and differentiation of hPSC on microcarriers. • Integration of expansion and differentiation of hPSC on microcarriers in one unit operation.« less

A highly efficient method for generation of therapeutic quality human pluripotent stem cells by using naive induced pluripotent stem cells nucleus for nuclear transfer.

PubMed

Sanal, Madhusudana Girija

2014-01-01

Even after several years since the discovery of human embryonic stem cells and induced pluripotent stem cells (iPSC), we are still unable to make any significant therapeutic benefits out of them such as cell therapy or generation of organs for transplantation. Recent success in somatic cell nuclear transfer (SCNT) made it possible to generate diploid embryonic stem cells, which opens up the way to make high-quality pluripotent stem cells. However, the process is highly inefficient and hence expensive compared to the generation of iPSC. Even with the latest SCNT technology, we are not sure whether one can make therapeutic quality pluripotent stem cell from any patient's somatic cells or by using oocytes from any donor. Combining iPSC technology with SCNT, that is, by using the nucleus of the candidate somatic cell which got reprogrammed to pluripotent state instead that of the unmodified nucleus of the candidate somatic cell, would boost the efficiency of the technique, and we would be able to generate therapeutic quality pluripotent stem cells. Induced pluripotent stem cell nuclear transfer (iPSCNT) combines the efficiency of iPSC generation with the speed and natural reprogramming environment of SCNT. The new technique may be called iPSCNT. This technique could prove to have very revolutionary benefits for humankind. This could be useful in generating organs for transplantation for patients and for reproductive cloning, especially for childless men and women who cannot have children by any other techniques. When combined with advanced gene editing techniques (such as CRISPR-Cas system) this technique might also prove useful to those who want to have healthy children but suffer from inherited diseases. The current code of ethics may be against reproductive cloning. However, this will change with time as it happened with most of the revolutionary scientific breakthroughs. After all, it is the right of every human to have healthy offspring and it is the question of reproductive freedom and existence.

Application of induced pluripotent stem cells to understand neurobiological basis of bipolar disorder and schizophrenia.

PubMed

Liu, Yao-Nan; Lu, Si-Yao; Yao, Jun

2017-09-01

The etiology of neuropsychiatric disorders, such as schizophrenia and bipolar disorder, usually involves complex combinations of genetic defects/variations and environmental impacts, which hindered, for a long time, research efforts based on animal models and patients' non-neuronal cells or post-mortem tissues. However, the development of human induced pluripotent stem cell (iPSC) technology by the Yamanaka group was immediately applied to establish cell research models for neuronal disorders. Since then, techniques to achieve highly efficient differentiation of different types of neural cells following iPSC modeling have made much progress. The fast-growing iPSC and neural differentiation techniques have brought valuable insights into the pathology and neurobiology of neuropsychiatric disorders. In this article, we first review the application of iPSC technology in modeling neuronal disorders and discuss the progress in the accompanying neural differentiation. Then, we summarize the progress in iPSC-based research that has been accomplished so far regarding schizophrenia and bipolar disorder. © 2017 The Authors. Psychiatry and Clinical Neurosciences © 2017 Japanese Society of Psychiatry and Neurology.

Application of biomaterials to advance induced pluripotent stem cell research and therapy

PubMed Central

Tong, Zhixiang; Solanki, Aniruddh; Hamilos, Allison; Levy, Oren; Wen, Kendall; Yin, Xiaolei; Karp, Jeffrey M

2015-01-01

Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation. PMID:25766254

CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

PubMed Central

Vaz, Candida; Tanavde, Vivek; Lakshmipathy, Uma

2014-01-01

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones. PMID:24416407

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.

Human Pluripotent Stem Cells Differentiated in Fully Defined Medium Generate Hematopoietic CD34+ and CD34− Progenitors with Distinct Characteristics

PubMed Central

Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

2011-01-01

Background Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. Methodology/Principal Findings ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34+ cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34+ cells. ESC-derived CD34+ cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34− cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Conclusions/Significance Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures. PMID:21364915

Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

PubMed

Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

2011-02-25

Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

Porcine induced pluripotent stem cells produce chimeric offspring.

PubMed

West, Franklin D; Terlouw, Steve L; Kwon, Dae Jin; Mumaw, Jennifer L; Dhara, Sujoy K; Hasneen, Kowser; Dobrinsky, John R; Stice, Steven L

2010-08-01

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform.

PubMed

Sivalingam, Jaichandran; Lam, Alan Tin-Lun; Chen, Hong Yu; Yang, Bin Xia; Chen, Allen Kuan-Liang; Reuveny, Shaul; Loh, Yuin-Han; Oh, Steve Kah-Weng

2016-08-01

In vitro generation of red blood cells (RBCs) from human embryonic stem cells and human induced pluripotent stem cells appears to be a promising alternate approach to circumvent shortages in donor-derived blood supplies for clinical applications. Conventional methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) rely on embryoid body (EB) formation and/or coculture with xenogeneic cell lines. However, most current methods for hPSC expansion and EB formation are not amenable for scale-up to levels required for large-scale RBC generation. Moreover, differentiation methods that rely on xenogenic cell lines would face obstacles for future clinical translation. In this study, we report the development of a serum-free and chemically defined microcarrier-based suspension culture platform for scalable hPSC expansion and EB formation. Improved survival and better quality EBs generated with the microcarrier-based method resulted in significantly improved mesoderm induction and, when combined with hematopoietic differentiation, resulted in at least a 6-fold improvement in hematopoietic precursor expansion, potentially culminating in a 80-fold improvement in the yield of RBC generation compared to a conventional EB-based differentiation method. In addition, we report efficient terminal maturation and generation of mature enucleated RBCs using a coculture system that comprised primary human mesenchymal stromal cells. The microcarrier-based platform could prove to be an appealing strategy for future scale-up of hPSC culture, EB generation, and large-scale generation of RBCs under defined and xeno-free conditions.

Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells.

PubMed

Gledhill, Karl; Guo, Zongyou; Umegaki-Arao, Noriko; Higgins, Claire A; Itoh, Munenari; Christiano, Angela M

2015-01-01

The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs) present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.

Human Induced Pluripotent Stem Cell-Derived Cardiac Cell Sheets Expressing Genetically Encoded Voltage Indicator for Pharmacological and Arrhythmia Studies.

PubMed

Shaheen, Naim; Shiti, Assad; Huber, Irit; Shinnawi, Rami; Arbel, Gil; Gepstein, Amira; Setter, Noga; Goldfracht, Idit; Gruber, Amit; Chorna, Snizhanna V; Gepstein, Lior

2018-06-05

Fulfilling the potential of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes for studying conduction and arrhythmogenesis requires development of multicellular models and methods for long-term repeated tissue phenotyping. We generated confluent hiPSC-derived cardiac cell sheets (hiPSC-CCSs), expressing the genetically encoded voltage indicator ArcLight. ArcLight-based optical mapping allowed generation of activation and action-potential duration (APD) maps, which were validated by mapping the same hiPSC-CCSs with the voltage-sensitive dye, Di-4-ANBDQBS. ArcLight mapping allowed long-term assessment of electrical remodeling in the hiPSC-CCSs and evaluation of drug-induced conduction slowing (carbenoxolone, lidocaine, and quinidine) and APD prolongation (quinidine and dofetilide). The latter studies also enabled step-by-step depiction of drug-induced arrhythmogenesis ("torsades de pointes in the culture dish") and its prevention by MgSO 4 and rapid pacing. Phase-mapping analysis allowed biophysical characterization of spiral waves induced in the hiPSC-CCSs and their termination by electrical cardioversion and overdrive pacing. In conclusion, ArcLight mapping of hiPSC-CCSs provides a powerful tool for drug testing and arrhythmia investigation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

PubMed Central

Bajpai, Vivek K.; Mistriotis, Panagiotis; Loh, Yuin-Han; Daley, George Q.; Andreadis, Stelios T.

2012-01-01

Aims Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC. Methods and results We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists. Conclusion We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology. PMID:22941255

Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease Specific Patterns of Cardiotoxicity

PubMed Central

Liang, Ping; Lan, Feng; Lee, Andrew S.; Gong, Tingyu; Sanchez-Freire, Veronica; Wang, Yongming; Diecke, Sebastian; Sallam, Karim; Knowles, Joshua W.; Wang, Paul J.; Nguyen, Patricia K.; Bers, Donald M.; Robbins, Robert C.; Wu, Joseph C.

2013-01-01

Background Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with pre-existing heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. Methods and Results Action potential duration (APD) and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome (LQT), familial hypertrophic cardiomyopathy (HCM), and familial dilated cardiomyopathy (DCM). Disease phenotypes were verified in LQT, HCM, and DCM iPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene (hERG) expressing human embryonic kidney (HEK293) cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in HEK293 cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by APD and quantification of drug-induced arrhythmias such as early after depolarizations (EADs) and delayed after depolarizations (DADs). Conclusions We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, LQT, HCM, and DCM patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than standard hERG test or healthy control hiPSC-CM/hESC-CM screening assays. PMID:23519760

Common genetic variation drives molecular heterogeneity in human iPSCs

PubMed Central

Leha, Andreas; Afzal, Vackar; Alasoo, Kaur; Ashford, Sofie; Bala, Sendu; Bensaddek, Dalila; Casale, Francesco Paolo; Culley, Oliver J; Danecek, Petr; Faulconbridge, Adam; Harrison, Peter W; Kathuria, Annie; McCarthy, Davis; McCarthy, Shane A; Meleckyte, Ruta; Memari, Yasin; Moens, Nathalie; Soares, Filipa; Mann, Alice; Streeter, Ian; Agu, Chukwuma A; Alderton, Alex; Nelson, Rachel; Harper, Sarah; Patel, Minal; White, Alistair; Patel, Sharad R; Clarke, Laura; Halai, Reena; Kirton, Christopher M; Kolb-Kokocinski, Anja; Beales, Philip; Birney, Ewan; Danovi, Davide; Lamond, Angus I; Ouwehand, Willem H; Vallier, Ludovic; Watt, Fiona M; Durbin, Richard

2017-01-01

Induced pluripotent stem cell (iPSC) technology has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterisation of many existing iPSC lines limits their potential use for research and therapy. Here, we describe the systematic generation, genotyping and phenotyping of 711 iPSC lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative (HipSci: http://www.hipsci.org). Our study outlines the major sources of genetic and phenotypic variation in iPSCs and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPSC phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of rare, genomic copy number mutations that are repeatedly observed in iPSC reprogramming and present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells. PMID:28489815

Cellular Models: HD Patient-Derived Pluripotent Stem Cells.

PubMed

Geater, Charlene; Hernandez, Sarah; Thompson, Leslie; Mattis, Virginia B

2018-01-01

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by expanded polyglutamine (polyQ)-encoding repeats in the Huntingtin (HTT) gene. Traditionally, HD cellular models consisted of either patient cells not affected by disease or rodent neurons expressing expanded polyQ repeats in HTT. As these models can be limited in their disease manifestation or proper genetic context, respectively, human HD pluripotent stem cells (PSCs) are currently under investigation as a way to model disease in patient-derived neurons and other neural cell types. This chapter reviews embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) models of disease, including published differentiation paradigms for neurons and their associated phenotypes, as well as current challenges to the field such as validation of the PSCs and PSC-derived cells. Highlighted are potential future technical advances to HD PSC modeling, including transdifferentiation, complex in vitro multiorgan/system reconstruction, and personalized medicine. Using a human HD patient model of the central nervous system, hopefully one day researchers can tease out the consequences of mutant HTT (mHTT) expression on specific cell types within the brain in order to identify and test novel therapies for disease.

Differentiation of human pluripotent stem cells into highly functional classical brown adipocytes.

PubMed

Nishio, Miwako; Saeki, Kumiko

2014-01-01

We describe a detailed method for directed differentiation of human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), into functional classical brown adipocytes (BAs) under serum-free and feeder-free conditions. It is a two-tiered culture system, based on very simple techniques, a floating culture and a subsequent adherent culture. It does not require gene transfer. The entire process can be carried out in about 10 days. The key point is the usage of our special hematopoietic cytokine cocktail. Almost all the differentiated cells express uncoupling protein 1, a BA-selective marker, as determined by immunostaining. The differentiated cells show characteristics of classical BA as assessed by morphology and gene/protein expression. Moreover, the expression of myoblast marker genes is transiently induced during the floating culture step. hESC/hiPSC-derived BAs show significantly higher oxygen consumption rates (OCRs) than white adipocytes generated from human mesenchymal stem cell. They also show responsiveness to adrenergic stimuli, with about twofold upregulation in OCR by β-adrenergic receptor (β-AR) agonist treatments. hESC/hiPSC-derived BAs exert in vivo calorigenic activities in response to β-AR agonist treatments as assessed by thermography. Finally, lipid and glucose metabolisms are significantly improved in hESC/hiPSC-derived BA-transplanted mice. Our system provides a highly feasible way to produce functional classical BA bearing metabolism-improving capacities from hESC/hiPSC under a feeder-free and serum-free condition without gene transfer. © 2014 Elsevier Inc. All rights reserved.

Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells

PubMed Central

Kidwai, Fahad; Edwards, Jessica; Zou, Li; Kaufman, Dan S.

2016-01-01

Pluripotent stem cells, both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), provide an important resource to produce specialized cells such as osteogenic cells for therapeutic applications such as repair or replacement of injured, diseased or damaged bone. hESCs and iPSCs can also be used to better define basic cellular and genetic mechanisms that regulate the earliest stages of human bone development. However, current strategies to mediate osteogenic differentiation of hESC and iPSC are typically limited by the use of xenogeneic components such as fetal bovine serum (FBS) that make defining specific agents that mediate human osteogenesis difficult. Runt-related transcription factor 2 (RUNX2) is a key regulator required for osteogenic differentiation. Here, we used a RUNX2-YFP reporter system to characterize the novel ability of fibrinogen to mediate human osteogenic development from hESC and iPSC in defined (serum-free) conditions. These studies demonstrate that fibrinogen mediates significant osteo-induction potential. Specifically, fibrinogen binds to the surface integrin (α9β1) to mediate RUNX2 gene expression through the SMAD1/5/8 signaling pathway. Additional studies characterize the fibrinogen-induced hESC/iPSC-derived osteogenic cells to demonstrate these osteogenic cells retain the capacity to express typical mature osteoblastic markers. Together, these studies define a novel fibrinogen-α9β1-SMAD1/5/8-RUNX2 signaling axis can efficiently induce osteogenic differentiation from hESCs and iPSCs. PMID:27331788

Safe and efficient method for cryopreservation of human induced pluripotent stem cell-derived neural stem and progenitor cells by a programmed freezer with a magnetic field.

PubMed

Nishiyama, Yuichiro; Iwanami, Akio; Kohyama, Jun; Itakura, Go; Kawabata, Soya; Sugai, Keiko; Nishimura, Soraya; Kashiwagi, Rei; Yasutake, Kaori; Isoda, Miho; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

2016-06-01

Stem cells represent a potential cellular resource in the development of regenerative medicine approaches to the treatment of pathologies in which specific cells are degenerated or damaged by genetic abnormality, disease, or injury. Securing sufficient supplies of cells suited to the demands of cell transplantation, however, remains challenging, and the establishment of safe and efficient cell banking procedures is an important goal. Cryopreservation allows the storage of stem cells for prolonged time periods while maintaining them in adequate condition for use in clinical settings. Conventional cryopreservation systems include slow-freezing and vitrification both have advantages and disadvantages in terms of cell viability and/or scalability. In the present study, we developed an advanced slow-freezing technique using a programmed freezer with a magnetic field called Cells Alive System (CAS) and examined its effectiveness on human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs). This system significantly increased cell viability after thawing and had less impact on cellular proliferation and differentiation. We further found that frozen-thawed hiPSC-NS/PCs were comparable with non-frozen ones at the transcriptome level. Given these findings, we suggest that the CAS is useful for hiPSC-NS/PCs banking for clinical uses involving neural disorders and may open new avenues for future regenerative medicine. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Therapeutic Application of Pluripotent Stem Cells: Challenges and Risks.

PubMed

Martin, Ulrich

2017-01-01

Stem-cell-based therapies are considered to be promising and innovative but complex approaches. Induced pluripotent stem cells (iPSCs) combine the advantages of adult stem cells with the hitherto unique characteristics of embryonic stem cells (ESCs). Major progress has already been achieved with regard to reprogramming technology, but also regarding targeted genome editing and scalable expansion and differentiation of iPSCs and ESCs, in some cases yielding highly enriched preparations of well-defined cell lineages at clinically required dimensions. It is noteworthy, however, that for many applications critical requirements such as the targeted specification into distinct cellular subpopulations and a proper cell maturation remain to be achieved. Moreover, current hurdles such as low survival rates and insufficient functional integration of cellular transplants remain to be overcome. Nevertheless, PSC technologies obviously have come of age and matured to a stage where various clinical applications of PSC-based cellular therapies have been initiated and are conducted.

X chromosome inactivation in human pluripotent stem cells as a model for human development: back to the drawing board?

PubMed

Geens, Mieke; Chuva De Sousa Lopes, Susana M

2017-09-01

Human pluripotent stem cells (hPSC), both embryonic and induced (hESC and hiPSC), are regarded as a valuable in vitro model for early human development. In order to fulfil this promise, it is important that these cells mimic as closely as possible the in vivo molecular events, both at the genetic and epigenetic level. One of the most important epigenetic events during early human development is X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female cells. XCI is important for proper development and aberrant XCI has been linked to several pathologies. Recently, novel data obtained using high throughput single-cell technology during human preimplantation development have suggested that the XCI mechanism is substantially different from XCI in mouse. It has also been suggested that hPSC show higher complexity in XCI than the mouse. Here we compare the available recent data to understand whether XCI during human preimplantation can be properly recapitulated using hPSC. We will summarize what is known on the timing and mechanisms of XCI during human preimplantation development. We will compare this to the XCI patterns that are observed during hPSC derivation, culture and differentiation, and comment on the cause of the aberrant XCI patterns observed in hPSC. Finally, we will discuss the implications of the aberrant XCI patterns on the applicability of hPSC as an in vitro model for human development and as cell source for regenerative medicine. Combinations of the following keywords were applied as search criteria in the PubMed database: X chromosome inactivation, preimplantation development, embryonic stem cells, induced pluripotent stem cells, primordial germ cells, differentiation. Recent single-cell RNASeq data have shed new light on the XCI process during human preimplantation development. These indicate a gradual inactivation on both XX chromosomes, starting from Day 4 of development and followed by a random choice to inactivate one of them, instead of the mechanism in mice where imprinted XCI is followed by random XCI. We have put these new findings in perspective using previous data obtained in human (and mouse) embryos. In addition, there is an ongoing discussion whether or not hPSC lines show X chromosome reactivation upon derivation, mimicking the earliest embryonic cells, and the XCI states observed during culture of hPSC are highly variable. Recent studies have shown that hPSC rapidly progress to highly aberrant XCI patterns and that this process is probably driven by suboptimal culture conditions. Importantly, these aberrant XCI states seem to be inherited by the differentiated hPSC-progeny. The aberrant XCI states (and epigenetic instability) observed in hPSC throw a shadow on their applicability as an in vitro model for development and disease modelling. Moreover, as the aberrant XCI states observed in hPSC seem to shift to a more malignant phenotype, this may also have important consequences for the safety aspect of using hPSC in the clinic. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells.

PubMed

Fonoudi, Hananeh; Ansari, Hassan; Abbasalizadeh, Saeed; Larijani, Mehran Rezaei; Kiani, Sahar; Hashemizadeh, Shiva; Zarchi, Ali Sharifi; Bosman, Alexis; Blue, Gillian M; Pahlavan, Sara; Perry, Matthew; Orr, Yishay; Mayorchak, Yaroslav; Vandenberg, Jamie; Talkhabi, Mahmood; Winlaw, David S; Harvey, Richard P; Aghdami, Nasser; Baharvand, Hossein

2015-12-01

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease. ©AlphaMed Press.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells

PubMed Central

Fonoudi, Hananeh; Ansari, Hassan; Abbasalizadeh, Saeed; Larijani, Mehran Rezaei; Kiani, Sahar; Hashemizadeh, Shiva; Zarchi, Ali Sharifi; Bosman, Alexis; Blue, Gillian M.; Pahlavan, Sara; Perry, Matthew; Orr, Yishay; Mayorchak, Yaroslav; Vandenberg, Jamie; Talkhabi, Mahmood; Winlaw, David S.; Harvey, Richard P.; Aghdami, Nasser

2015-01-01

Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. Significance Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease. PMID:26511653

Inflammatory Responses and Barrier Function of Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

PubMed

Halaidych, Oleh V; Freund, Christian; van den Hil, Francijna; Salvatori, Daniela C F; Riminucci, Mara; Mummery, Christine L; Orlova, Valeria V

2018-05-08

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

PubMed

Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

2016-01-01

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

SIRT1 Overexpression Maintains Cell Phenotype and Function of Endothelial Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Jiang, Bin; Jen, Michele; Perrin, Louisiane; Wertheim, Jason A; Ameer, Guillermo A

2015-12-01

Endothelial cells (ECs) that are differentiated from induced pluripotent stem cells (iPSCs) can be used in establishing disease models for personalized drug discovery or developing patient-specific vascularized tissues or organoids. However, a number of technical challenges are often associated with iPSC-ECs in culture, including instability of the endothelial phenotype and limited cell proliferative capacity over time. Early senescence is believed to be the primary mechanism underlying these limitations. Sirtuin1 (SIRT1) is an NAD(+)-dependent deacetylase involved in the regulation of cell senescence, redox state, and inflammatory status. We hypothesize that overexpression of the SIRT1 gene in iPSC-ECs will maintain EC phenotype, function, and proliferative capacity by overcoming early cell senescence. SIRT1 gene was packaged into a lentiviral vector (LV-SIRT1) and transduced into iPSC-ECs at passage 4. Beginning with passage 5, iPSC-ECs exhibited a fibroblast-like morphology, whereas iPSC-ECs overexpressing SIRT1 maintained EC cobblestone morphology. SIRT1 overexpressing iPSC-ECs also exhibited a higher percentage of canonical markers of endothelia (LV-SIRT1 61.8% CD31(+) vs. LV-empty 31.7% CD31(+), P < 0.001; LV-SIRT1 46.3% CD144(+) vs. LV-empty 20.5% CD144(+), P < 0.02), with a higher nitric oxide synthesis, lower β-galactosidase production indicating decreased senescence (3.4% for LV-SIRT1 vs. 38.6% for LV-empty, P < 0.001), enhanced angiogenesis, increased deacetylation activity, and higher proliferation rate. SIRT1 overexpressing iPSC-ECs continued to proliferate through passage 9 with high purity of EC-like characteristics, while iPSC-ECs without SIRT1 overexpression became senescent after passage 5. Taken together, SIRT1 overexpression in iPSC-ECs maintains EC phenotype, improves EC function, and extends cell lifespan, overcoming critical hurdles associated with the use of iPSC-ECs in translational research.

Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

PubMed Central

Frith, Jessica E.; Frith, Thomas J.R.; Ovchinnikov, Dmitry A.; Cooper-White, Justin J.; Wolvetang, Ernst J.

2014-01-01

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases. PMID:25055193

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells

PubMed Central

Merling, Randall K.; Sweeney, Colin L.; Choi, Uimook; De Ravin, Suk See; Myers, Timothy G.; Otaizo-Carrasquero, Francisco; Pan, Jason; Linton, Gilda; Chen, Lifeng; Koontz, Sherry; Theobald, Narda L.; Malech, Harry L.

2013-01-01

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34+ hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34+ HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60+, yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60+), with variable yield (6 to >500 TRA-1-60+ iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP–reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34+ HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14+ monocytes do not yield iPSC colonies under these reprogramming conditions. PMID:23386128

Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

PubMed Central

Veerman, Christiaan C.; Zegers, Jan G.; Mengarelli, Isabella; Bezzina, Connie R.

2017-01-01

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs. PMID:28867785

Differentiation of Swine iPSC into Rod Photoreceptors and Their Integration into the Retina

PubMed Central

Zhou, Liang; Wang, Wei; Liu, Yongqing; de Castro, Juan Fernandez; Ezashi, Toshihiko; Telugu, Bhanu Prakash V.L.; Roberts, R. Michael; Kaplan, Henry J.; Dean, Douglas C.

2014-01-01

Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments utilizing stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with swine induced pluripotent stem cells (iPSC). Here, we subjected swine iPSC to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of RHO and ROM1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that swine iPSC can differentiate into photoreceptors in culture and these cells can integrate into the damaged swine neural retina thus laying a foundation for future studies using the pig as a model for retinal stem cell transplantation. PMID:21491544

Somatic cell reprogramming informed by the oocyte.

PubMed

Gonzalez-Munoz, Elena; Cibelli, Jose B

2018-05-08

The successful production of animals and embryonic stem cells (ESCs) using somatic cell nuclear transfer (SCNT) has demonstrated the unmatched nuclear reprogramming capacity of the oocyte and helped prove the degree of plasticity of differentiated cells. The introduction of transcription factors to generate induced pluripotent stem cells (iPSCs) displaced SCNT and, due to its ease of implementation, became the method of choice for cell reprogramming. Nonetheless, iPSC derivation remains inefficient and stochastic. This review article focuses on using the oocyte as a source of reprogramming factors, comparing the SCNT and iPSC mechanisms for remodeling chromatin and acquiring pluripotency.

Novel therapeutic approaches: Rett syndrome and human induced pluripotent stem cell technology

PubMed Central

Gomathi, Mohan

2017-01-01

Recent advances in induced pluripotent stem cell (iPSC) technology target screening and discovering of therapeutic agents for the possible cure of human diseases. Human induced pluripotent stem cells (hiPSC) are the right kind of platform for testing potency of specific active compounds. Ayurveda, the Indian traditional system of medicine developed between 2,500 and 500 BC, is a science involving the intelligent formulations of herbs and minerals. It can serve as a “goldmine” for novel neuroprotective agents used for centuries to treat neurological disorders. This review discusses limitations in screening drugs for neurological disorders and the advantages offered by hiPSC integrated with Indian traditional system of medicine. We begin by describing the current state of hiPSC technology in research on Rett syndrome (RTT) followed by the current controversies in RTT research combined with the emergence of patient-specific hiPSC that indicate an urgent need for researchers to understand the etiology and drug mechanism. We conclude by offering recommendations to reinforce the screening of active compounds present in the ayurvedic medicines using the human induced pluripotent neural model system for research involving drug discovery for RTT. This integrative approach will fill the current knowledge gap in the traditional medicines and drug discovery. PMID:28447035

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

Gene and Cell Therapy for β-Thalassemia and Sickle Cell Disease with Induced Pluripotent Stem Cells (iPSCs): The Next Frontier.

PubMed

Papapetrou, Eirini P

2017-01-01

In recent years, breakthroughs in human pluripotent stem cell (hPSC) research, namely cellular reprogramming and the emergence of sophisticated genetic engineering technologies, have opened new frontiers for cell and gene therapy. The prospect of using hPSCs, either autologous or histocompatible, as targets of genetic modification and their differentiated progeny as cell products for transplantation, presents a new paradigm of regenerative medicine of potential tremendous value for the treatment of blood disorders, including beta-thalassemia (BT) and sickle cell disease (SCD). Despite advances at a remarkable pace and great promise, many roadblocks remain before clinical translation can be realistically considered. Here we discuss the theoretical advantages of cell therapies utilizing hPSC derivatives, recent proof-of-principle studies and the main challenges towards realizing the potential of hPSC therapies in the clinic.

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.

Pharyngeal satellite cells undergo myogenesis under basal conditions and are required for pharyngeal muscle maintenance

PubMed Central

Randolph, Matthew E.; Phillips, Brittany L.; Choo, Hyo-Jung; Vest, Katherine E.; Vera, Yandery; Pavlath, Grace K.

2015-01-01

The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging. PMID:26178867

Introduction to thematic minireview series: Development of human therapeutics based on induced pluripotent stem cell (iPSC) technology.

PubMed

Rao, Mahendra; Gottesfeld, Joel M

2014-02-21

With the advent of human induced pluripotent stem cell (hiPSC) technology, it is now possible to derive patient-specific cell lines that are of great potential in both basic research and the development of new therapeutics for human diseases. Not only do hiPSCs offer unprecedented opportunities to study cellular differentiation and model human diseases, but the differentiated cell types obtained from iPSCs may become therapeutics themselves. These cells can also be used in the screening of therapeutics and in toxicology assays for potential liabilities of therapeutic agents. The remarkable achievement of transcription factor reprogramming to generate iPSCs was recognized by the award of the Nobel Prize in Medicine to Shinya Yamanaka in 2012, just 6 years after the first publication of reprogramming methods to generate hiPSCs (Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Cell 131, 861-872). This minireview series highlights both the promises and challenges of using iPSC technology for disease modeling, drug screening, and the development of stem cell therapeutics.

Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium.

PubMed

Salomonis, Nathan; Dexheimer, Phillip J; Omberg, Larsson; Schroll, Robin; Bush, Stacy; Huo, Jeffrey; Schriml, Lynn; Ho Sui, Shannan; Keddache, Mehdi; Mayhew, Christopher; Shanmukhappa, Shiva Kumar; Wells, James; Daily, Kenneth; Hubler, Shane; Wang, Yuliang; Zambidis, Elias; Margolin, Adam; Hide, Winston; Hatzopoulos, Antonis K; Malik, Punam; Cancelas, Jose A; Aronow, Bruce J; Lutzko, Carolyn

2016-07-12

The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Induced adult stem (iAS) cells and induced transit amplifying progenitor (iTAP) cells-a possible alternative to induced pluripotent stem (iPS) cells?

PubMed

Heng, Boon Chin; Richards, Mark; Ge, Zigang; Shu, Yimin

2010-02-01

The successful derivation of iPSC lines effectively demonstrates that it is possible to reset the 'developmental clock' of somatic cells all the way back to the initial embryonic state. Hence, it is plausible that this clock may instead be turned back half-way to a less immature developmental stage that is more directly applicable to clinical therapeutic applications or for in vitro pharmacology/toxicology screening assays. Such a suitable developmental state is postulated to be either the putative transit amplifying progenitor stage or adult stem cell stage. It is hypothetically possible to reprogram mature and terminally differentiated somatic cells back to the adult stem cell or transit amplifying progenitor stage, in a manner similar to the derivation of iPSC. It is proposed that the terminology 'Induced Adult Stem Cells' (iASC) or 'Induced Transit Amplifying Progenitor Cells' (iTAPC) be used to described such reprogrammed somatic cells. Of particular interest, is the possibility of resetting the developmental clock of mature differentiated somatic cells of the mesenchymal lineage, explanted from adipose tissue, bone marrow and cartilage. The putative adult stem cell sub-population from which these cells are derived, commonly referred to as 'mesenchymal stem cells', are highly versatile and hold much therapeutic promise in regenerative medicine, as attested to by numerous human clinical trials and animal studies. Perhaps it may be appropriate to term such reprogrammed cells as 'Induced Mesenchymal Stem Cells' (iMSC) or as 'Induced Mesenchumal Progenitor Cells' (iMPC). Given that cells from the same organ/tissue will share some commonalities in gene expression, we hypothesize that the generation of iASC or iTAPC would be more efficient as compared to iPSC generation, since a common epigenetic program must exist between the reprogrammed cells, adult stem cell or progenitor cell types and terminally differentiated cell types from the same organ/tissue.

Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels

PubMed Central

Zanotelli, Matthew R.; Ardalani, Hamisha; Zhang, Jue; Hou, Zhonggang; Nguyen, Eric H.; Swanson, Scott; Nguyen, Bao Kim; Bolin, Jennifer; Elwell, Angela; Bischel, Lauren L.; Xie, Angela W.; Stewart, Ron; Beebe, David J.; Thomson, James A.; Schwartz, Michael P.; Murphy, William L.

2016-01-01

Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14 days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. PMID:26945632

Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells

PubMed Central

Gledhill, Karl; Guo, Zongyou; Umegaki-Arao, Noriko; Higgins, Claire A.; Itoh, Munenari; Christiano, Angela M.

2015-01-01

The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs) present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes. PMID:26308443

Pluripotent stem cells: the last 10 years.

PubMed

Kimbrel, Erin A; Lanza, Robert

2016-12-01

Pluripotent stem cells (PSCs) can differentiate into virtually any cell type in the body, making them attractive for both regenerative medicine and drug discovery. Over the past 10 years, technological advances and innovative platforms have yielded first-in-man PSC-based clinical trials and opened up new approaches for disease modeling and drug development. Induced PSCs have become the foremost alternative to embryonic stem cells and accelerated the development of disease-in-a-dish models. Over the years and with each new discovery, PSCs have proven to be extremely versatile. This review article highlights key advancements in PSC research, from 2006 to 2016, and how they will guide the direction of the field over the next decade.

Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts.

PubMed

Questa, María; Romorini, Leonardo; Blüguermann, Carolina; Solari, Claudia María; Neiman, Gabriel; Luzzani, Carlos; Scassa, María Élida; Sevlever, Gustavo Emilio; Guberman, Alejandra Sonia; Miriuka, Santiago Gabriel

2016-03-01

Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls. Copyright © 2015 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs).

PubMed

Sala, Luca; Ward-van Oostwaard, Dorien; Tertoolen, Leon G J; Mummery, Christine L; Bellin, Milena

2017-05-12

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Advances in Microfluidic Platforms for Analyzing and Regulating Human Pluripotent Stem Cells

PubMed Central

Qian, Tongcheng; Shusta, Eric V.; Palecek, Sean P.

2015-01-01

Microfluidic devices employ submillimeter length scale control of flow to achieve high-resolution spatial and temporal control over the microenvironment, providing powerful tools to elucidate mechanisms of human pluripotent stem cell (hPSC) regulation and to elicit desired hPSC fates. In addition, microfluidics allow control of paracrine and juxtracrine signaling, thereby enabling fabrication of microphysiological systems comprised of multiple cell types organized into organs-on-a-chip. Microfluidic cell culture systems can also be integrated with actuators and sensors, permitting construction of high-density arrays of cell-based biosensors for screening applications. This review describes recent advances in using microfluidics to understand mechanisms by which the microenvironment regulates hPSC fates and applications of microfluidics to realize the potential of hPSCs for in vitro modeling and screening applications. PMID:26313850

Cryopreservation of Brain Endothelial Cells Derived from Human Induced Pluripotent Stem Cells Is Enhanced by Rho-Associated Coiled Coil-Containing Kinase Inhibition.

PubMed

Wilson, Hannah K; Faubion, Madeline G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V

2016-12-01

The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently, human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However, the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages, including enabling production of larger scale lots, decreasing lead time to generate purified iPSC-BMEC cultures, and facilitating use of iPSC-BMECs in large-scale screening. In this study, we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability, express standard endothelial and BBB markers, and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ω·cm 2 , equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs, as well as stabilized TEER above 800 Ω·cm 2 out to 7 days post-thaw. Overall, cryopreservation will ease handling and storage of high-quality iPSC-BMECs, reducing a key barrier to greater implementation of these cells in modeling the human BBB.

Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor.

PubMed

Sivarapatna, Amogh; Ghaedi, Mahboobe; Le, Andrew V; Mendez, Julio J; Qyang, Yibing; Niklason, Laura E

2015-01-01

Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Safety and immune regulatory properties of canine induced pluripotent stem cell-derived mesenchymal stem cells.

PubMed

Chow, Lyndah; Johnson, Valerie; Regan, Dan; Wheat, William; Webb, Saiphone; Koch, Peter; Dow, Steven

2017-12-01

Mesenchymal stem cells (MSCs) exhibit broad immune modulatory activity in vivo and can suppress T cell proliferation and dendritic cell activation in vitro. Currently, most MSC for clinical usage are derived from younger donors, due to ease of procurement and to the superior immune modulatory activity. However, the use of MSC from multiple unrelated donors makes it difficult to standardize study results and compare outcomes between different clinical trials. One solution is the use of MSC derived from induced pluripotent stem cells (iPSC); as iPSC-derived MSC have nearly unlimited proliferative potential and exhibit in vitro phenotypic stability. Given the value of dogs as a spontaneous disease model for pre-clinical evaluation of stem cell therapeutics, we investigated the functional properties of canine iPSC-derived MSC (iMSC), including immune modulatory properties and potential for teratoma formation. We found that canine iMSC downregulated expression of pluripotency genes and appeared morphologically similar to conventional MSC. Importantly, iMSC retained a stable phenotype after multiple passages, did not form teratomas in immune deficient mice, and did not induce tumor formation in dogs following systemic injection. We concluded therefore that iMSC were phenotypically stable, immunologically potent, safe with respect to tumor formation, and represented an important new source of cells for therapeutic modulation of inflammatory disorders. Copyright © 2017. Published by Elsevier B.V.

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

PubMed

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

2015-06-01

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

High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.

PubMed

Sharma, Arun; Burridge, Paul W; McKeithan, Wesley L; Serrano, Ricardo; Shukla, Praveen; Sayed, Nazish; Churko, Jared M; Kitani, Tomoya; Wu, Haodi; Holmström, Alexandra; Matsa, Elena; Zhang, Yuan; Kumar, Anusha; Fan, Alice C; Del Álamo, Juan C; Wu, Sean M; Moslehi, Javid J; Mercola, Mark; Wu, Joseph C

2017-02-15

Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling. Copyright © 2017, American Association for the Advancement of Science.

From pericytes to perivascular tumours: correlation between pathology, stem cell biology, and tissue engineering.

PubMed

Mravic, Marco; Asatrian, Greg; Soo, Chia; Lugassy, Claire; Barnhill, Raymond L; Dry, Sarah M; Peault, Bruno; James, Aaron W

2014-09-01

Pericytes were once thought only to aid in angiogenesis and blood pressure control. Gradually, the known functions of pericytes and other perivascular stem cells (PSC) have broadly increased. The following review article will summarize the known functions and importance of pericytes across disciplines of pathology, stem cell biology, and tissue engineering. A literature review was performed for studies examining the importance of pericytes in pathology, stem cell biology, and tissue engineering. The importance of pericytes most prominently includes the identification of the perivascular identity of mesenchymal stem cells (or MSC). Now, pericytes and other PSC are known to display surface markers and multilineage differentiation potential of MSC. Accordingly, interest in the purification and use of PSC for mesenchymal tissue formation and regeneration has increased. Significant demonstration of in vivo efficacy in bone and muscle regeneration has been made in laboratory animals. Contemporaneously with the uncovering of an MSC identity for pericytes, investigators in tumour biology have found biologically relevant roles for pericytes in tumor formation, lymphovascular invasion, and perivascular tumor spread. As well, the contribution of pericytes to perivascular tumors has been examined (and debated), including glomus tumour, myopericytoma and solitary fibrous tumour/hemangiopericytoma. In addition, an expanding recognition of pericyte mimicry and perivascular tumour invasion has occurred, encompassing common malignancies of the brain and skin. In summary, pericytes have a wide range of roles in health and disease. Pericytes are being increasingly studied for their role in tumour formation, growth and invasion. Likewise, the application of pericytes/PSC for mesenchymal tissue engineering is an expanding field of interest.

Systematic evaluation of markers used for the identification of human induced pluripotent stem cells

PubMed Central

Bharathan, Sumitha Prameela; Manian, Kannan Vrindavan; Aalam, Syed Mohammed Musheer; Palani, Dhavapriya; Deshpande, Prashant Ajit; Pratheesh, Mankuzhy Damodaran; Srivastava, Alok

2017-01-01

ABSTRACT Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4+ and TRA-1-60+ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs). PMID:28089995

Efficient stage-specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells.

PubMed

Mellough, Carla B; Sernagor, Evelyne; Moreno-Gimeno, Inmaculada; Steel, David H W; Lako, Majlinda

2012-04-01

Recent successes in the stem cell field have identified some of the key chemical and biological cues which drive photoreceptor derivation from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC); however, the efficiency of this process is variable. We have designed a three-step photoreceptor differentiation protocol combining previously published methods that direct the differentiation of hESC and hiPSC toward a retinal lineage, which we further modified with additional supplements selected on the basis of reports from the eye field and retinal development. We report that hESC and hiPSC differentiating under our regimen over a 60 day period sequentially acquire markers associated with neural, retinal field, retinal pigmented epithelium and photoreceptor cells, including mature photoreceptor markers OPN1SW and RHODOPSIN with a higher efficiency than previously reported. In addition, we report the ability of hESC and hiPSC cultures to generate neural and retinal phenotypes under minimal culture conditions, which may be linked to their ability to endogenously upregulate the expression of a range of factors important for retinal cell type specification. However, cultures that were differentiated with full supplementation under our photoreceptor-induction regimen achieve this within a significantly shorter time frame and show a substantial increase in the expression of photoreceptor-specific markers in comparison to cultures differentiated under minimal conditions. Interestingly, cultures supplemented only with B27 and/or N2 displayed comparable differentiation efficiency to those under full supplementation, indicating a key role for B27 and N2 during the differentiation process. Furthermore, our data highlight an important role for Dkk1 and Noggin in enhancing the differentiation of hESC and hiPSC toward retinal progenitor cells and photoreceptor precursors during the early stages of differentiation, while suggesting that further maturation of these cells into photoreceptors may not require additional factors and can ensue under minimal culture conditions. Copyright © 2012 AlphaMed Press.

Enhanced Therapeutic and Long-Term Dynamic Vascularization Effects of Human Pluripotent Stem Cell-Derived Endothelial Cells Encapsulated in a Nanomatrix Gel.

PubMed

Lee, Shin-Jeong; Sohn, Young-Doug; Andukuri, Adinarayana; Kim, Sangsung; Byun, Jaemin; Han, Ji Woong; Park, In-Hyun; Jun, Ho-Wook; Yoon, Young-Sup

2017-11-14

Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3β inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5 + cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis. The resultant hPSC-derived CDH5 + cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs. © 2017 American Heart Association, Inc.

Human pluripotent stem cell-derived erythropoietin-producing cells ameliorate renal anemia in mice.

PubMed

Hitomi, Hirofumi; Kasahara, Tomoko; Katagiri, Naoko; Hoshina, Azusa; Mae, Shin-Ichi; Kotaka, Maki; Toyohara, Takafumi; Rahman, Asadur; Nakano, Daisuke; Niwa, Akira; Saito, Megumu K; Nakahata, Tatsutoshi; Nishiyama, Akira; Osafune, Kenji

2017-09-27

The production of erythropoietin (EPO) by the kidneys, a principal hormone for the hematopoietic system, is reduced in patients with chronic kidney disease (CKD), eventually resulting in severe anemia. Although recombinant human EPO treatment improves anemia in patients with CKD, returning to full red blood cell production without fluctuations does not always occur. We established a method to generate EPO-producing cells from human induced pluripotent stem cells (hiPSCs) by modifying previously reported hepatic differentiation protocols. These cells showed increased EPO expression and secretion in response to low oxygen conditions, prolyl hydroxylase domain-containing enzyme inhibitors, and insulin-like growth factor 1. The EPO protein secreted from hiPSC-derived EPO-producing (hiPSC-EPO) cells induced the erythropoietic differentiation of human umbilical cord blood progenitor cells in vitro. Furthermore, transplantation of hiPSC-EPO cells into mice with CKD induced by adenine treatment improved renal anemia. Thus, hiPSC-EPO cells may be a useful tool for clarifying the mechanisms of EPO production and may be useful as a therapeutic strategy for treating renal anemia. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Big Animal Cloning Using Transgenic Induced Pluripotent Stem Cells: A Case Study of Goat Transgenic Induced Pluripotent Stem Cells.

PubMed

Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Wang, Ziyu; Wang, Feng

2016-02-01

Using of embryonic stem cells (ESCs) could improve production traits and disease resistance by improving the efficiency of somatic cell nuclear transfer (SCNT) technology. However, robust ESCs have not been established from domestic ungulates. In the present study, we generated goat induced pluripotent stem cells (giPSCs) and transgenic cloned dairy goat induced pluripotent stem cells (tgiPSCs) from dairy goat fibroblasts (gFs) and transgenic cloned dairy goat fibroblasts (tgFs), respectively, using lentiviruses that contained hOCT4, hSOX2, hMYC, and hKLF4 without chemical compounds. The giPSCs and tgiPSCs expressed endogenous pluripotent markers, including OCT4, SOX2, MYC, KLF4, and NANOG. Moreover, they were able to maintain a normal karyotype and differentiate into derivatives from all three germ layers in vitro and in vivo. Using SCNT, tgFs and tgiPSCs were used as donor cells to produce embryos, which were named tgF-Embryos and tgiPSC-Embryos. The fusion rates and cleavage rates had no significant differences between tgF-Embryos and tgiPSC-Embryos. However, the expression of IGF-2, which is an important gene associated with embryonic development, was significantly lower in tgiPSC-Embryos than in tgF-Embryos and was not significantly different from vivo-Embryos.

Generation of induced pluripotent stem cells from a patient with X-linked juvenile retinoschisis.

PubMed

Peng, Chi-Hsien; Huang, Kang-Chieh; Lu, Huai-En; Syu, Shih-Han; Yarmishyn, Aliaksandr A; Lu, Jyh-Feng; Buddhakosai, Waradee; Lin, Tai-Chi; Hsu, Chih-Chien; Hwang, De-Kuang; Shen, Chia-Ning; Chen, Shih-Jen; Chiou, Shih-Hwa

2018-05-01

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal dystrophy manifested as splitting of anatomical layers of retina. In this report, we generated a patient-specific induced pluripotent stem cell (iPSC) line, TVGH-iPSC-013-05, from the peripheral blood mononuclear cells of a male patient with XLRS by using the Sendai-virus delivery system. We believe that XLRS patient-specific iPSCs provide a powerful in vitro model for evaluating the pathological phenotypes of the disease. Copyright © 2018. Published by Elsevier B.V.

Behavior of Xeno-Transplanted Undifferentiated Human Induced Pluripotent Stem Cells Is Impacted by Microenvironment Without Evidence of Tumors.

PubMed

Martínez-Cerdeño, Veronica; Barrilleaux, Bonnie L; McDonough, Ashley; Ariza, Jeanelle; Yuen, Benjamin T K; Somanath, Priyanka; Le, Catherine T; Steward, Craig; Horton-Sparks, Kayla; Knoepfler, Paul S

2017-10-01

Human pluripotent stem cells (hPSC) have great clinical potential through the use of their differentiated progeny, a population in which there is some concern over risks of tumorigenicity or other unwanted cellular behavior due to residual hPSC. Preclinical studies using human stem cells are most often performed within a xenotransplant context. In this study, we sought to measure how undifferentiated hPSC behave following xenotransplant. We directly transplanted undifferentiated human induced pluripotent stem cells (hIPSC) and human embryonic stem cells (hESC) into the adult mouse brain ventricle and analyzed their fates. No tumors or precancerous lesions were present at more than one year after transplantation. This result differed with the tumorigenic capacity we observed after allotransplantation of mouse ESC into the mouse brain. A substantial population of cellular derivatives of undifferentiated hESC and hIPSC engrafted, survived, and migrated within the mouse brain parenchyma. Within brain structures, transplanted cell distribution followed a very specific pattern, suggesting the existence of distinct microenvironments that offer different degrees of permissibility for engraftment. Most of the transplanted hESC and hIPSC that developed into brain cells were NeuN+ neuronal cells, and no astrocytes were detected. Substantial cell and nuclear fusion occurred between host and transplanted cells, a phenomenon influenced by microenvironment. Overall, hIPSC appear to be largely functionally equivalent to hESC in vivo. Altogether, these data bring new insights into the behavior of stem cells without prior differentiation following xenotransplantation into the adult brain.

Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism

PubMed Central

Gu, Mingxia; Mordwinkin, Nicholas M.; Kooreman, Nigel G.; Lee, Jaecheol; Wu, Haodi; Hu, Shijun; Churko, Jared M.; Diecke, Sebastian; Burridge, Paul W.; He, Chunjiang; Barron, Frances E.; Ong, Sang-Ging; Gold, Joseph D.; Wu, Joseph C.

2015-01-01

Aims High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays. Methods and results Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, Nω-nitro-l-arginine methyl ester. Conclusion This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population. PMID:25368203

Vascular Smooth Muscle Cells From Hypertensive Patient-Derived Induced Pluripotent Stem Cells to Advance Hypertension Pharmacogenomics.

PubMed

Biel, Nikolett M; Santostefano, Katherine E; DiVita, Bayli B; El Rouby, Nihal; Carrasquilla, Santiago D; Simmons, Chelsey; Nakanishi, Mahito; Cooper-DeHoff, Rhonda M; Johnson, Julie A; Terada, Naohiro

2015-12-01

Studies in hypertension (HTN) pharmacogenomics seek to identify genetic sources of variable antihypertensive drug response. Genetic association studies have detected single-nucleotide polymorphisms (SNPs) that link to drug responses; however, to understand mechanisms underlying how genetic traits alter drug responses, a biological interface is needed. Patient-derived induced pluripotent stem cells (iPSCs) provide a potential source for studying otherwise inaccessible tissues that may be important to antihypertensive drug response. The present study established multiple iPSC lines from an HTN pharmacogenomics cohort. We demonstrated that established HTN iPSCs can robustly and reproducibly differentiate into functional vascular smooth muscle cells (VSMCs), a cell type most relevant to vasculature tone control. Moreover, a sensitive traction force microscopy assay demonstrated that iPSC-derived VSMCs show a quantitative contractile response on physiological stimulus of endothelin-1. Furthermore, the inflammatory chemokine tumor necrosis factor α induced a typical VSMC response in iPSC-derived VSMCs. These studies pave the way for a large research initiative to decode biological significance of identified SNPs in hypertension pharmacogenomics. Treatment of hypertension remains suboptimal, and a pharmacogenomics approach seeks to identify genetic biomarkers that could be used to guide treatment decisions; however, it is important to understand the biological underpinnings of genetic associations. Mouse models do not accurately recapitulate individual patient responses based on their genetics, and hypertension-relevant cells are difficult to obtain from patients. Induced pluripotent stem cell (iPSC) technology provides a great interface to bring patient cells with their genomic data into the laboratory and to study hypertensive responses. As an initial step, the present study established an iPSC bank from patients with primary hypertension and demonstrated an effective and reproducible method of generating functional vascular smooth muscle cells. ©AlphaMed Press.

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 induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

PubMed

Ishii, Misawa Niki; Yamamoto, Koji; Shoji, Masanobu; Asami, Asano; Kawamata, Yuji

2017-08-15

Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform activity in response to convulsant drugs. Our study indicates the possibility that co-culture system of hiPSC-derived neurons with hiPSC-derived astrocytes could be useful in the risk assessment of drug-induced seizure. Copyright © 2017 Elsevier B.V. All rights reserved.

DSG2 Is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells.

PubMed

Park, Jongjin; Son, Yeonsung; Lee, Na Geum; Lee, Kyungmin; Lee, Dong Gwang; Song, Jinhoi; Lee, Jaemin; Kim, Seokho; Cho, Min Ji; Jang, Ju-Hong; Lee, Jangwook; Park, Jong-Gil; Kim, Yeon-Gu; Kim, Jang-Seong; Lee, Jungwoon; Cho, Yee Sook; Park, Young-Jun; Han, Baek Soo; Bae, Kwang-Hee; Han, Seungmin; Kang, Byunghoon; Haam, Seungjoo; Lee, Sang-Hyun; Lee, Sang Chul; Min, Jeong-Ki

2018-06-08

Pluripotent stem cells (PSCs) represent the most promising clinical source for regenerative medicine. However, given the cellular heterogeneity within cultivation and safety concerns, the development of specific and efficient tools to isolate a pure population and eliminate all residual undifferentiated PSCs from differentiated derivatives is a prerequisite for clinical applications. In this study, we raised a monoclonal antibody and identified its target antigen as desmoglein-2 (DSG2). DSG2 co-localized with human PSC (hPSC)-specific cell surface markers, and its expression was rapidly downregulated upon differentiation. The depletion of DSG2 markedly decreased hPSC proliferation and pluripotency marker expression. In addition, DSG2-negative population in hPSCs exhibited a notable suppression in embryonic body and teratoma formation. The actions of DSG2 in regulating the self-renewal and pluripotency of hPSCs were predominantly exerted through the regulation of β-catenin/Slug-mediated epithelial-to-mesenchymal transition. Our results demonstrate that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Generation of Viable Mice from Induced Pluripotent Stem Cells (iPSCs) Through Tetraploid Complementation.

PubMed

Kang, Lan; Gao, Shaorong

2015-01-01

Tetraploid complementation assay is the most rigorous criteria for pluripotency characterization of pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Pluripotent stem cells could complement the developmental deficiency of tetraploid embryos and thus support the full-term mice development. Here we describe the protocol for tetraploid complementation using iPSCs to produce viable all-iPSC mice.

Present and future challenges of induced pluripotent stem cells.

PubMed

Ohnuki, Mari; Takahashi, Kazutoshi

2015-10-19

Growing old is our destiny. However, the mature differentiated cells making up our body can be rejuvenated to an embryo-like fate called pluripotency which is an ability to differentiate into all cell types by enforced expression of defined transcription factors. The discovery of this induced pluripotent stem cell (iPSC) technology has opened up unprecedented opportunities in regenerative medicine, disease modelling and drug discovery. In this review, we introduce the applications and future perspectives of human iPSCs and we also show how iPSC technology has evolved along the way. © 2015 The Author(s).

Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers.

PubMed

Li, Junjun; Minami, Itsunari; Yu, Leqian; Tsuji, Kiyotaka; Nakajima, Minako; Qiao, Jing; Suzuki, Masato; Shimono, Ken; Nakatsuji, Norio; Kotera, Hitetoshi; Liu, Li; Chen, Yong

2016-01-01

Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays.

Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers

PubMed Central

Minami, Itsunari; Yu, Leqian; Nakajima, Minako; Qiao, Jing; Shimono, Ken; Nakatsuji, Norio; Kotera, Hitetoshi; Chen, Yong

2016-01-01

Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) hold high potential for use in drug assessment and myocardial regeneration. To create tissue-like constructs of CMs for extracellular monitoring, we placed aligned fibers (AFs) on the surface of a microelectrode array and then seeded hiPSC-CMs for subsequent monitoring for 14 days. As expected, the CMs organized into anisotropic and matured tissue and the extracellular recordings showed reduced premature beating higher signal amplitude and a higher probability of T-wave detection as compared to the culture without fibers. The CMs on the aligned fibers samples also exhibited anisotropic propagation of the field potential. These results therefore suggest that the hiPSC-CMs cultured on AFs can be used more reliably for cell based assays. PMID:27446217

Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

PubMed Central

Yanagi, Satoshi; Kato, Chika; Takashima, Ryokichi; Kobayashi, Eiji; Hagiwara, Keitaro; Ochiya, Takahiro

2015-01-01

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation. PMID:25875613

Generation of functional human pancreatic β cells in vitro

PubMed Central

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

2015-01-01

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

Human induced pluripotent stem cell line with cytochrome P450 enzyme polymorphism (CYP2C19*2/CYP3A5*3C) generated from lymphoblastoid cells.

PubMed

Lee, Jaehun; Woo, Dong-Hun; Park, Han-Jin; Park, Kijung; Ko, Duck Sung; Kim, Jong-Hoon

2018-03-01

Cytochrome P450 (CYP) comprises a superfamily of monooxygenase responsible for the metabolism of xenobiotics and approximately 75% of drugs in use today. Thus, genetic polymorphisms in CYP genes contribute to interindividual differences in hepatic metabolism of drugs, affecting on individual drug efficacy and may cause adverse effects. Here, we generated a human induced pluripotent stem cell (hiPSC) line with pharmacologically important traits (CYP2C19*2/CYP3A5*3C), which are highly polymorphic in Asian from lymphoblastoid cells. This hiPSC line could be a valuable source for predicting individual drug responses in the drug screening process that uses hiPSC-derived somatic cells, including hepatocytes. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Attenuation of Hind-Limb Ischemia in Mice with Endothelial-Like Cells Derived from Different Sources of Human Stem Cells

PubMed Central

Chan, Yau-Chi; Ng, Joyce H. L.; Au, Ka-Wing; Wong, Lai-Yung; Siu, Chung-Wah; Tse, Hung-Fat

2013-01-01

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as “off-the-shelf” format for the treatment of tissue ischemia. PMID:23472116

Stem cell-derived kidney cells and organoids: Recent breakthroughs and emerging applications.

PubMed

Chuah, Jacqueline Kai Chin; Zink, Daniele

The global rise in the numbers of kidney patients and the shortage in transplantable organs have led to an increasing interest in kidney-specific regenerative therapies, renal disease modelling and bioartificial kidneys. Sources for large quantities of high-quality renal cells and tissues would be required, also for applications in in vitro platforms for compound safety and efficacy screening. Stem cell-based approaches for the generation of renal-like cells and tissues would be most attractive, but such methods were not available until recently. This situation has drastically changed since 2013, and various protocols for the generation of renal-like cells and precursors from pluripotent stem cells (PSC) have been established. The most recent breakthroughs were related to the establishment of various protocols for the generation of PSC-derived kidney organoids. In combination with recent advances in genome editing, bioprinting and the establishment of predictive renal screening platforms this results in exciting new possibilities. This review will give a comprehensive overview over current PSC-based protocols for the generation of renal-like cells, precursors and organoids, and their current and potential applications in regenerative medicine, compound screening, disease modelling and bioartificial organs. Copyright © 2016 Elsevier Inc. All rights reserved.

An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells.

PubMed

Tcw, Julia; Wang, Minghui; Pimenova, Anna A; Bowles, Kathryn R; Hartley, Brigham J; Lacin, Emre; Machlovi, Saima I; Abdelaal, Rawan; Karch, Celeste M; Phatnani, Hemali; Slesinger, Paul A; Zhang, Bin; Goate, Alison M; Brennand, Kristen J

2017-08-08

Growing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (<30 days) method to generate populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.

PubMed

Yang, Xiulan; Pabon, Lil; Murry, Charles E

2014-01-31

The discovery of human pluripotent stem cells (hPSCs), including both human embryonic stem cells and human-induced pluripotent stem cells, has opened up novel paths for a wide range of scientific studies. The capability to direct the differentiation of hPSCs into functional cardiomyocytes has provided a platform for regenerative medicine, development, tissue engineering, disease modeling, and drug toxicity testing. Despite exciting progress, achieving the optimal benefits has been hampered by the immature nature of these cardiomyocytes. Cardiac maturation has long been studied in vivo using animal models; however, finding ways to mature hPSC cardiomyocytes is only in its initial stages. In this review, we discuss progress in promoting the maturation of the hPSC cardiomyocytes, in the context of our current knowledge of developmental cardiac maturation and in relation to in vitro model systems such as rodent ventricular myocytes. Promising approaches that have begun to be examined in hPSC cardiomyocytes include long-term culturing, 3-dimensional tissue engineering, mechanical loading, electric stimulation, modulation of substrate stiffness, and treatment with neurohormonal factors. Future studies will benefit from the combinatorial use of different approaches that more closely mimic nature's diverse cues, which may result in broader changes in structure, function, and therapeutic applicability.

Overexpression of Polysialylated Neural Cell Adhesion Molecule Improves the Migration Capacity of Induced Pluripotent Stem Cell-Derived Oligodendrocyte Precursors

PubMed Central

Czepiel, Marcin; Leicher, Lasse; Becker, Katja; Boddeke, Erik

2014-01-01

Cell replacement therapy aiming at the compensation of lost oligodendrocytes and restoration of myelination in acquired or congenital demyelination disorders has gained considerable interest since the discovery of induced pluripotent stem cells (iPSCs). Patient-derived iPSCs provide an inexhaustible source for transplantable autologous oligodendrocyte precursors (OPCs). The first transplantation studies in animal models for demyelination with iPSC-derived OPCs demonstrated their survival and remyelinating capacity, but also revealed their limited migration capacity. In the present study, we induced overexpression of the polysialylating enzyme sialyltransferase X (STX) in iPSC-derived OPCs to stimulate the production of polysialic acid-neuronal cell adhesion molecules (PSA-NCAMs), known to promote and facilitate the migration of OPCs. The STX-overexpressing iPSC-derived OPCs showed a normal differentiation and maturation pattern and were able to downregulate PSA-NCAMs when they became myelin-forming oligodendrocytes. After implantation in the demyelinated corpus callosum of cuprizone-fed mice, STX-expressing iPSC-derived OPCs demonstrated a significant increase in migration along the axons. Our findings suggest that the reach and efficacy of iPSC-derived OPC transplantation can be improved by stimulating the OPC migration potential via specific gene modulation. PMID:25069776

Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform.

PubMed

Denning, Chris; Borgdorff, Viola; Crutchley, James; Firth, Karl S A; George, Vinoj; Kalra, Spandan; Kondrashov, Alexander; Hoang, Minh Duc; Mosqueira, Diogo; Patel, Asha; Prodanov, Ljupcho; Rajamohan, Divya; Skarnes, William C; Smith, James G W; Young, Lorraine E

2016-07-01

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Study familial hypertrophic cardiomyopathy using patient-specific induced pluripotent stem cells

PubMed Central

Han, Lu; Li, Yang; Tchao, Jason; Kaplan, Aaron D.; Lin, Bo; Li, You; Mich-Basso, Jocelyn; Lis, Agnieszka; Hassan, Narmeen; London, Barry; Bett, Glenna C.L.; Tobita, Kimimasa; Rasmusson, Randall L.; Yang, Lei

2014-01-01

Aims Familial hypertrophic cardiomyopathy (HCM) is one the most common heart disorders, with gene mutations in the cardiac sarcomere. Studying HCM with patient-specific induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs) would benefit the understanding of HCM mechanism, as well as the development of personalized therapeutic strategies. Methods and results To investigate the molecular mechanism underlying the abnormal CM functions in HCM, we derived iPSCs from an HCM patient with a single missense mutation (Arginine442Glycine) in the MYH7 gene. CMs were next enriched from HCM and healthy iPSCs, followed with whole transcriptome sequencing and pathway enrichment analysis. A widespread increase of genes responsible for ‘Cell Proliferation’ was observed in HCM iPSC-CMs when compared with control iPSC-CMs. Additionally, HCM iPSC-CMs exhibited disorganized sarcomeres and electrophysiological irregularities. Furthermore, disease phenotypes of HCM iPSC-CMs were attenuated with pharmaceutical treatments. Conclusion Overall, this study explored the possible patient-specific and mutation-specific disease mechanism of HCM, and demonstrates the potential of using HCM iPSC-CMs for future development of therapeutic strategies. Additionally, the whole methodology established in this study could be utilized to study mechanisms of other human-inherited heart diseases. PMID:25209314

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

PubMed

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

PubMed Central

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics

PubMed Central

Handel, Adam E.; Chintawar, Satyan; Lalic, Tatjana; Whiteley, Emma; Vowles, Jane; Giustacchini, Alice; Argoud, Karene; Sopp, Paul; Nakanishi, Mahito; Bowden, Rory; Cowley, Sally; Newey, Sarah; Akerman, Colin; Ponting, Chris P.; Cader, M. Zameel

2016-01-01

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells. PMID:26740550

Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy.

PubMed

Cho, Gun-Sik; Lee, Dong I; Tampakakis, Emmanouil; Murphy, Sean; Andersen, Peter; Uosaki, Hideki; Chelko, Stephen; Chakir, Khalid; Hong, Ingie; Seo, Kinya; Chen, Huei-Sheng Vincent; Chen, Xiongwen; Basso, Cristina; Houser, Steven R; Tomaselli, Gordon F; O'Rourke, Brian; Judge, Daniel P; Kass, David A; Kwon, Chulan

2017-01-10

Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy

PubMed Central

Cho, Gun-Sik; Lee, Dong I.; Tampakakis, Emmanouil; Murphy, Sean; Andersen, Peter; Uosaki, Hideki; Chelko, Stephen; Chakir, Khalid; Hong, Ingie; Seo, Kinya; Vincent Chen, Huei-Sheng; Chen, Xiongwen; Basso, Cristina; Houser, Steven R.; Tomaselli, Gordon F.; O’Rourke, Brian; Judge, Daniel P.; Kass, David A.; Kwon, Chulan

2016-01-01

SUMMARY Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of the transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which predominantly manifests in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases. PMID:28076798

Comprehensive protocols for CRISPR/Cas9-based gene editing in human pluripotent stem cells

PubMed Central

Santos, David P.; Kiskinis, Evangelos; Eggan, Kevin; Merkle, Florian T.

2016-01-01

Application of the CRISPR/Cas9 system to edit the genomes of human pluripotent stem cells (hPSCs) has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. PMID:27532820

Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

PubMed Central

Guo, Zongyou; Liu, Liang; Higgins, Claire A.; Christiano, Angela M.

2013-01-01

Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells. PMID:24147053

Fabrication of micropatterned alginate-gelatin and k-carrageenan hydrogels of defined shapes using simple wax mould method as a platform for stem cell/induced Pluripotent Stem Cells (iPSC) culture.

PubMed

Vignesh, S; Gopalakrishnan, Aswathi; M R, Poorna; Nair, Shantikumar V; Jayakumar, R; Mony, Ullas

2018-06-01

Micropatterning techniques involve soft lithography, which is laborious, expensive and restricted to a narrow spectrum of biomaterials. In this work we report, first time employment of patterned wax moulds for generation of micropatterned alginate-gelatin and κ-carrageenan (κ-CRG) hydrogel systems by a novel, simple and cost effective method. We generated and characterized uniform and reproducible micropatterned hydrogels of varying sizes and shapes such as square projections, square grooves, and circular grids and crisscrossed hillocks. The rheological analysis showed that κ-carrageenan hydrogels had higher gel strength when compared to alginate-gelatin hydrogels. Human Mesenchymal stem cells (hMSCs) and Human Induced Pluripotent Stem Cells (hiPSCs) were found to be cytocompatible with these hydrogels. This micropatterned hydrogel system may have potential application in tissue engineering and also in understanding the basic biology behind the stem cell/iPSC fate. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel Surface-Enhanced Raman Scattering-based Assays for Ultra-sensitive Detection of Human Pluripotent Stem Cells

PubMed Central

Han, Jingjia; Qian, Ximei; Wu, Qingling; Jha, Rajneesh; Duan, Jinshuai; Yang, Zhou; Maher, Kevin O.; Nie, Shuming; Xu, Chunhui

2017-01-01

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 106 cells, a sensitivity (0.0001%) which was ~2,000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5+ and TRA-1-60+ cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications. PMID:27509304

Lymphoblast-derived integration-free iPSC line AD-TREM2-3 from a 74 year-old Alzheimer's disease patient expressing the TREM2 p.R47H variant.

PubMed

Martins, Soraia; Yigit, Hatice; Bohndorf, Martina; Graffmann, Nina; Fiszl, Aurelian Robert; Wruck, Wasco; Sleegers, Kristel; Van Broeckhoven, Christine; Adjaye, James

2018-06-01

Human lymphoblast cells from a male diagnosed with Alzheimer's disease (AD) expressing the TREM2 p.R47H variant were used to generate integration-free induced pluripotent stem cells (iPSCs) by over-expressing episomal-based plasmids harbouring OCT4, SOX2, KLF4, LIN28, L-MYC and p53 shRNA. The derived iPSC line - AD-TREM2-3 was defined as pluripotent based on (i) expression of pluripotency-associated markers (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptome of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.940. Copyright © 2018. Published by Elsevier B.V.

Human Embryonic and Induced Pluripotent Stem Cells Express TRAIL Receptors and Can Be Sensitized to TRAIL-Induced Apoptosis

PubMed Central

Vinarsky, Vladimir; Krivanek, Jan; Rankel, Liina; Nahacka, Zuzana; Barta, Tomas; Jaros, Josef; Andera, Ladislav

2013-01-01

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis. PMID:23806100

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Bradycardiac Effects Caused by Co-Administration of Sofosbuvir and Amiodarone.

PubMed

Yu, Yankun; Liu, Feng; He, Liuming; Ramakrishna, Seeram; Zheng, Monica; Bu, Lei; Xu, Ying

2018-05-30

Co-administration of sofosbuvir, an anti-hepatitis C virus medication, and antiarrhythmic amiodarone causes symptomatic severe bradycardia in patients and animal models. However, in a few in vitro studies, the combination of sofosbuvir and amiodarone resulted in tachycardiac effects in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). This discrepancy may be attributable to the use of immature hiPSC-CMs in the in vitro studies. To address this, we evaluated the ability of our in-house hiPSC-CMs to assess the interactions between sofosbuvir and amiodarone in vitro. We performed whole-cell patch recordings on hiPSC-CMs to examine the cardiac effect of sofosbuvir and amiodarone, alone or in combination. We found that sofosbuvir and amiodarone caused bradycardiac effects (the beating rate decreased to 75% of the vehicle control, P < 0.001) on our hiPSC-CMs when applied in combination, but they had no significant effect when applied alone. Furthermore, the bradycardiac effect was membrane potential dependent: it increased with depolarization. This raised the possibility that the bradycardiac effects in vivo may originate in nodal cells, which have a more depolarized resting membrane potential compared with ventricular cells. The bradycardiac effects of sofosbuvir plus amiodarone in vitro are consistent with the clinical phenotype and suggest that our hiPSC-CMs may serve as a useful tool in assessing cardiac safety during drug discovery and development process.

Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

PubMed

Wang, Juan; Hao, Jie; Bai, Donghui; Gu, Qi; Han, Weifang; Wang, Lei; Tan, Yuanqing; Li, Xia; Xue, Ke; Han, Pencheng; Liu, Zhengxin; Jia, Yundan; Wu, Jun; Liu, Lei; Wang, Liu; Li, Wei; Liu, Zhonghua; Zhou, Qi

2015-11-12

Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents. Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III". We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. The clinical-grade hiPSC lines therefore could be valuable sources for future hiPSC-based clinical trials or therapies and for drug screening.

Improved cell therapy protocol for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC and non-human primate iPSC-derived DA neurons

PubMed Central

Maria, Sundberg; Helle, Bogetofte; Tristan, Lawson; Gaynor, Smith; Arnar, Astradsson; Michele, Moore; Teresia, Osborn; Oliver, Cooper; Roger, Spealman; Penelope, Hallett; Ole, Isacson

2013-01-01

The main motor symptoms of Parkinson’s disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson’s disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA-neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for pre-clinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate-iPSC (PiPSC)-derived DA neurons. According to our results, NCAM+/CD29low sorting enriched VM DA-neurons from pluripotent stem cell-derived neural cell populations. NCAM+/CD29low DA-neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM+/CD29low DA-neurons were able to restore motor function of 6-OHDA lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future. PMID:23666606

FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

PubMed

Liu, Yanying; Qiao, Fangfang; Leiferman, Patricia C; Ross, Alan; Schlenker, Evelyn H; Wang, Hongmin

2017-11-15

Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Age Is Relative-Impact of Donor Age on Induced Pluripotent Stem Cell-Derived Cell Functionality.

PubMed

Strässler, Elisabeth Tamara; Aalto-Setälä, Katriina; Kiamehr, Mostafa; Landmesser, Ulf; Kränkel, Nicolle

2018-01-01

Induced pluripotent stem cells (iPSCs) avoid many of the restrictions that hamper the application of human embryonic stem cells: limited availability of source material due to legal restrictions in some countries, immunogenic rejection and ethical concerns. Also, the donor's clinical phenotype is often known when working with iPSCs. Therefore, iPSCs seem ideal to tackle the two biggest tasks of regenerative medicine: degenerative diseases with genetic cause (e.g., Duchenne's muscular dystrophy) and organ replacement in age-related diseases (e.g., end-stage heart or renal failure), especially in combination with recently developed gene-editing tools. In the setting of autologous transplantation in elderly patients, donor age becomes a potentially relevant factor that needs to be assessed. Here, we review and critically discuss available data pertinent to the questions: How does donor age influence the reprogramming process and iPSC functionality? Would it even be possible to reprogram senescent somatic cells? How does donor age affect iPSC differentiation into specialised cells and their functionality? We also identify research needs, which might help resolve current unknowns. Until recently, most hallmarks of ageing were attributed to an accumulation of DNA damage over time, and it was thus expected that DNA damage from a somatic cell would accumulate in iPSCs and the cells derived from them. In line with this, a decreased lifespan of cloned organisms compared with the donor was also observed in early cloning experiments. Therefore, it was questioned for a time whether iPSC derived from an old individual's somatic cells would suffer from early senescence and, thus, may not be a viable option either for disease modelling nor future clinical applications. Instead, typical signs of cellular ageing are reverted in the process of iPSC reprogramming, and iPSCs from older donors do not show diminished differentiation potential nor do iPSC-derived cells from older donors suffer early senescence or show functional impairments when compared with those from younger donors. Thus, the data would suggest that donor age does not limit iPSC application for modelling genetic diseases nor regenerative therapies. However, open questions remain, e.g., regarding the potential tumourigenicity of iPSC-derived cells and the impact of epigenetic pattern retention.

Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells.

PubMed

Sotthibundhu, Areechun; McDonagh, Katya; von Kriegsheim, Alexander; Garcia-Munoz, Amaya; Klawiter, Agnieszka; Thompson, Kerry; Chauhan, Kapil Dev; Krawczyk, Janusz; McInerney, Veronica; Dockery, Peter; Devine, Michael J; Kunath, Tilo; Barry, Frank; O'Brien, Timothy; Shen, Sanbing

2016-11-15

Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined. Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment. We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation. High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.

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.

Characterization of Induced Pluripotent Stem Cell Microvesicle Genesis, Morphology and Pluripotent Content

PubMed Central

Zhou, Jing; Ghoroghi, Shima; Benito-Martin, Alberto; Wu, Hao; Unachukwu, Uchenna John; Einbond, Linda Saxe; Guariglia, Sara; Peinado, Hector; Redenti, Stephen

2016-01-01

Microvesicles (MVs) are lipid bilayer-covered cell fragments that range in diameter from 30 nm–1uM and are released from all cell types. An increasing number of studies reveal that MVs contain microRNA, mRNA and protein that can be detected in the extracellular space. In this study, we characterized induced pluripotent stem cell (iPSC) MV genesis, content and fusion to retinal progenitor cells (RPCs) in vitro. Nanoparticle tracking revealed that iPSCs released approximately 2200 MVs cell/hour in the first 12 hrs with an average diameter of 122 nm. Electron and light microscopic analysis of iPSCs showed MV release via lipid bilayer budding. The mRNA content of iPSC MVs was characterized and revealed the presence of the transcription factors Oct-3/4, Nanog, Klf4, and C-Myc. The protein content of iPSCs MVs, detected by immunogold electron microscopy, revealed the presence of the Oct-3/4 and Nanog. Isolated iPSC MVs were shown to fuse with RPCs in vitro at multiple points along the plasma membrane. These findings demonstrate that the mRNA and protein cargo in iPSC MVs have established roles in maintenance of pluripotency. Building on this work, iPSC derived MVs may be shown to be involved in maintaining cellular pluripotency and may have application in regenerative strategies for neural tissue. PMID:26797168

3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

PubMed

Correia, Cláudia; Koshkin, Alexey; Duarte, Patrícia; Hu, Dongjian; Carido, Madalena; Sebastião, Maria J; Gomes-Alves, Patrícia; Elliott, David A; Domian, Ibrahim J; Teixeira, Ana P; Alves, Paula M; Serra, Margarida

2018-03-01

Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs. © 2017 Wiley Periodicals, Inc.

Dynamic and social behaviors of human pluripotent stem cells.

PubMed

Phadnis, Smruti M; Loewke, Nathan O; Dimov, Ivan K; Pai, Sunil; Amwake, Christine E; Solgaard, Olav; Baer, Thomas M; Chen, Bertha; Reijo Pera, Renee A

2015-09-18

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored.

Dynamic and social behaviors of human pluripotent stem cells

PubMed Central

Phadnis, Smruti M.; Loewke, Nathan O.; Dimov, Ivan K.; Pai, Sunil; Amwake, Christine E.; Solgaard, Olav; Baer, Thomas M.; Chen, Bertha; Pera, Renee A. Reijo

2015-01-01

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored. PMID:26381699

Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells.

PubMed

Simara, Pavel; Tesarova, Lenka; Rehakova, Daniela; Farkas, Simon; Salingova, Barbara; Kutalkova, Katerina; Vavreckova, Eva; Matula, Pavel; Matula, Petr; Veverkova, Lenka; Koutna, Irena

2018-01-01

New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

Human Perivascular Stem Cells Show Enhanced Osteogenesis and Vasculogenesis with Nel-Like Molecule I Protein

PubMed Central

Askarinam, Asal; James, Aaron W.; Zara, Janette N.; Goyal, Raghav; Corselli, Mirko; Pan, Angel; Liang, Pei; Chang, Le; Rackohn, Todd; Stoker, David; Zhang, Xinli; Ting, Kang; Péault, Bruno

2013-01-01

An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34−, and CD45−), which surround capillaries and microvessels, and (2) adventitial cells (CD146−, CD34+, and CD45−), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 μg/μL), hPSC (2.5×105 cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC+NELL-1 in the development of strategies for vascularized bone regeneration. PMID:23406369

The effects of gentamicin and penicillin/streptomycin on the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes in manual patch clamp and multi-electrode array system.

PubMed

Hyun, Soo-Wang; Kim, Bo-Ram; Lin, Dan; Hyun, Sung-Ae; Yoon, Seong Shoon; Seo, Joung-Wook

Cell culture media usually contains antibiotics including gentamicin or penicillin/streptomycin (PS) to protect cells from bacterial contamination. However, little is known about the effects of antibiotics on action potential and field potential parameters in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The present study examined the effects of gentamicin (10, 25, and 50μg/ml) and PS (50, 100, and 200U/μg/ml) on electrophysiological activity in spontaneously beating hiPSC-CMs using manual patch clamp and multi-electrode array. We also measured mRNA expression of cardiac ion channels in hiPSC-CMs grown in media with or without gentamicin (25μg/ml) using reverse transcription-polymerase chain reaction. We recorded action potential and field potential of hiPSC-CMs grown in the presence or absence of gentamicin or PS. We also observed action potential parameters in hiPSC-CMs after short-term treatment with these antibiotics. Changes in action potential and field potential parameters were observed in hiPSC-CMs grown in media containing gentamicin or PS. Treatment with PS also affected action potential parameters in hiPSC-CMs. In addition, the mRNA expression of cardiac sodium and potassium ion channels was significantly attenuated in hiPSC-CMs grown in the presence of gentamicin (25μg/ml). The present findings suggested that gentamicin should not be used in the culture media of hiPSC-CMs used for the measurement of electrophysiological parameters. Our findings also suggest that 100U/100μg/ml of PS are the maximum appropriate concentrations of these antibiotics for recording action potential waveform, because they did not influence action potential parameters in these cells. Copyright © 2017. Published by Elsevier Inc.

Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

2014-01-01

Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection

PubMed Central

Yuan, Lunzhi; Liu, Xuan; Zhang, Liang; Li, Xiaoling; Zhang, Yali; Wu, Kun; Chen, Yao; Cao, Jiali; Hou, Wangheng; Zhang, Jun; Zhu, Hua; Yuan, Quan; Tang, Qiyi; Cheng, Tong; Xia, Ningshao

2018-01-01

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs. Highlights:    (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver.    (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle.    (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo. Research in Context  To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses. PMID:29867819

Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.

PubMed

Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S; Kim, Dong H; Deng, Wenbin; Liu, Ying

2015-12-15

Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.

Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System

PubMed Central

Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S.; Kim, Dong H.; Deng, Wenbin

2015-01-01

Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼33% correctly targeted clones) compared to conventional targeting protocol (∼3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations. PMID:26414932

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.

Engineering of a Potent Recombinant Lectin-Toxin Fusion Protein to Eliminate Human Pluripotent Stem Cells.

PubMed

Tateno, Hiroaki; Saito, Sayoko

2017-07-10

The use of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in regenerative medicine is hindered by their tumorigenic potential. Previously, we developed a recombinant lectin-toxin fusion protein of the hPSC-specific lectin rBC2LCN, which has a 23 kDa catalytic domain (domain III) of Pseudomonas aeruginosa exotoxin A (rBC2LCN-PE23). This fusion protein could selectively eliminate hPSCs following its addition to the cell culture medium. Here we conjugated rBC2LCN lectin with a 38 kDa domain of exotoxin A containing domains Ib and II in addition to domain III (PE38). The developed rBC2LCN-PE38 fusion protein could eliminate 50% of 201B7 hPSCs at a concentration of 0.003 μg/mL (24 h incubation), representing an approximately 556-fold higher activity than rBC2LCN-PE23. Little or no effect on human fibroblasts, human mesenchymal stem cells, and hiPSC-derived hepatocytes was observed at concentrations lower than 1 μg/mL. Finally, we demonstrate that rBC2LCN-PE38 selectively eliminates hiPSCs from a mixed culture of hiPSCs and hiPSC-derived hepatocytes. Since rBC2LCN-PE38 can be prepared from soluble fractions of E. coli culture at a yield of 9 mg/L, rBC2LCN-PE38 represents a practical reagent to remove human pluripotent stem cells residing in cultured cells destined for transplantation.

Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays.

PubMed

Navarrete, Enrique G; Liang, Ping; Lan, Feng; Sanchez-Freire, Verónica; Simmons, Chelsey; Gong, Tingyu; Sharma, Arun; Burridge, Paul W; Patlolla, Bhagat; Lee, Andrew S; Wu, Haodi; Beygui, Ramin E; Wu, Sean M; Robbins, Robert C; Bers, Donald M; Wu, Joseph C

2013-09-10

Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities. Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls. We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

PubMed

Abecasis, Bernardo; Aguiar, Tiago; Arnault, Émilie; Costa, Rita; Gomes-Alves, Patricia; Aspegren, Anders; Serra, Margarida; Alves, Paula M

2017-03-20

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day -1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×10 6 cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. Copyright © 2017 Elsevier B.V. All rights reserved.

Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells.

PubMed

Santos, David P; Kiskinis, Evangelos; Eggan, Kevin; Merkle, Florian T

2016-08-17

Genome editing of human pluripotent stem cells (hPSCs) with the CRISPR/Cas9 system has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

PSA-NCAM-Negative Neural Crest Cells Emerging during Neural Induction of Pluripotent Stem Cells Cause Mesodermal Tumors and Unwanted Grafts

PubMed Central

Lee, Dongjin R.; Yoo, Jeong-Eun; Lee, Jae Souk; Park, Sanghyun; Lee, Junwon; Park, Chul-Yong; Ji, Eunhyun; Kim, Han-Soo; Hwang, Dong-Youn; Kim, Dae-Sung; Kim, Dong-Wook

2015-01-01

Summary Tumorigenic potential of human pluripotent stem cells (hPSCs) is an important issue in clinical applications. Despite many efforts, PSC-derived neural precursor cells (NPCs) have repeatedly induced tumors in animal models even though pluripotent cells were not detected. We found that polysialic acid-neural cell adhesion molecule (PSA-NCAM)− cells among the early NPCs caused tumors, whereas PSA-NCAM+ cells were nontumorigenic. Molecular profiling, global gene analysis, and multilineage differentiation of PSA-NCAM− cells confirm that they are multipotent neural crest stem cells (NCSCs) that could differentiate into both ectodermal and mesodermal lineages. Transplantation of PSA-NCAM− cells in a gradient manner mixed with PSA-NCAM+ cells proportionally increased mesodermal tumor formation and unwanted grafts such as PERIPHERIN+ cells or pigmented cells in the rat brain. Therefore, we suggest that NCSCs are a critical target for tumor prevention in hPSC-derived NPCs, and removal of PSA-NCAM− cells eliminates the tumorigenic potential originating from NCSCs after transplantation. PMID:25937368

Biomarker Discovery by Modeling Behçet's Disease with Patient-Specific Human Induced Pluripotent Stem Cells.

PubMed

Son, Mi-Young; Kim, Young-Dae; Seol, Binna; Lee, Mi-Ok; Na, Hee-Jun; Yoo, Bin; Chang, Jae-Suk; Cho, Yee Sook

2017-01-15

Behçet's disease (BD) is a chronic inflammatory and multisystemic autoimmune disease of unknown etiology. Due to the lack of a specific test for BD, its diagnosis is very difficult and therapeutic options are limited. Induced pluripotent stem cell (iPSC) technology, which provides inaccessible disease-relevant cell types, opens a new era for disease treatment. In this study, we generated BD iPSCs from patient somatic cells and differentiated them into hematopoietic precursor cells (BD iPSC-HPCs) as BD model cells. Based on comparative transcriptome analysis using our BD model cells, we identified eight novel BD-specific genes, AGTR2, CA9, CD44, CXCL1, HTN3, IL-2, PTGER4, and TSLP, which were differentially expressed in BD patients compared with healthy controls or patients with other immune diseases. The use of CXCL1 as a BD biomarker was further validated at the protein level using both a BD iPSC-HPC-based assay system and BD patient serum samples. Furthermore, we show that our BD iPSC-HPC-based drug screening system is highly effective for testing CXCL1 BD biomarkers, as determined by monitoring the efficacy of existing anti-inflammatory drugs. Our results shed new light on the usefulness of patient-specific iPSC technology in the development of a benchmarking platform for disease-specific biomarkers, phenotype- or target-driven drug discovery, and patient-tailored therapies.

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

PubMed

D'Antonio, Matteo; Woodruff, Grace; Nathanson, Jason L; D'Antonio-Chronowska, Agnieszka; Arias, Angelo; Matsui, Hiroko; Williams, Roy; Herrera, Cheryl; Reyna, Sol M; Yeo, Gene W; Goldstein, Lawrence S B; Panopoulos, Athanasia D; Frazer, Kelly A

2017-04-11

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost. Published by Elsevier Inc.

Comparability of automated human induced pluripotent stem cell culture: a pilot study.

PubMed

Archibald, Peter R T; Chandra, Amit; Thomas, Dave; Chose, Olivier; Massouridès, Emmanuelle; Laâbi, Yacine; Williams, David J

2016-12-01

Consistent and robust manufacturing is essential for the translation of cell therapies, and the utilisation automation throughout the manufacturing process may allow for improvements in quality control, scalability, reproducibility and economics of the process. The aim of this study was to measure and establish the comparability between alternative process steps for the culture of hiPSCs. Consequently, the effects of manual centrifugation and automated non-centrifugation process steps, performed using TAP Biosystems' CompacT SelecT automated cell culture platform, upon the culture of a human induced pluripotent stem cell (hiPSC) line (VAX001024c07) were compared. This study, has demonstrated that comparable morphologies and cell diameters were observed in hiPSCs cultured using either manual or automated process steps. However, non-centrifugation hiPSC populations exhibited greater cell yields, greater aggregate rates, increased pluripotency marker expression, and decreased differentiation marker expression compared to centrifugation hiPSCs. A trend for decreased variability in cell yield was also observed after the utilisation of the automated process step. This study also highlights the detrimental effect of the cryopreservation and thawing processes upon the growth and characteristics of hiPSC cultures, and demonstrates that automated hiPSC manufacturing protocols can be successfully transferred between independent laboratories.

Current Advances and Limitations in Modeling ALS/FTD in a Dish Using Induced Pluripotent Stem Cells

PubMed Central

Guo, Wenting; Fumagalli, Laura; Prior, Robert; Van Den Bosch, Ludo

2017-01-01

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two age-dependent multifactorial neurodegenerative disorders, which are typically characterized by the selective death of motor neurons and cerebral cortex neurons, respectively. These two diseases share many clinical, genetic and pathological aspects. During the past decade, cell reprogramming technologies enabled researchers to generate human induced pluripotent stem cells (iPSCs) from somatic cells. This resulted in the unique opportunity to obtain specific neuronal and non-neuronal cell types from patients which could be used for basic research. Moreover, these in vitro models can mimic not only the familial forms of ALS/FTD, but also sporadic cases without known genetic cause. At present, there have been extensive technical advances in the generation of iPSCs, as well as in the differentiation procedures to obtain iPSC-derived motor neurons, cortical neurons and non-neuronal cells. The major challenge at this moment is to determine whether these iPSC-derived cells show relevant phenotypes that recapitulate complex diseases. In this review, we will summarize the work related to iPSC models of ALS and FTD. In addition, we will discuss potential drawbacks and solutions for establishing more trustworthy iPSC models for both ALS and FTD. PMID:29326542

Genome Editing in Induced Pluripotent Stem Cells using CRISPR/Cas9.

PubMed

Ben Jehuda, Ronen; Shemer, Yuval; Binah, Ofer

2018-06-01

The development of the reprogramming technology led to generation of induced Pluripotent Stem Cells (iPSC) from a variety of somatic cells. Ever since, fast growing knowledge of different efficient protocols enabled the differentiation of these iPSCs into different cells types utilized for disease modeling. Indeed, iPSC-derived cells have been increasingly used for investigating molecular and cellular pathophysiological mechanisms underlying inherited diseases. However, a major barrier in the field of iPSC-based disease modeling relies on discriminating between the effects of the causative mutation and the genetic background of these cells. In the past decade, researchers have made great improvement in genome editing techniques, with one of the latest being CRISPR/Cas9. Using a single non-sequence specific protein combined with a small guiding RNA molecule, this state-of-the-art approach enables modifications of genes with high efficiency and accuracy. By so doing, this technique enables the generation of isogenic controls or isogenic mutated cell lines in order to focus on the pathologies caused by a specific mutation. In this article, we review the latest studies combining iPSC and CRISPR/Cas9 technologies for the investigation of the molecular and cellular mechanisms underlying inherited diseases including immunological, metabolic, hematological, neurodegenerative and cardiac diseases.

Modeling and study of the mechanism of dilated cardiomyopathy using induced pluripotent stem cells derived from individuals with Duchenne muscular dystrophy.

PubMed

Lin, Bo; Li, Yang; Han, Lu; Kaplan, Aaron D; Ao, Ying; Kalra, Spandan; Bett, Glenna C L; Rasmusson, Randall L; Denning, Chris; Yang, Lei

2015-05-01

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), and is characterized by progressive weakness in skeletal and cardiac muscles. Currently, dilated cardiomyopathy due to cardiac muscle loss is one of the major causes of lethality in late-stage DMD patients. To study the molecular mechanisms underlying dilated cardiomyopathy in DMD heart, we generated cardiomyocytes (CMs) from DMD and healthy control induced pluripotent stem cells (iPSCs). DMD iPSC-derived CMs (iPSC-CMs) displayed dystrophin deficiency, as well as the elevated levels of resting Ca(2+), mitochondrial damage and cell apoptosis. Additionally, we found an activated mitochondria-mediated signaling network underlying the enhanced apoptosis in DMD iPSC-CMs. Furthermore, when we treated DMD iPSC-CMs with the membrane sealant Poloxamer 188, it significantly decreased the resting cytosolic Ca(2+) level, repressed caspase-3 (CASP3) activation and consequently suppressed apoptosis in DMD iPSC-CMs. Taken together, using DMD patient-derived iPSC-CMs, we established an in vitro model that manifests the major phenotypes of dilated cardiomyopathy in DMD patients, and uncovered a potential new disease mechanism. Our model could be used for the mechanistic study of human muscular dystrophy, as well as future preclinical testing of novel therapeutic compounds for dilated cardiomyopathy in DMD patients. © 2015. Published by The Company of Biologists Ltd.

Induced pluripotent stem cell technology for modelling and therapy of cerebellar ataxia

PubMed Central

Watson, Lauren M.; Wong, Maggie M. K.; Becker, Esther B. E.

2015-01-01

Induced pluripotent stem cell (iPSC) technology has emerged as an important tool in understanding, and potentially reversing, disease pathology. This is particularly true in the case of neurodegenerative diseases, in which the affected cell types are not readily accessible for study. Since the first descriptions of iPSC-based disease modelling, considerable advances have been made in understanding the aetiology and progression of a diverse array of neurodegenerative conditions, including Parkinson's disease and Alzheimer's disease. To date, however, relatively few studies have succeeded in using iPSCs to model the neurodegeneration observed in cerebellar ataxia. Given the distinct neurodevelopmental phenotypes associated with certain types of ataxia, iPSC-based models are likely to provide significant insights, not only into disease progression, but also to the development of early-intervention therapies. In this review, we describe the existing iPSC-based disease models of this heterogeneous group of conditions and explore the challenges associated with generating cerebellar neurons from iPSCs, which have thus far hindered the expansion of this research. PMID:26136256

Murine pluripotent stem cells derived scaffold-free cartilage grafts from a micro-cavitary hydrogel platform.

PubMed

He, Pengfei; Fu, Jiayin; Wang, Dong-An

2016-04-15

By means of appropriate cell type and scaffold, tissue-engineering approaches aim to construct grafts for cartilage repair. Pluripotent stem cells especially induced pluripotent stem cells (iPSCs) are of promising cell candidates due to the pluripotent plasticity and abundant cell source. We explored three dimensional (3D) culture and chondrogenesis of murine iPSCs (miPSCs) on an alginate-based micro-cavity hydrogel (MCG) platform in pursuit of fabricating synthetic-scaffold-free cartilage grafts. Murine embryonic stem cells (mESCs) were employed in parallel as the control. Chondrogenesis was fulfilled using a consecutive protocol via mesoderm differentiation followed by chondrogenic differentiation; subsequently, miPSC and mESC-seeded constructs were further respectively cultured in chondrocyte culture (CC) medium. Alginate phase in the constructs was then removed to generate a graft only comprised of induced chondrocytic cells and cartilaginous extracellular matrix (ECMs). We found that from the mESC-seeded constructs, formation of intact grafts could be achieved in greater sizes with relatively fewer chondrocytic cells and abundant ECMs; from miPSC-seeded constructs, relatively smaller sized cartilaginous grafts could be formed by cells with chondrocytic phenotype wrapped by abundant and better assembled collagen type II. This study demonstrated successful creation of pluripotent stem cells-derived cartilage/chondroid graft from a 3D MCG interim platform. By the support of materials and methodologies established from this study, particularly given the autologous availability of iPSCs, engineered autologous cartilage engraftment may be potentially fulfilled without relying on the limited and invasive autologous chondrocytes acquisition. In this study, we explored chondrogenic differentiation of pluripotent stem cells on a 3D micro-cavitary hydrogel interim platform and creation of pluripotent stem cells-derived cartilage/chondroid graft via a consecutive procedure. Our results demonstrated chondrogenic differentiation could be realized on the platform via mesoderm differentiation. The mESCs/miPSCs derived chondrocytic cells were further cultured to finally generate a pluripotent stem cells-derived scaffold-free construct based on the micro-cavitary hydrogel platform, in which alginate hydrogel could be removed finally. Our results showed that miPSC-derived graft could be formed by cells with chondrocytic phenotype wrapped by abundant and assembled collagen type II. To our knowledge, this study is the first study that initials from pluripotent stem cell seeding on 3D scaffold environment and ends with a scaffold-free chondrogenic micro-tissue. By the support of materials and methodologies established from this study, engineered autologous iPSC-derived cartilage engraftment may be potentially developed instead of autologous chondrocytes grafts that have limited source. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

PubMed

Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

2016-01-01

The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

HOXB7 overexpression in lung cancer is a hallmark of acquired stem-like phenotype.

PubMed

Monterisi, Simona; Lo Riso, Pietro; Russo, Karin; Bertalot, Giovanni; Vecchi, Manuela; Testa, Giuseppe; Di Fiore, Pier Paolo; Bianchi, Fabrizio

2018-03-26

HOXB7 is a homeodomain (HOX) transcription factor involved in regional body patterning of invertebrates and vertebrates. We previously identified HOXB7 within a ten-gene prognostic signature for lung adenocarcinoma, where increased expression of HOXB7 was associated with poor prognosis. This raises the question of how HOXB7 overexpression can influence the metastatic behavior of lung adenocarcinoma. Here, we analyzed publicly available microarray and RNA-seq lung cancer expression datasets and found that HOXB7-overexpressing tumors are enriched in gene signatures characterizing adult and embryonic stem cells (SC), and induced pluripotent stem cells (iPSC). Experimentally, we found that HOXB7 upregulates several canonical SC/iPSC markers and sustains the expansion of a subpopulation of cells with SC characteristics, through modulation of LIN28B, an emerging cancer gene and pluripotency factor, which we discovered to be a direct target of HOXB7. We validated this new circuit by showing that HOXB7 enhances reprogramming to iPSC with comparable efficiency to LIN28B or its target c-MYC, which is a canonical reprogramming factor.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

PubMed

D'Aiuto, Leonardo; Zhi, Yun; Kumar Das, Dhanjit; Wilcox, Madeleine R; Johnson, Jon W; McClain, Lora; MacDonald, Matthew L; Di Maio, Roberto; Schurdak, Mark E; Piazza, Paolo; Viggiano, Luigi; Sweet, Robert; Kinchington, Paul R; Bhattacharjee, Ayantika G; Yolken, Robert; Nimgaonka, Vishwajit L; Nimgaonkar, Vishwajit L

2014-01-01

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET.

PubMed

Shalom-Feuerstein, Ruby; Serror, Laura; Aberdam, Edith; Müller, Franz-Josef; van Bokhoven, Hans; Wiman, Klas G; Zhou, Huiqing; Aberdam, Daniel; Petit, Isabelle

2013-02-05

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.

A Simple and Robust Method for Culturing Human-Induced Pluripotent Stem Cells in an Undifferentiated State Using Botulinum Hemagglutinin.

PubMed

Kim, Mee-Hae; Matsubara, Yoshifumi; Fujinaga, Yukako; Kino-Oka, Masahiro

2018-02-01

Clinical and industrial applications of human-induced pluripotent stem cells (hiPSCs) is hindered by the lack of robust culture strategies capable of sustaining a culture in an undifferentiated state. Here, a simple and robust hiPSC-culture-propagation strategy incorporating botulinum hemagglutinin (HA)-mediated selective removal of cells deviating from an undifferentiated state is developed. After HA treatment, cell-cell adhesion is disrupted, and deviated cells detached from the central region of the colony to subsequently form tight monolayer colonies following prolonged incubation. The authors find that the temporal and dose-dependent activity of HA regulated deviated-cell removal and recoverability after disruption of cell-cell adhesion in hiPSC colonies. The effects of HA are confirmed under all culture conditions examined, regardless of hiPSC line and feeder-dependent or -free culture conditions. After routine application of our HA-treatment paradigm for serial passages, hiPSCs maintains expression of pluripotent markers and readily forms embryoid bodies expressing markers for all three germ-cell layers. This method enables highly efficient culturing of hiPSCs and use of entire undifferentiated portions without having to pick deviated cells manually. This simple and readily reproducible culture strategy is a potentially useful tool for improving the robust and scalable maintenance of undifferentiated hiPSC cultures. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermo-responsive polymeric nanoparticles for enhancing neuronal differentiation of human induced pluripotent stem cells.

PubMed

Seo, Hye In; Cho, Ann-Na; Jang, Jiho; Kim, Dong-Wook; Cho, Seung-Woo; Chung, Bong Geun

2015-10-01

We report thermo-responsive retinoic acid (RA)-loaded poly(N-isopropylacrylamide)-co-acrylamide (PNIPAM-co-Am) nanoparticles for directing human induced pluripotent stem cell (hiPSC) fate. Fourier transform infrared spectroscopy and (1)H nuclear magnetic resonance analysis confirmed that RA was efficiently incorporated into PNIAPM-co-Am nanoparticles (PCANs). The size of PCANs dropped with increasing temperatures (300-400 nm at room temperature, 80-90 nm at 37°C) due to its phase transition from hydrophilic to hydrophobic. Due to particle shrinkage caused by this thermo-responsive property of PCANs, RA could be released from nanoparticles in the cells upon cellular uptake. Immunocytochemistry and quantitative real-time polymerase chain reaction analysis demonstrated that neuronal differentiation of hiPSC-derived neuronal precursors was enhanced after treatment with 1-2 μg/ml RA-loaded PCANs. Therefore, we propose that this PCAN could be a potentially powerful carrier for effective RA delivery to direct hiPSC fate to neuronal lineage. The use of induced pluripotent stem cells (iPSCs) has been at the forefront of research in the field of regenerative medicine, as these cells have the potential to differentiate into various terminal cell types. In this article, the authors utilized a thermo-responsive polymer, Poly(N-isopropylacrylamide) (PNIPAM), as a delivery platform for retinoic acid. It was shown that neuronal differentiation could be enhanced in hiPSC-derived neuronal precursor cells. This method may pave a way for future treatment of neuronal diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

PubMed

Guo, Liang; Eldridge, Sandy; Furniss, Mike; Mussio, Jodie; Davis, Myrtle

2015-09-01

There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. Copyright © 2015 John Wiley & Sons, Inc.

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

PubMed Central

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

2017-01-01

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

Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix—A Comparative Analysis of Bioartificial Liver Microenvironments

PubMed Central

Wang, Bo; Jakus, Adam E.; Baptista, Pedro M.; Soker, Shay; Soto-Gutierrez, Alejandro; Abecassis, Michael M.; Shah, Ramille N.

2016-01-01

Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-l-lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a “deconstructed” control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, α-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery. Significance Through the use of novel technology to develop three-dimensional (3D) scaffolds, the present study demonstrated that hepatocyte-like cells derived via induced pluripotent stem cell (iPSC) technology mature on 3D extracellular matrix scaffolds as a result of 3D matrix structure and scaffold biology. The result is an improved hepatic phenotype with increased synthetic and catalytic potency, an improvement on the blunted phenotype of iPSC-derived hepatocytes, a critical limitation of iPSC technology. These findings provide insight into the influence of 3D microenvironments on the viability, proliferation, and function of iPSC hepatocytes to yield a more mature population of cells for cell toxicity studies and disease modeling. PMID:27421950

Novel surface-enhanced Raman scattering-based assays for ultra-sensitive detection of human pluripotent stem cells.

PubMed

Han, Jingjia; Qian, Ximei; Wu, Qingling; Jha, Rajneesh; Duan, Jinshuai; Yang, Zhou; Maher, Kevin O; Nie, Shuming; Xu, Chunhui

2016-10-01

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 10(6) cells, a sensitivity (0.0001%) which was ∼2000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5(+) and TRA-1-60(+) cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

PubMed Central

Ma, Xuanyi; Qu, Xin; Zhu, Wei; Li, Yi-Shuan; Yuan, Suli; Zhang, Hong; Liu, Justin; Wang, Pengrui; Lai, Cheuk Sun Edwin; Zanella, Fabian; Feng, Gen-Sheng; Sheikh, Farah; Chien, Shu; Chen, Shaochen

2016-01-01

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling. PMID:26858399

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting.

PubMed

Ma, Xuanyi; Qu, Xin; Zhu, Wei; Li, Yi-Shuan; Yuan, Suli; Zhang, Hong; Liu, Justin; Wang, Pengrui; Lai, Cheuk Sun Edwin; Zanella, Fabian; Feng, Gen-Sheng; Sheikh, Farah; Chien, Shu; Chen, Shaochen

2016-02-23

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.

Induced Accelerated Aging in Induced Pluripotent Stem Cell Lines from Patients with Parkinson’s Disease

DTIC Science & Technology

2014-07-01

after manipulation of the cells prohibited this approach. 2. Differentiation into oligoprecursor cells ( OPCs ) and oligodendrocytes As we have...Jing Bian, PhD and Birgitt Schuele, MD 18 Development of expandable OPCs from human iPSC derived...Neri et al., 2010) + + + + Human iPSC derived OPCs and Oligos (Wang et al., 2013) + + + + mEpsc derived OPCs and Oligos (Najm et al

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

PubMed Central

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-01-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

PubMed

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-10-14

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

Exosomes from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stromal Cells (hiPSC-MSCs) Protect Liver against Hepatic Ischemia/ Reperfusion Injury via Activating Sphingosine Kinase and Sphingosine-1-Phosphate Signaling Pathway.

PubMed

Du, Yingdong; Li, Dawei; Han, Conghui; Wu, Haoyu; Xu, Longmei; Zhang, Ming; Zhang, Jianjun; Chen, Xiaosong

2017-01-01

This study aimed to evaluate the effects of exosomes produced by human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs-Exo) on hepatic ischemia-reperfusion (I/R) injury, as well as the underlying mechanisms. Exosomes derived from hiPSC-MSCs were isolated and characterized both biochemically and biophysically. hiPSC-MSCs-Exo were injected systemically into a murine ischemia/reperfusion injury model via the inferior vena cava, and then the therapeutic effects were evaluated. The serum levels of transaminases (aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as histological changes were examined. Primary hepatocytes and human hepatocyte cell line HL7702 were used to test whether exosomes could induce hepatocytes proliferation in vitro. In addition, the expression levels of proliferation markers (proliferation cell nuclear antigen, PCNA; Phosphohistone-H3, PHH3) were measured by immunohistochemistry and Western blot. Moreover, SK inhibitor (SKI-II) and S1P1 receptor antagonist (VPC23019) were used to investigate the role of sphingosine kinase and sphingosine-1-phosphate-dependent pathway in the effects of hiPSC-MSCs-Exo on hepatocytes. hiPSCs were efficiently induced into hiPSC-MSCs that had typical MSC characteristics. hiPSC-MSCs-Exo had diameters ranging from 100 to 200 nm and expressed exosome markers (Alix, CD63 and CD81). After hiPSC-MSCs-Exo administration, hepatocyte necrosis and sinusoidal congestion were markedly suppressed in the ischemia/reperfusion injury model, with lower histopathological scores. The levels of hepatocyte injury markers AST and ALT were significantly lower in the treatment group compared to control, and the expression levels of proliferation markers (PCNA and PHH3) were greatly induced after hiPSC-MSCs-Exo administration. Moreover, hiPSC-MSCs-Exo also induced primary hepatocytes and HL7702 cells proliferation in vitro in a dose-dependent manner. We found that hiPSC-MSCs-Exo could directly fuse with target hepatocytes or HL7702 cells and increase the activity of sphingosine kinase and synthesis of sphingosine-1-phosphate (S1P). Furthermore, the inhibition of SK1 or S1P1 receptor completely abolished the protective and proliferative effects of hiPSC-MSCs-Exo on hepatocytes, both in vitro and in vivo. Our results demonstrated that hiPSC-MSCs-Exo could alleviate hepatic I/R injury via activating sphingosine kinase and sphingosine-1-phosphate pathway in hepatocytes and promote cell proliferation. These findings represent a novel mechanism that potentially contributes to liver regeneration and have important implications for new therapeutic approaches to acute liver disease. © 2017 The Author(s). Published by S. Karger AG, Basel.

New medium used in the differentiation of human pluripotent stem cells to retinal cells is comparable to fetal human eye tissue.

PubMed

Wang, Xiaobing; Xiong, Kai; Lin, Cong; Lv, Lei; Chen, Jing; Xu, Chongchong; Wang, Songtao; Gu, Dandan; Zheng, Hua; Yu, Hurong; Li, Yan; Xiao, Honglei; Zhou, Guomin

2015-06-01

Human pluripotent stem cells (hPSCs) have the potential to differentiate along the retinal lineage. However, most induction systems are dependent on multiple small molecular compounds such as Dkk-1, Lefty-A, and retinoic acid. In the present study, we efficiently differentiated hPSCs into retinal cells using a retinal differentiation medium (RDM) without the use of small molecular compounds. This novel differentiation system recapitulates retinal morphogenesis in humans, i.e. hPSCs gradually differentiate into optic vesicle-shaped spheres, followed by optic cup-shaped spheres and, lastly, retinal progenitor cells. Furthermore, at different stages, hPSC-derived retinal cells mirror the transcription factor expression profiles seen in their counterparts during human embryogenesis. Most importantly, hinge epithelium was found between the hPSC-derived neural retina (NR) and retinal pigment epithelium (RPE). These data suggest that our culture system provides a new method for generating hPSC-derived retinal cells that, for the first time, might be used in human transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tailoring Mathematical Models to Stem-Cell Derived Cardiomyocyte Lines Can Improve Predictions of Drug-Induced Changes to Their Electrophysiology.

PubMed

Lei, Chon Lok; Wang, Ken; Clerx, Michael; Johnstone, Ross H; Hortigon-Vinagre, Maria P; Zamora, Victor; Allan, Andrew; Smith, Godfrey L; Gavaghan, David J; Mirams, Gary R; Polonchuk, Liudmila

2017-01-01

Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in disease modeling, cell therapy, drug screening and personalized medicine. Computational models can be used to interpret experimental findings in iPSC-CMs, provide mechanistic insights, and translate these findings to adult cardiomyocyte (CM) electrophysiology. However, different cell lines display different expression of ion channels, pumps and receptors, and show differences in electrophysiology. In this exploratory study, we use a mathematical model based on iPSC-CMs from Cellular Dynamic International (CDI, iCell), and compare its predictions to novel experimental recordings made with the Axiogenesis Cor.4U line. We show that tailoring this model to the specific cell line, even using limited data and a relatively simple approach, leads to improved predictions of baseline behavior and response to drugs. This demonstrates the need and the feasibility to tailor models to individual cell lines, although a more refined approach will be needed to characterize individual currents, address differences in ion current kinetics, and further improve these results.

Modeling Viral Infectious Diseases and Development of Antiviral Therapies Using Human Induced Pluripotent Stem Cell-Derived Systems.

PubMed

Trevisan, Marta; Sinigaglia, Alessandro; Desole, Giovanna; Berto, Alessandro; Pacenti, Monia; Palù, Giorgio; Barzon, Luisa

2015-07-13

The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host-pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

PubMed Central

Hawkins, Kate E.; Joy, Shona; Delhove, Juliette M.K.M.; Kotiadis, Vassilios N.; Fernandez, Emilio; Fitzpatrick, Lorna M.; Whiteford, James R.; King, Peter J.; Bolanos, Juan P.; Duchen, Michael R.; Waddington, Simon N.; McKay, Tristan R.

2016-01-01

Summary The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation. PMID:26904936

Integrity of Induced Pluripotent Stem Cell (iPSC) Derived Megakaryocytes as Assessed by Genetic and Transcriptomic Analysis

PubMed Central

Kammers, Kai; Taub, Margaret A.; Ruczinski, Ingo; Martin, Joshua; Yanek, Lisa R.; Frazee, Alyssa; Gao, Yongxing; Hoyle, Dixie; Faraday, Nauder; Becker, Diane M.; Cheng, Linzhao; Wang, Zack Z.; Leek, Jeff T.; Becker, Lewis C.; Mathias, Rasika A.

2017-01-01

Previously, we have described our feeder-free, xeno-free approach to generate megakaryocytes (MKs) in culture from human induced pluripotent stem cells (iPSCs). Here, we focus specifically on the integrity of these MKs using: (1) genotype discordance between parent cell DNA to iPSC cell DNA and onward to the differentiated MK DNA; (2) genomic structural integrity using copy number variation (CNV); and (3) transcriptomic signatures of the derived MK lines compared to the iPSC lines. We detected a very low rate of genotype discordance; estimates were 0.0001%-0.01%, well below the genotyping error rate for our assay (0.37%). No CNVs were generated in the iPSCs that were subsequently passed on to the MKs. Finally, we observed highly biologically relevant gene sets as being upregulated in MKs relative to the iPSCs: platelet activation, blood coagulation, megakaryocyte development, platelet formation, platelet degranulation, and platelet aggregation. These data strongly support the integrity of the derived MK lines. PMID:28107356

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).

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Stem Cell Banking: A Global View.

PubMed

Stacey, Glyn

2017-01-01

Stem cell banking has been a topic of discussion and debate for more than a decade since the first public services to supply human embryonic stem cells (hESCs) were established in the USA and the UK. This topic has received a recent revival with numerous ambitious programmes announced to deliver large collections of human induced pluripotency cell (hiPSC) lines. This chapter will provide a brief overview charting the development of stem cell banks, their value, and their likely role in the future.

Generation of Induced Pluripotent Stem Cells and Neural Stem/Progenitor Cells from Newborns with Spina Bifida Aperta.

PubMed

Bamba, Yohei; Nonaka, Masahiro; Sasaki, Natsu; Shofuda, Tomoko; Kanematsu, Daisuke; Suemizu, Hiroshi; Higuchi, Yuichiro; Pooh, Ritsuko K; Kanemura, Yonehiro; Okano, Hideyuki; Yamasaki, Mami

2017-12-01

We established induced pluripotent stem cells (iPSCs) and neural stem/progenitor cells (NSPCs) from three newborns with spina bifida aperta (SBa) using clinically practical methods. We aimed to develop stem cell lines derived from newborns with SBa for future therapeutic use. SBa is a common congenital spinal cord abnormality that causes defects in neurological and urological functions. Stem cell transplantation therapies are predicted to provide beneficial effects for patients with SBa. However, the availability of appropriate cell sources is inadequate for clinical use because of their limited accessibility and expandability, as well as ethical issues. Fibroblast cultures were established from small fragments of skin obtained from newborns with SBa during SBa repair surgery. The cultured cells were transfected with episomal plasmid vectors encoding reprogramming factors necessary for generating iPSCs. These cells were then differentiated into NSPCs by chemical compound treatment, and NSPCs were expanded using neurosphere technology. We successfully generated iPSC lines from the neonatal dermal fibroblasts of three newborns with SBa. We confirmed that these lines exhibited the characteristics of human pluripotent stem cells. We successfully generated NSPCs from all SBa newborn-derived iPSCs with a combination of neural induction and neurosphere technology. We successfully generated iPSCs and iPSC-NSPCs from surgical samples obtained from newborns with SBa with the goal of future clinical use in patients with SBa.

Patient-Specific Human Induced Pluripotent Stem Cell Model Assessed with Electrical Pacing Validates S107 as a Potential Therapeutic Agent for Catecholaminergic Polymorphic Ventricular Tachycardia

PubMed Central

Sasaki, Kenichi; Makiyama, Takeru; Yoshida, Yoshinori; Wuriyanghai, Yimin; Kamakura, Tsukasa; Nishiuchi, Suguru; Hayano, Mamoru; Harita, Takeshi; Yamamoto, Yuta; Kohjitani, Hirohiko; Hirose, Sayako; Chen, Jiarong; Kawamura, Mihoko; Ohno, Seiko; Itoh, Hideki; Takeuchi, Ayako; Matsuoka, Satoshi; Miura, Masaru; Sumitomo, Naokata; Horie, Minoru; Yamanaka, Shinya; Kimura, Takeshi

2016-01-01

Introduction Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies. Method and Results We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 μM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05). Conclusions We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs. PMID:27764147

Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing.

PubMed

Tasnim, Farah; Toh, Yi-Chin; Qu, Yinghua; Li, Huan; Phan, Derek; Narmada, Balakrishnan C; Ananthanarayanan, Abhishek; Mittal, Nikhil; Meng, Ryan Q; Yu, Hanry

2016-06-06

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications.

Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration

PubMed Central

Masumoto, Hidetoshi; Ikuno, Takeshi; Takeda, Masafumi; Fukushima, Hiroyuki; Marui, Akira; Katayama, Shiori; Shimizu, Tatsuya; Ikeda, Tadashi; Okano, Teruo; Sakata, Ryuzo; Yamashita, Jun K.

2014-01-01

To realize cardiac regeneration using human induced pluripotent stem cells (hiPSCs), strategies for cell preparation, tissue engineering and transplantation must be explored. Here we report a new protocol for the simultaneous induction of cardiomyocytes (CMs) and vascular cells [endothelial cells (ECs)/vascular mural cells (MCs)], and generate entirely hiPSC-engineered cardiovascular cell sheets, which showed advantageous therapeutic effects in infarcted hearts. The protocol adds to a previous differentiation protocol of CMs by using stage-specific supplementation of vascular endothelial cell growth factor for the additional induction of vascular cells. Using this cell sheet technology, we successfully generated physically integrated cardiac tissue sheets (hiPSC-CTSs). HiPSC-CTS transplantation to rat infarcted hearts significantly improved cardiac function. In addition to neovascularization, we confirmed that engrafted human cells mainly consisted of CMs in >40% of transplanted rats four weeks after transplantation. Thus, our HiPSC-CTSs show promise for cardiac regenerative therapy. PMID:25336194

Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

PubMed

Maruotti, Julien; Sripathi, Srinivas R; Bharti, Kapil; Fuller, John; Wahlin, Karl J; Ranganathan, Vinod; Sluch, Valentin M; Berlinicke, Cynthia A; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z; Bhutto, Imran; Lutty, Gerard A; Zack, Donald J

2015-09-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

PubMed Central

Maruotti, Julien; Sripathi, Srinivas R.; Bharti, Kapil; Fuller, John; Wahlin, Karl J.; Ranganathan, Vinod; Sluch, Valentin M.; Berlinicke, Cynthia A.; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z.; Bhutto, Imran; Lutty, Gerard A.; Zack, Donald J.

2015-01-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. PMID:26269569

Pompe Disease Results in a Golgi-based Glycosylation Deficit in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes*

PubMed Central

Raval, Kunil K.; Tao, Ran; White, Brent E.; De Lange, Willem J.; Koonce, Chad H.; Yu, Junying; Kishnani, Priya S.; Thomson, James A.; Mosher, Deane F.; Ralphe, John C.; Kamp, Timothy J.

2015-01-01

Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation. PMID:25488666

The potential of induced pluripotent stem cells as a tool to study skeletal dysplasias and cartilage-related pathologic conditions.

PubMed

Liu, H; Yang, L; Yu, F F; Wang, S; Wu, C; Qu, C; Lammi, M J; Guo, X

2017-05-01

The development of induced pluripotent stem cells (iPSCs) technology has opened up new horizons for development of new research tools especially for skeletal dysplasias, which often lack human disease models. Regenerative medicine and tissue engineering could be the next areas to benefit from refinement of iPSC methods to repair focal cartilage defects, while applications for osteoarthritis (OA) and drug screening have evolved rather slowly. Although the advances in iPSC research of skeletal dysplasias and repair of focal cartilage lesions are not directly relevant to OA, they can be considered to pave the way to future prospects and solutions to OA research, too. The same problems which face the present cell-based treatments of cartilage injuries concern also the iPSC-based ones. However, established iPSC lines, which have no genomic aberrations and which efficiently differentiate into extracellular matrix secreting chondrocytes, could be an invaluable cell source for cell transplantations in the future. The safety issues concerning the recipient risks of teratoma formation and immune response still have to be solved before the potential use of iPSCs in cartilage repair of focal cartilage defects and OA. Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Pluripotent stem cells.

PubMed

Verfaillie, C

2009-05-01

The isolation of human embryonic stem cells (ESC) in 1998 has created the hope that stem cells will one day be used to regenerate tissues and organs, even though it is obvious that a number of hurdles will need to be overcome for such therapies to become reality. The cloning of "Dolly" in 1997, more than 40 years after the first frogs were cloned, combined with the very fast progress made in our understanding of the molecular processes that govern the pluripotency of ESC has lead to the ability of scientists to recreate a pluripotent state in fibroblasts and other cells from mouse, rat and man, named induced pluripotent stem cells (iPSC). This feat makes it theoretically possible to create patient specific pluripotent stem cells whose differentiated progeny could be used in an autologous manner obviating the need for immunosuppression that would be needed to use allogeneic ESC-derived differentiated cells. In addition, the ability to generate custom made pluripotent stem cells will no doubt lead to the development of protein or small molecule drugs that can induce differentiation not only of iPSC or ESC to mature tissue cells, but also endogenous tissue stem cells. Moreover, it allows scientists to create models of human diseases and may aid the pharmaceutical industry in testing more rigorously toxicity of drugs for human differentiated cells. Thus, there is little doubt that progress in stem cell biology will change many aspects of medicine as we know it in the next one to two decades.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor.

PubMed

Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2016-01-01

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 10(6) hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

PubMed Central

Freyer, Nora; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Schrade, Petra; Bachmann, Sebastian; Damm, Georg; Seehofer, Daniel; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2016-01-01

Abstract The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p < 0.05) higher in 2D cultures, while secretion of albumin, a typical characteristic for mature hepatocytes, was higher after hepatic differentiation of hiPSC in 3D bioreactors. Functional analysis of multiple cytochrome P450 (CYP) isoenzymes showed activity of CYP1A2, CYP2B6, and CYP3A4 in both groups, although at a lower level compared to primary human hepatocytes (PHH). CYP2B6 activities were significantly (p < 0.05) higher in 3D bioreactors compared with 2D cultures, which is in line with results from gene expression. Immunofluorescence staining showed that the majority of cells was positive for albumin, cytokeratin 18 (CK18), and hepatocyte nuclear factor 4-alpha (HNF4A) at the end of the differentiation process. In addition, cytokeratin 19 (CK19) staining revealed the formation of bile duct-like structures in 3D bioreactors similar to native liver tissue. The results indicate a better maturation of hiPSC in the 3D bioreactor system compared to 2D cultures and emphasize the potential of dynamic 3D culture systems in stem cell differentiation approaches for improved formation of differentiated tissue structures. PMID:27610270

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

PubMed

Canfield, Scott G; Stebbins, Matthew J; Morales, Bethsymarie Soto; Asai, Shusaku W; Vatine, Gad D; Svendsen, Clive N; Palecek, Sean P; Shusta, Eric V

2017-03-01

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843. © 2016 International Society for Neurochemistry.

Improvement of Carbon Tetrachloride-Induced Acute Hepatic Failure by Transplantation of Induced Pluripotent Stem Cells without Reprogramming Factor c-Myc

PubMed Central

Chang, Hua-Ming; Liao, Yi-Wen; Chiang, Chih-Hung; Chen, Yi-Jen; Lai, Ying-Hsiu; Chang, Yuh-Lih; Chen, Hen-Li; Jeng, Shaw-Yeu; Hsieh, Jung-Hung; Peng, Chi-Hsien; Li, Hsin-Yang; Chien, Yueh; Chen, Szu-Yu; Chen, Liang-Kung; Huo, Teh-Ia

2012-01-01

The only curative treatment for hepatic failure is liver transplantation. Unfortunately, this treatment has several major limitations, as for example donor organ shortage. A previous report demonstrated that transplantation of induced pluripotent stem cells without reprogramming factor c-Myc (3-genes iPSCs) attenuates thioacetamide-induced hepatic failure with minimal incidence of tumorigenicity. In this study, we investigated whether 3-genes iPSC transplantation is capable of rescuing carbon tetrachloride (CCl4)-induced fulminant hepatic failure and hepatic encephalopathy in mice. Firstly, we demonstrated that 3-genes iPSCs possess the capacity to differentiate into hepatocyte-like cells (iPSC-Heps) that exhibit biological functions and express various hepatic specific markers. 3-genes iPSCs also exhibited several antioxidant enzymes that prevented CCl4-induced reactive oxygen species production and cell death. Intraperitoneal transplantation of either 3-genes iPSCs or 3-genes iPSC-Heps significantly reduced hepatic necrotic areas, improved hepatic functions, and survival rate in CCl4-treated mice. CCl4-induced hepatic encephalopathy was also improved by 3-genes iPSC transplantation. Hoechst staining confirmed the successful engraftment of both 3-genes iPSCs and 3-genes iPSC-Heps, indicating the homing properties of these cells. The most pronounced hepatoprotective effect of iPSCs appeared to originate from the highest antioxidant activity of 3-gene iPSCs among all transplanted cells. In summary, our findings demonstrated that 3-genes iPSCs serve as an available cell source for the treatment of an experimental model of acute liver diseases. PMID:22489170

Long-Distance Axonal Growth from Human Induced Pluripotent Stem Cells After Spinal Cord Injury

PubMed Central

Lu, Paul; Woodruff, Grace; Wang, Yaozhi; Graham, Lori; Hunt, Matt; Wu, Di; Boehle, Eileen; Ahmad, Ruhel; Poplawski, Gunnar; Brock, John; Goldstein, Lawrence S. B.; Tuszynski, Mark H.

2014-01-01

Human induced pluripotent stem cells (iPSCs) from a healthy 86 year-old male were differentiated into neural stem cells and grafted into adult immunodeficient rats after spinal cord injury. Three months after C5 lateral hemisections, iPSCs survived and differentiated into neurons and glia, and extended tens of thousands of axons from the lesion site over virtually the entire length of the rat central nervous system. These iPSC-derived axons extended through adult white matter of the injured spinal cord, frequently penetrating gray matter and forming synapses with rat neurons. In turn, host supraspinal motor axons penetrated human iPSC grafts and formed synapses. These findings indicate that intrinsic neuronal mechanisms readily overcome the inhibitory milieu of the adult injured spinal cord to extend many axons over very long distances; these capabilities persist even in neurons reprogrammed from very aged human cells. PMID:25123310

Potential of Induced Pluripotent Stem Cells (iPSCs) for Treating Age-Related Macular Degeneration (AMD).

PubMed

Fields, Mark; Cai, Hui; Gong, Jie; Del Priore, Lucian

2016-12-08

The field of stem cell biology has rapidly evolved in the last few decades. In the area of regenerative medicine, clinical applications using stem cells hold the potential to be a powerful tool in the treatment of a wide variety of diseases, in particular, disorders of the eye. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are promising technologies that can potentially provide an unlimited source of cells for cell replacement therapy in the treatment of retinal degenerative disorders such as age-related macular degeneration (AMD), Stargardt disease, and other disorders. ESCs and iPSCs have been used to generate retinal pigment epithelium (RPE) cells and their functional behavior has been tested in vitro and in vivo in animal models. Additionally, iPSC-derived RPE cells provide an autologous source of cells for therapeutic use, as well as allow for novel approaches in disease modeling and drug development platforms. Clinical trials are currently testing the safety and efficacy of these cells in patients with AMD. In this review, the current status of iPSC disease modeling of AMD is discussed, as well as the challenges and potential of this technology as a viable option for cell replacement therapy in retinal degeneration.

Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells

PubMed Central

Eggenschwiler, Reto; Wichmann, Christian; Buhmann, Raymund; Cantz, Tobias

2017-01-01

Kindlin-2 is a multidomain intracellular protein that can be recruited to β-integrin domains to activate signaling, initiate transcriptional programs, and bind to E-cadherin. To explore its involvement in cell fate decisions in mesenchymal cells, we studied the effects of Kindlin-2 modification (overexpression/knockdown) in induced pluripotent cell-derived mesenchymal stromal cells (iPSC-MSCs). Kindlin-2 overexpression resulted in increased proliferation and reduced apoptosis of iPSC-MSCs, as well as inhibition of their differentiation towards osteocytes, adipocytes, and chondrocytes. In contrast, siRNA-mediated Kindlin-2 knockdown induced increased apoptosis and increased differentiation response in iPSC-MSCs. The ability of iPSC-MSCs to adhere to VCAM-1/SDF-1α under shear stress and to migrate in a wound scratch assay was significantly increased after Kindlin-2 overexpression. In contrast, inhibition of mixed lymphocyte reaction (MLR) was generally independent of Kindlin-2 modulation in iPSC-MSCs, except for decreased production of interleukin-2 (IL-2) after Kindlin-2 overexpression in iPS-MSCs. Thus, Kindlin-2 upregulates survival, proliferation, stemness, and migration potential in iPSC-MSCs and may therefore be beneficial in optimizing performance of iPSC-MSC in therapies. PMID:28163724

Nanowires and Electrical Stimulation Synergistically Improve Functions of hiPSC Cardiac Spheroids.

PubMed

Richards, Dylan J; Tan, Yu; Coyle, Robert; Li, Yang; Xu, Ruoyu; Yeung, Nelson; Parker, Arran; Menick, Donald R; Tian, Bozhi; Mei, Ying

2016-07-13

The advancement of human induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology has shown promising potential to provide a patient-specific, regenerative cell therapy strategy to treat cardiovascular disease. Despite the progress, the unspecific, underdeveloped phenotype of hiPSC-CMs has shown arrhythmogenic risk and limited functional improvements after transplantation. To address this, tissue engineering strategies have utilized both exogenous and endogenous stimuli to accelerate the development of hiPSC-CMs. Exogenous electrical stimulation provides a biomimetic pacemaker-like stimuli that has been shown to advance the electrical properties of tissue engineered cardiac constructs. Recently, we demonstrated that the incorporation of electrically conductive silicon nanowires to hiPSC cardiac spheroids led to advanced structural and functional development of hiPSC-CMs by improving the endogenous electrical microenvironment. Here, we reasoned that the enhanced endogenous electrical microenvironment of nanowired hiPSC cardiac spheroids would synergize with exogenous electrical stimulation to further advance the functional development of nanowired hiPSC cardiac spheroids. For the first time, we report that the combination of nanowires and electrical stimulation enhanced cell-cell junction formation, improved development of contractile machinery, and led to a significant decrease in the spontaneous beat rate of hiPSC cardiac spheroids. The advancements made here address critical challenges for the use of hiPSC-CMs in cardiac developmental and translational research and provide an advanced cell delivery vehicle for the next generation of cardiac repair.

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

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

Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana

2014-10-15

According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro.more » Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.« less

The stem cell laboratory: design, equipment, and oversight.

PubMed

Wesselschmidt, Robin L; Schwartz, Philip H

2011-01-01

This chapter describes some of the major issues to be considered when setting up a laboratory for the culture of human pluripotent stem cells (hPSCs). The process of establishing a hPSC laboratory can be divided into two equally important parts. One is completely administrative and includes developing protocols, seeking approval, and establishing reporting processes and documentation. The other part of establishing a hPSC laboratory involves the physical plant and includes design, equipment and personnel. Proper planning of laboratory operations and proper design of the physical layout of the stem cell laboratory so that meets the scope of planned operations is a major undertaking, but the time spent upfront will pay long-term returns in operational efficiency and effectiveness. A well-planned, organized, and properly equipped laboratory supports research activities by increasing efficiency and reducing lost time and wasted resources.

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

PubMed

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

2017-01-01

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

Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

PubMed

Stacey, Glyn N; Coecke, Sandra; Price, Anna-Bal; Healy, Lyn; Jennings, Paul; Wilmes, Anja; Pinset, Christian; Ingelman-Sundberg, Magnus; Louisse, Jochem; Haupt, Simone; Kidd, Darren; Robitski, Andrea; Jahnke, Heinz-Georg; Lemaitre, Gilles; Myatt, Glenn

Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes. Also addressed is the development of hPSC-based assays for the tissue and cell types of greatest interest in drug toxicology. The chapter draws on a range of expert opinion within the European Commission/Cosmetics Europe-funded alternative testing cluster SEURAT-1 and consensus from international groups delivering this guidance such as the International Stem Cell Banking Initiative. Accordingly, the chapter summarizes the most up-date best practices in the use and quality control of human Pluripotent Stem Cell lines in the development of in vitro toxicity assays from leading experts in the field.

Establishment of induced pluripotent stem cell (iPSC) line from 55-year old male patient with hemorrhagic Moyamoya disease.

PubMed

Cardano, Marina; Marsoner, Fabio; Marcatili, Matteo; Karnavas, Thodoris; Zasso, Jacopo; Lanterna, Luigi Andrea; Conti, Luciano

2016-11-01

Peripheral blood mononuclear cells (PBMCs) were collected from 55-year old male patient with a confirmed diagnosis of hemorrhagic Moyamoya disease (MMD). PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. A footprint-free hiPSC line was characterized by the expression of pluripotency markers and a normal karyotype. These cells were able to give rise to Embryoid Bodies and to a progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a suitable tool for modelling in vitro MMD disease to investigate the cellular mechanisms underlying the occurrence of this pathology. Copyright © 2016. Published by Elsevier B.V.

Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats.

PubMed

Xiao, Mei; An, LiLong; Yang, XueYi; Ge, Xin; Qiao, Hai; Zhao, Ting; Ma, XiaoFei; Fan, JingZhuang; Zhu, MengYang; Dou, ZhongYing

2008-09-01

The major obstacle in using pancreatic islet transplantation to cure type I and some type II diabetes is the shortage of the donors. One of ways to overcome such obstacle is to isolate and clone pancreatic stem cells as "seed cells" and induce their differentiation into functional islets as an abundant transplantation source. In this study, a monoclonal human pancreatic stem cell (mhPSC) line was obtained from abortive fetal pancreatic tissues. Pancreatic tissues were taken from abortive fetus by sterile procedures, and digested into single cells and cell clusters with 0.1% type IV collagenase. Cultured in modified glucose-low DMEM with 10% fetal bovine serum (FBS), these single cells and cell clusters adhered to culture dishes, and then primary epidermal-like pancreatic stem cells started to clone. After digesting with 0.25% trypsin and 0.04% EDTA, fibroblasts and other cells were gradually eliminated and epithelioid pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the mhPSCs were obtained. After addition of 10 ng/mL epidermal growth factor (EGF) in cell culture medium, the mhPSCs quickly grew and formed a gravelstone-like monolayer. Continuously proliferated, a mhPSC line, which was derived from a male abortive fetus of 4 months old, has been passed through 50 generations. More than 1 x 10(9) mhPSCs were cryo-preserved in liquid nitrogen. Karyotype analysis showed that the chromosome set of the mhPSC line was normal diploid. Immunocytochemistry results demonstrated that the mhPSC line was positive for the pdx1, glucagon, nestin and CK19, and negative for the insulin, CD34, CD44 and CD45 protein expression. RT-PCR revealed further that the mhPSCs expressed transcription factors of the pdx1, glucagon, nestin and CK19. Also, in vitro induced with beta-mercaptoethanol, the mhPSCs differentiated into nerve cells that expressed the NF protein. Induced with nicotinamide, the mhPSCs differentiated into functional islet-like clusters, as identified by dithizone staining, which expressed the transcription factor of the insulin and secreted the insulin and C-peptide. Furthermore, the transplantation of mhPSCs-induced pancreatic islets into the subcapsular region of the kidney in streptozotocin-induced diabetic rats could reduce blood glucose levels and prolong the life time.

Induced Pluripotent Stem Cells: Generation, Characterization, and Differentiation--Methods and Protocols.

PubMed

Graversen, Veronica Kon; Chavala, Sai H

2016-01-01

Reprogramming fibroblasts into induced pluripotent stem cells (iPSC) remains a promising technique for cell replacement therapy. Diverse populations of somatic cells have been examined for their reprogramming potential. Recently, ocular ciliary body epithelial cells (CECs) have been reprogrammed with high reprogramming efficiency and single transcription factor reprogramming, making them an exciting candidate for cellular reprogramming strategies.

Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies.

PubMed

Abbasalizadeh, Saeed; Baharvand, Hossein

2013-12-01

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing. © 2013.

Induced Pluripotent Stem Cells from Patients with Huntington’s Disease Show CAG Repeat Expansion Associated Phenotypes

PubMed Central

Mattis, Virginia B; Svendsen, Soshana P; Ebert, Allison; Svendsen, Clive N; King, Alvin R; Casale, Malcolm; Winokur, Sara T; Batugedara, Gayani; Vawter, Marquis; Donovan, Peter J; Lock, Leslie F; Thompson, Leslie M; Zhu, Yu; Fossale, Elisa; Singh Atwal, Ranjit; Gillis, Tammy; Mysore, Jayalakshmi; Li, Jian-hong; Seong, IhnSik; Shen, Yiping; Chen, Xiaoli; Wheeler, Vanessa C; MacDonald, Marcy E; Gusella, James F; Akimov, Sergey; Arbez, Nicolas; Juopperi, Tarja; Ratovitski, Tamara; Chiang, Jason H; Kim, Woon Roung; Chighladze, Eka; Watkin, Erin; Zhong, Chun; Makri, Georgia; Cole, Robert N; Margolis, Russell L; Song, Hongjun; Ming, Guoli; Ross, Christopher A; Kaye, Julia A; Daub, Aaron; Sharma, Punita; Mason, Amanda R; Finkbeiner, Steven; Yu, Junying; Thomson, James A; Rushton, David; Brazier, Stephen P; Battersby, Alysia A; Redfern, Amanda; Tseng, Hsui-Er; Harrison, Alexander W; Kemp, Paul J; Allen, Nicholas D; Onorati, Marco; Castiglioni, Valentina; Cattaneo, Elena; Arjomand, Jamshid

2013-01-01

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, the HD consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a novel human stem cell platform for screening new candidate therapeutics. PMID:22748968

Comparing ESC and iPSC-Based Models for Human Genetic Disorders.

PubMed

Halevy, Tomer; Urbach, Achia

2014-10-24

Traditionally, human disorders were studied using animal models or somatic cells taken from patients. Such studies enabled the analysis of the molecular mechanisms of numerous disorders, and led to the discovery of new treatments. Yet, these systems are limited or even irrelevant in modeling multiple genetic diseases. The isolation of human embryonic stem cells (ESCs) from diseased blastocysts, the derivation of induced pluripotent stem cells (iPSCs) from patients' somatic cells, and the new technologies for genome editing of pluripotent stem cells have opened a new window of opportunities in the field of disease modeling, and enabled studying diseases that couldn't be modeled in the past. Importantly, despite the high similarity between ESCs and iPSCs, there are several fundamental differences between these cells, which have important implications regarding disease modeling. In this review we compare ESC-based models to iPSC-based models, and highlight the advantages and disadvantages of each system. We further suggest a roadmap for how to choose the optimal strategy to model each specific disorder.

Cultural relativism: maintenance of genomic imprints in pluripotent stem cell culture systems.

PubMed

Greenberg, Maxim Vc; Bourc'his, Déborah

2015-04-01

Pluripotent stem cells (PSCs) in culture have become a widely used model for studying events occurring during mammalian development; they also present an exciting avenue for therapeutics. However, compared to their in vivo counterparts, cultured PSC derivatives have unique properties, and it is well established that their epigenome is sensitive to medium composition. Here we review the specific effects on genomic imprints in various PSC types and culture systems. Imprinted gene regulation is developmentally important, and imprinting defects have been associated with several human diseases. Therefore, imprint abnormalities in PSCs may have considerable consequences for downstream applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience.

PubMed

De Sousa, Paul A; Steeg, Rachel; Wachter, Elisabeth; Bruce, Kevin; King, Jason; Hoeve, Marieke; Khadun, Shalinee; McConnachie, George; Holder, Julie; Kurtz, Andreas; Seltmann, Stefanie; Dewender, Johannes; Reimann, Sascha; Stacey, Glyn; O'Shea, Orla; Chapman, Charlotte; Healy, Lyn; Zimmermann, Heiko; Bolton, Bryan; Rawat, Trisha; Atkin, Isobel; Veiga, Anna; Kuebler, Bernd; Serano, Blanca Miranda; Saric, Tomo; Hescheler, Jürgen; Brüstle, Oliver; Peitz, Michael; Thiele, Cornelia; Geijsen, Niels; Holst, Bjørn; Clausen, Christian; Lako, Majlinda; Armstrong, Lyle; Gupta, Shailesh K; Kvist, Alexander J; Hicks, Ryan; Jonebring, Anna; Brolén, Gabriella; Ebneth, Andreas; Cabrera-Socorro, Alfredo; Foerch, Patrik; Geraerts, Martine; Stummann, Tina C; Harmon, Shawn; George, Carol; Streeter, Ian; Clarke, Laura; Parkinson, Helen; Harrison, Peter W; Faulconbridge, Adam; Cherubin, Luca; Burdett, Tony; Trigueros, Cesar; Patel, Minal J; Lucas, Christa; Hardy, Barry; Predan, Rok; Dokler, Joh; Brajnik, Maja; Keminer, Oliver; Pless, Ole; Gribbon, Philip; Claussen, Carsten; Ringwald, Annette; Kreisel, Beate; Courtney, Aidan; Allsopp, Timothy E

2017-04-01

A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. ETOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

PubMed

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B Cicero; Zarzoso, Manuel; Ramirez, Rafael J; Sener, Michelle F; Mundada, Lakshmi V; Klos, Matthew; Devaney, Eric J; Vikstrom, Karen L; Herron, Todd J; Jalife, José

2013-11-01

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. © 2013.

Self-organized amniogenesis by human pluripotent stem cells in a biomimetic implantation-like niche

NASA Astrophysics Data System (ADS)

Shao, Yue; Taniguchi, Kenichiro; Gurdziel, Katherine; Townshend, Ryan F.; Xue, Xufeng; Yong, Koh Meng Aw; Sang, Jianming; Spence, Jason R.; Gumucio, Deborah L.; Fu, Jianping

2017-04-01

Amniogenesis--the development of amnion--is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine.

Pluripotent cell models of fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors.

PubMed

Suzuki, Naoya M; Niwa, Akira; Yabe, Miharu; Hira, Asuka; Okada, Chihiro; Amano, Naoki; Watanabe, Akira; Watanabe, Ken-Ichiro; Heike, Toshio; Takata, Minoru; Nakahata, Tatsutoshi; Saito, Megumu K

2015-04-01

Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities, and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six patients with FA and FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF. ©AlphaMed Press.

Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

PubMed

Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

2016-05-15

Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. Copyright © 2015 Elsevier Inc. All rights reserved.

Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform.

PubMed

Sharma, Arun; Marceau, Caleb; Hamaguchi, Ryoko; Burridge, Paul W; Rajarajan, Kuppusamy; Churko, Jared M; Wu, Haodi; Sallam, Karim I; Matsa, Elena; Sturzu, Anthony C; Che, Yonglu; Ebert, Antje; Diecke, Sebastian; Liang, Ping; Red-Horse, Kristy; Carette, Jan E; Wu, Sean M; Wu, Joseph C

2014-08-29

Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. This study examined whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonβ1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonβ1 treatment. This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion. © 2014 American Heart Association, Inc.

Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential

PubMed Central

Hohenstein Elliott, Kristi A.; Peterson, Cory; Soundararajan, Anuradha; Kan, Natalia; Nelson, Brandon; Spiering, Sean; Mercola, Mark; Bright, Gary R.

2012-01-01

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications. PMID:22701128

Livestock models for exploiting the promise of pluripotent stem cells.

PubMed

Roberts, R Michael; Yuan, Ye; Genovese, Nicholas; Ezashi, Toshihiko

2015-01-01

Livestock species are widely used as biomedical models. Pigs, in particular, are beginning to have a significant role in regenerative medicine for testing the applicability, success, and safety of grafts derived from induced pluripotent stem cells. Animal testing must always be performed before any clinical trials are performed in humans, and pigs may sometimes be the species of choice because of their physiological and anatomical similarities to humans. Induced pluripotent stem cells (iPSC) have been generated with some success from livestock species by a variety of reprogramming procedures, but authenticated embryonic stem cells (ESC) have not. There are now several studies in which porcine iPSC have been tested for their ability to provide functional grafts in pigs. Pigs have also served as recipients for grafts derived from human iPSC. There have also been recent advances in creating pigs with severe combined immunodeficiency (SCID). Like SCID mice, these pigs are expected to be graft tolerant. Additionally, chimeric, partially humanized pigs could be sources of human organs. Another potential application of pluripotent stem cells from livestock is for the purpose of differentiating the cells into skeletal muscle, which, in turn, could be used either to produce cultured meat or to engraft into damaged muscle. None of these technologies has advanced to a stage that they have become mainstream, however. Despite the value of livestock models in regenerative medicine, only a limited number of institutions are able to use these animals. © The Author 2015. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Human Perivascular Stem Cell-Based Bone Graft Substitute Induces Rat Spinal Fusion

PubMed Central

Chung, Choon G.; James, Aaron W.; Asatrian, Greg; Chang, Le; Nguyen, Alan; Le, Khoi; Bayani, Georgina; Lee, Robert; Stoker, David; Zhang, Xinli

2014-01-01

Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair. PMID:25154782

Disease characterization using LQTS-specific induced pluripotent stem cells.

PubMed

Egashira, Toru; Yuasa, Shinsuke; Suzuki, Tomoyuki; Aizawa, Yoshiyasu; Yamakawa, Hiroyuki; Matsuhashi, Tomohiro; Ohno, Yohei; Tohyama, Shugo; Okata, Shinichiro; Seki, Tomohisa; Kuroda, Yusuke; Yae, Kojiro; Hashimoto, Hisayuki; Tanaka, Tomofumi; Hattori, Fumiyuki; Sato, Toshiaki; Miyoshi, Shunichiro; Takatsuki, Seiji; Murata, Mitsushige; Kurokawa, Junko; Furukawa, Tetsushi; Makita, Naomasa; Aiba, Takeshi; Shimizu, Wataru; Horie, Minoru; Kamiya, Kaichiro; Kodama, Itsuo; Ogawa, Satoshi; Fukuda, Keiichi

2012-09-01

Long QT syndrome (LQTS) is an inheritable and life-threatening disease; however, it is often difficult to determine disease characteristics in sporadic cases with novel mutations, and more precise analysis is necessary for the successful development of evidence-based clinical therapies. This study thus sought to better characterize ion channel cardiac disorders using induced pluripotent stem cells (iPSCs). We reprogrammed somatic cells from a patient with sporadic LQTS and from controls, and differentiated them into cardiomyocytes through embryoid body (EB) formation. Electrophysiological analysis of the LQTS-iPSC-derived EBs using a multi-electrode array (MEA) system revealed a markedly prolonged field potential duration (FPD). The IKr blocker E4031 significantly prolonged FPD in control- and LQTS-iPSC-derived EBs and induced frequent severe arrhythmia only in LQTS-iPSC-derived EBs. The IKs blocker chromanol 293B did not prolong FPD in the LQTS-iPSC-derived EBs, but significantly prolonged FPD in the control EBs, suggesting the involvement of IKs disturbance in the patient. Patch-clamp analysis and immunostaining confirmed a dominant-negative role for 1893delC in IKs channels due to a trafficking deficiency in iPSC-derived cardiomyocytes and human embryonic kidney (HEK) cells. This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.

Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro.

PubMed

Hyysalo, Anu; Ristola, Mervi; Mäkinen, Meeri E-L; Häyrynen, Sergei; Nykter, Matti; Narkilahti, Susanna

2017-10-01

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511-E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also for the production of clinical grade hPSC-derived cells for regenerative medicine applications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

PubMed

Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

2017-10-02

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

PubMed

O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

2017-03-01

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Periodontal Biology: Stem Cells, Bmp2 Gene, Transcriptional Enhancers, and Use of Sclerostin Antibody and Pth for Treatment of Periodontal Disease and Bone Loss

PubMed Central

Harris, Stephen E; Rediske, Michael; Neitzke, Rebecca; Rakian, Audrey

2017-01-01

The periodontium is a complex tissue with epithelial components and a complex set of mesodermal derived alveolar bone, cellular and a cellular cementum, and tendon like ligaments (PDL). The current evidence demonstrates that the major pool of periodontal stem cells is derived from a population of micro vascular associated aSMA-positive stem/progenitor (PSC) cells that by lineage tracing form all three major mesodermal derived components of the periodontium. With in vitro aSMA+ stem cells, transcriptome and chip- seq experiments, the gene network and enhancer maps were determined at several differentiation states of the PSC. Current work on the role of the Bmp2 gene in the periodontal stem cell differentiation demonstrated that this Wnt regulated gene, Bmp2, is necessary for differentiation to all three major mesodermal derived component of the periodontium. The mechanism and use of Sclerostin antibody as an activator of Wnt signaling and Bmp2 gene as a potential route to treat craniofacial bone loss is discussed. As well, the mechanism and use of Pth in the treatment of periodontal bone loss or other craniofacial bone loss is presented in this review. PMID:29457146

Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells as an Individual-Specific and Renewable Source of Adult Stem Cells.

PubMed

Sequiera, Glen Lester; Saravanan, Sekaran; Dhingra, Sanjiv

2017-01-01

This chapter deals with the employment of human-induced pluripotent stem cells (hiPSCs) as a candidate to differentiate into mesenchymal stem cells (MSCs). This would enable to help establish a regular source of human MSCs with the aim of avoiding the problems associated with procuring the MSCs either from different healthy individuals or patients, limited extraction potentials, batch-to-batch variations or from diverse sources such as bone marrow or adipose tissue. The procedures described herein allow for a guided and ensured approach for the regular maintenance of hiPSCs and their subsequent differentiation into MSCs using the prescribed medium. Subsequently, an easy protocol for the successive isolation and purification of the hiPSC-differentiated MSCs is outlined, which is carried out through passaging and can be further sorted through flow cytometry. Further, the maintenance and expansion of the resultant hiPSC-differentiated MSCs using appropriate characterization techniques, i.e., Reverse-transcription PCR and immunostaining is also elaborated. The course of action has been deliberated keeping in mind the awareness and the requisites available to even beginner researchers who mostly have access to regular consumables and medium components found in the general laboratory.

Sensitivity of hiPSC-derived neural stem cells (NSC) to Pyrroloquinoline quinone depends on their developmental stage.

PubMed

Augustyniak, J; Lenart, J; Zychowicz, M; Lipka, G; Gaj, P; Kolanowska, M; Stepien, P P; Buzanska, L

2017-12-01

Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the "developmental window" of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point. Copyright © 2017 Elsevier Ltd. All rights reserved.

Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

PubMed

El Harane, Nadia; Kervadec, Anaïs; Bellamy, Valérie; Pidial, Laetitia; Neametalla, Hany J; Perier, Marie-Cécile; Lima Correa, Bruna; Thiébault, Léa; Cagnard, Nicolas; Duché, Angéline; Brunaud, Camille; Lemitre, Mathilde; Gauthier, Jeanne; Bourdillon, Alexandra T; Renault, Marc P; Hovhannisyan, Yeranuhi; Paiva, Solenne; Colas, Alexandre R; Agbulut, Onnik; Hagège, Albert; Silvestre, Jean-Sébastien; Menasché, Philippe; Renault, Nisa K E

2018-05-21

We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways. Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.

Self-Organized Cerebellar Tissue from Human Pluripotent Stem Cells and Disease Modeling with Patient-Derived iPSCs.

PubMed

Muguruma, Keiko

2018-02-01

Recent advances in the techniques that differentiate induced pluripotent stem cells (iPSCs) into specific types of cells enabled us to establish in vitro cell-based models as a platform for drug discovery. iPSC-derived disease models are advantageous to generation of a large number of cells required for high-throughput screening. Furthermore, disease-relevant cells differentiated from patient-derived iPSCs are expected to recapitulate the disorder-specific pathogenesis and physiology in vitro. Such disease-relevant cells will be useful for developing effective therapies. We demonstrated that cerebellar tissues are generated from human PSCs (hPSCs) in 3D culture systems that recapitulate the in vivo microenvironments associated with the isthmic organizer. Recently, we have succeeded in generation of spinocerebellar ataxia (SCA) patient-derived Purkinje cells by combining the iPSC technology and the self-organizing stem cell 3D culture technology. We demonstrated that SCA6-derived Purkinje cells exhibit vulnerability to triiodothyronine depletion, which is suppressed by treatment with thyrotropin-releasing hormone and Riluzole. We further discuss applications of patient-specific iPSCs to intractable cerebellar disease.

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.

Gingival Fibroblasts as Autologous Feeders for Induced Pluripotent Stem Cells.

PubMed

Yu, G; Okawa, H; Okita, K; Kamano, Y; Wang, F; Saeki, M; Yatani, H; Egusa, H

2016-01-01

Human gingival fibroblasts (hGFs) present an attractive source of induced pluripotent stem cells (iPSCs), which are expected to be a powerful tool for regenerative dentistry. However, problems to be addressed prior to clinical application include the use of animal-derived feeder cells for cultures. The aim of this study was to establish an autologous hGF-derived iPSC (hGF-iPSC) culture system by evaluating the feeder ability of hGFs. In both serum-containing and serum-free media, hGFs showed higher proliferation than human dermal fibroblasts (hDFs). Three hGF strains were isolated under serum-free conditions, although 2 showed impaired proliferation. When hGF-iPSCs were transferred onto mitomycin C-inactivated hGFs, hDFs, or mouse-derived SNL feeders, hGF and SNL feeders were clearly hGF-iPSC supportive for more than 50 passages, whereas hDF feeders were only able to maintain undifferentiated hGF-iPSC growth for a few passages. After 20 passages on hGF feeders, embryonic stem cell marker expression and CpG methylation at the NANOG and OCT3/4 promoters were similar for hGF-iPSCs cultured on hGF and SNL feeder cells. Long-term cultures of hGF-iPSCs on hGF feeders sustained their normal karyotype and pluripotency. On hGF feeders, hGF-iPSC colonies were surrounded by many colony-derived fibroblast-like cells, and the size of intact colonies at 7 d after passage was significantly larger than that on SNL feeders. Allogeneic hGF strains also maintained hGF-iPSCs for 10 passages. Compared with hDFs, hGFs showed a higher production of laminin-332, laminin α5 chain, and insulin-like growth factor-II, which have been reported to sustain the long-term self-renewal of pluripotent stem cells. These results suggest that hGFs possess an excellent feeder capability and thus can be used as alternatives to conventional mouse-derived SNL and hDF feeders. In addition, our findings suggest that hGF feeders are promising candidates for animal component-free ex vivo expansion of autologous hGF-iPSCs, thus providing an important step toward the future therapeutic application of hGF-iPSCs. © International & American Associations for Dental Research 2015.

The Stem Cell Laboratory: Design, Equipment, and Oversight

PubMed Central

Wesselschmidt, Robin L.; Schwartz, Philip H.

2013-01-01

This chapter describes some of the major issues to be considered when setting up a laboratory for the culture of human pluripotent stem cells (hPSCs). The process of establishing a hPSC laboratory can be divided into two equally important parts. One is completely administrative and includes developing protocols, seeking approval, and establishing reporting processes and documentation. The other part of establishing a hPSC laboratory involves the physical plant and includes design, equipment and personnel. Proper planning of laboratory operations and proper design of the physical layout of the stem cell laboratory so that meets the scope of planned operations is a major undertaking, but the time spent upfront will pay long-term returns in operational efficiency and effectiveness. A well-planned, organized, and properly equipped laboratory supports research activities by increasing efficiency and reducing lost time and wasted resources. PMID:21822863

Comparison of electrophysiological data from human-induced pluripotent stem cell-derived cardiomyocytes to functional preclinical safety assays.

PubMed

Harris, Kate; Aylott, Mike; Cui, Yi; Louttit, James B; McMahon, Nicholas C; Sridhar, Arun

2013-08-01

Human-induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are a potential source to develop assays for predictive electrophysiological safety screening. Published studies show that the relevant physiology and pharmacology exist but does not show the translation between stem cell cardiomyocyte assays and other preclinical safety screening assays, which is crucial for drug discovery and safety scientists and the regulators. Our studies are the first to show the pharmacology of ion channel blockade and compare them with existing functional cardiac electrophysiology studies. Ten compounds (a mixture of pure hERG [E-4031 and Cisapride], hERG and sodium [Flecainide, Mexiletine, Quinidine, and Terfenadine], calcium channel blockers [Nifedipine and Verapamil], and two proprietary compounds [GSK A and B]) were tested, and results from hiPSC-CMs studied on multielectrode arrays (MEA) were compared with other preclincial models and clinical drug concentrations and effects using integrated risk assessment plots. All ion channel blockers produced (1) functional effects on repolarization and depolarization around the IC25 and IC50 values and (2) excessive blockade of hERG and/or blockade of sodium current precipitated arrhythmias. Our MEA data show that hiPSC-CMs demonstrate relevant pharmacology and show excellent correlations to current functional cardiac electrophysiological studies. Based on these results, MEA assays using iPSC-CMs offer a reliable, cost effective, and surrogate to preclinical in vitro testing, in addition to the 3Rs (refine, reduce, and replace animals in research) benefit.

Electrophysiological characteristics and pharmacological sensitivity of two lines of human induced pluripotent stem cell derived cardiomyocytes coming from two different suppliers.

PubMed

Goineau, Sonia; Castagné, Vincent

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as preclinical tool for predicting drug-induced QT prolongation and arrhythmias. This study was conducted to assess the electrophysiological characteristics and the pharmacological sensitivity of two commercialized hiPSC-CMs. The baseline electrophysiological characteristics measured with a multi-electrode array (MEA) technology differ between Cor.4U and iCell 2 : higher beat rate (+32bpm) and shorter field potential duration (FPD, -201ms) for Cor.4U. The FPD lengthening after cisapride (100nM: +65% versus +18%), quinidine (10μM: +65% versus +31%), sotalol (30μM: +90% versus +47%) or flecainide (3μM: +76% versus +22%) application appeared earlier in iCell 2 as compared to Cor.4U. Arrhythmia occurrence also appeared earlier in iCell 2 as compared to Cor.4U for the 3 substances mentioned above. The FPD shortening recorded after verapamil or nifedipine application was similar in both hiPSC-CMs. In conclusion, Cor.4U and iCell 2 hiPSC-CMs are both sensitive enough to detect drug-induced delayed or shortened repolarization and arrhythmia and can provide useful predictive cardiac electrophysiology data. Arrhythmias occurred at concentrations higher than clinical free maximum plasma concentrations with an overestimation of the risk with cisapride. However, quantitative differences of baseline electrophysiological characteristics or pharmacological sensitivity of both cell types have to be considered with caution during the interpretation of data. The new chemical entities included within a given drug development program should be evaluated in hiPSC-CMs coming from a single supplier. Copyright © 2017 Elsevier Inc. All rights reserved.

Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

PubMed

Herron, Todd J; Rocha, Andre Monteiro Da; Campbell, Katherine F; Ponce-Balbuena, Daniela; Willis, B Cicero; Guerrero-Serna, Guadalupe; Liu, Qinghua; Klos, Matt; Musa, Hassan; Zarzoso, Manuel; Bizy, Alexandra; Furness, Jamie; Anumonwo, Justus; Mironov, Sergey; Jalife, José

2016-04-01

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers. © 2016 American Heart Association, Inc.

Generation of induced pluripotent stem cells from a patient with Best Dystrophy carrying 11q12.3 (BEST1 (VMD2)) mutation.

PubMed

Hsu, Chih-Chien; Lu, Huai-En; Chuang, Jen-Hua; Ko, Yu-Ling; Tsai, Yi-Ching; Tai, Hsiao-Yun; Yarmishyn, Aliaksandr A; Hwang, De-Kuang; Wang, Mong-Lien; Yang, Yi-Ping; Chen, Shih-Jen; Peng, Chi-Hsien; Chiou, Shih-Hwa; Lin, Tai-Chi

2018-04-03

Best disease (BD), also termed Best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and central visual loss. In this report, we generated an induced pluripotent stem cell (iPSC) line, TVGH-iPSC-012-04, from the peripheral blood mononuclear cells of a female patient with BD by using the Sendai virus delivery system. The resulting iPSCs retained the disease-causing DNA mutation, expressed pluripotent markers and could differentiate into three germ layers. We believe that BD patient-specific iPSCs provide a powerful in vitro model for evaluating the pathological phenotypes of the disease. Copyright © 2018. Published by Elsevier B.V.

The iCRISPR platform for rapid genome editing in human pluripotent stem cells.

PubMed

Zhu, Zengrong; González, Federico; Huangfu, Danwei

2014-01-01

Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), "scarless" introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single-stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.

Economic 3D-printing approach for transplantation of human stem cell-derived β-like cells

PubMed Central

Song, Jiwon; Millman, Jeffrey R.

2016-01-01

Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing β cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived β (SC-β) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-β cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-β cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies. PMID:27906687

Slow Cooling Cryopreservation Optimized to Human Pluripotent Stem Cells.

PubMed

Miyazaki, Takamichi; Suemori, Hirofumi

2016-01-01

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cells that form all three germ layers. Cryopreservation is one of the key processes for successful applications of hPSCs, because it allows semi-permanent preservation of cells and their easy transportation. Most animal cell lines, including mouse embryonic stem cells, are standardly cryopreserved by slow cooling; however, hPSCs have been difficult to preserve and their cell viability has been extremely low whenever cryopreservation has been attempted.Here, we investigate the reasons for failure of slow cooling in hPSC cryopreservation. Cryopreservation involves a series of steps and is not a straightforward process. Cells may die due to various reasons during cryopreservation. Indeed, hPSCs preserved by traditional methods often suffer necrosis during the freeze-thawing stages, and the colony state of hPSCs prior to cryopreservation is a major factor contributing to cell death.It has now become possible to cryopreserve hPSCs using conventional cryopreservation methods without any specific equipment. This review summarizes the advances in this area and discusses the optimization of slow cooling cryopreservation for hPSC storage.

Economic 3D-printing approach for transplantation of human stem cell-derived β-like cells.

PubMed

Song, Jiwon; Millman, Jeffrey R

2016-12-01

Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing β cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived β (SC-β) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-β cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-β cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.

Analysis of Induced Pluripotent Stem Cells from a BRCA1 Mutant Family

PubMed Central

Soyombo, Abigail A.; Wu, Yipin; Kolski, Lauren; Rios, Jonathan J.; Rakheja, Dinesh; Chen, Alice; Kehler, James; Hampel, Heather; Coughran, Alanna; Ross, Theodora S.

2013-01-01

Summary Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability. PMID:24319668

Inhibition of Focal Adhesion Kinase Signaling by Integrin α6β1 Supports Human Pluripotent Stem Cell Self-Renewal.

PubMed

Villa-Diaz, Luis G; Kim, Jin Koo; Laperle, Alex; Palecek, Sean P; Krebsbach, Paul H

2016-07-01

Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6β1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of β1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin α6 functions in inactivation of integrin β1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6β1. Knockdown of laminin α5 resulted in reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6β1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. Stem Cells 2016;34:1753-1764. © 2016 AlphaMed Press.

Conserved Role of bFGF and a Divergent Role of LIF for Pluripotency Maintenance and Survival in Canine Pluripotent Stem Cells.

PubMed

Luo, Jiesi; Cibelli, Jose B

2016-09-19

Dogs have been widely used as a preclinical model for human disease. With the successful generation of canine induced pluripotent stem cells (ciPSCs), the biomedical community has a unique opportunity to study therapeutic interventions using autologous stem cells that can benefit dogs and humans. Unlike mice and human pluripotent cells, which are leukemia inhibitory factor (LIF)- and basic fibroblast growth factor (bFGF)-dependent, respectively, dog iPSCs require both growth factors simultaneously. In an effort to elucidate the role of each factor in the control of ciPSC self-renewal, we performed a series of experiments aiming at understanding the signaling pathways activated by them. We found that bFGF regulates pluripotency by indirectly activating the SMAD2/3 pathway in the presence of feeder cells, exclusively targeting NANOG expression, and inhibiting spontaneous differentiation toward ectoderm and mesoderm. LIF activates the JAK-STAT3 pathway but does not function in the typical manner described in mouse naïve embryonic stem cells. These results show that a unique mechanism for maintenance of pluripotency is present in ciPSC. These findings should be taken into account when establishing stem cell differentiation protocols and may provide more insight into pluripotency regulation in species other than mice and humans.

The European General Data Protection Regulation: challenges and considerations for iPSC researchers and biobanks

PubMed Central

Morrison, Michael; Bell, Jessica; George, Carol; Harmon, Shawn; Munsie, Megan; Kaye, Jane

2017-01-01

Increasingly, human induced pluripotent stem cells (iPSC) and their associated genetic and clinical information are being used in a wide range of applications, with large biobanks being established to support and increase their scientific use. The new European General Data Protection Regulations, which comes into effect in 2018, will have implications for biobanks that generate, store and allow research access to iPSC. This paper describes some of the challenges that iPSC biobanks face and suggests some points for the development of appropriate governance structures to address these new requirements. These suggestions also have implications for iPSC research in general. PMID:28976812

CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.

PubMed

Soh, Chew-Li; Huangfu, Danwei

2017-01-01

The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images.

PubMed

Joutsijoki, Henry; Haponen, Markus; Rasku, Jyrki; Aalto-Setälä, Katriina; Juhola, Martti

2016-01-01

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies.

Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro

PubMed Central

Kayashima, Hiroki; Miura, Jiro; Uraguchi, Shinya; Wang, Fangfang; Okawa, Hiroko; Sasaki, Jun-Ichi; Saeki, Makio; Matsumoto, Takuya; Yatani, Hirofumi

2014-01-01

Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and are, therefore, expected to be useful for bone regenerative medicine; however, the characteristics of iPSC-derived osteogenic cells remain unclear. Here, we provide a direct in vitro comparison of the osteogenic differentiation process in mesenchymal stem cells (MSCs) and iPSCs from adult C57BL/6J mice. After 30 days of culture in osteogenic medium, both MSCs and iPSCs produced robustly mineralized bone nodules that contained abundant calcium phosphate with hydroxyapatite crystal formation. Mineral deposition was significantly higher in iPSC cultures than in MSC cultures. Scanning electron microscopy revealed budding matrix vesicles in early osteogenic iPSCs; subsequently, the vesicles propagated to exhibit robust mineralization without rich fibrous structures. Early osteogenic MSCs showed deposition of many matrix vesicles in abundant collagen fibrils that became solid mineralized structures. Both cell types demonstrated increased expression of osteogenic marker genes, such as runx2, osterix, dlx5, bone sialoprotein (BSP), and osteocalcin, during osteogenesis; however, real-time reverse transcription–polymerase chain reaction array analysis revealed that osteogenesis-related genes encoding mineralization-associated molecules, bone morphogenetic proteins, and extracellular matrix collagens were differentially expressed between iPSCs and MSCs. These data suggest that iPSCs are capable of differentiation into mature osteoblasts whose associated hydroxyapatite has a crystal structure similar to that of MSC-associated hydroxyapatite; however, the transcriptional differences between iPSCs and MSCs could result in differences in the mineral and matrix environments of the bone nodules. Determining the biological mechanisms underlying cell-specific differences in mineralization during in vitro iPSC osteogenesis may facilitate the development of clinically effective engineered bone. PMID:24625139

Generation of Integration-Free Induced Pluripotent Stem Cells from Urine-Derived Cells Isolated from Individuals with Down Syndrome.

PubMed

M Lee, Young; Zampieri, Bruna L; Scott-McKean, Jonah J; Johnson, Mark W; Costa, Alberto C S

2017-06-01

Down syndrome (DS) is a genetic disorder caused by trisomy 21 (T21). Over the past two decades, the use of mouse models has led to significant advances in the understanding of mechanisms underlying various phenotypic features and comorbidities secondary to T21 and even informed the design of clinical trials aimed at enhancing the cognitive abilities of persons with DS. In spite of its success, this approach has been plagued by all the typical limitations of rodent modeling of human disorders and diseases. Recently, several laboratories have succeeded in producing T21 human induced pluripotent stem cells (T21-iPSCs) from individuals with DS, which is emerging as a promising complementary tool for the study of DS. Here, we describe the method by which we generated 10 T21-iPSC lines from epithelial cells in urine samples, presumably from kidney epithelial origin, using nonintegrating episomal vectors. We also show that these iPSCs maintain chromosomal stability for well over 20 passages and are more sensitive to proteotoxic stress than euploid iPSCs. Furthermore, these iPSC lines can be differentiated into glutamatergic neurons and cardiomyocytes. By culturing urine-derived cells and maximizing the efficiency of episomal vector transfection, we have been able to generate iPSCs noninvasively and effectively from participants with DS in an ongoing clinical trial, and thus address most shortcomings of previously generated T21-iPSC lines. These techniques should extend the application of iPSCs in modeling DS and other neurodevelopmental and neurodegenerative disorders, and may lead to future human cell-based platforms for high-throughput drug screening. Stem Cells Translational Medicine 2017;6:1465-1476. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

An Induced Pluripotent Stem Cell Patient Specific Model of Complement Factor H (Y402H) Polymorphism Displays Characteristic Features of Age-Related Macular Degeneration and Indicates a Beneficial Role for UV Light Exposure.

PubMed

Hallam, Dean; Collin, Joseph; Bojic, Sanja; Chichagova, Valeria; Buskin, Adriana; Xu, Yaobo; Lafage, Lucia; Otten, Elsje G; Anyfantis, George; Mellough, Carla; Przyborski, Stefan; Alharthi, Sameer; Korolchuk, Viktor; Lotery, Andrew; Saretzki, Gabriele; McKibbin, Martin; Armstrong, Lyle; Steel, David; Kavanagh, David; Lako, Majlinda

2017-11-01

Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells

PubMed Central

Belair, David G.; Whisler, Jordan A.; Valdez, Jorge; Velazquez, Jeremy; Molenda, James A.; Vickerman, Vernella; Lewis, Rachel; Daigh, Christine; Hansen, Tyler D.; Mann, David A.; Thomson, James A.; Griffith, Linda G.; Kamm, Roger D.; Schwartz, Michael P.; Murphy, William L.

2015-01-01

Here we describe a strategy to model blood vessel development using a well-defined iPSC-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats. PMID:25190668

Development of a Monitoring Method for Nonlabeled Human Pluripotent Stem Cell Growth by Time-Lapse Image Analysis.

PubMed

Suga, Mika; Kii, Hiroaki; Niikura, Keiichi; Kiyota, Yasujiro; Furue, Miho K

2015-07-01

: Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy. This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs. ©AlphaMed Press.

Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes

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

Lee, Sujeong, E-mail: crystalee@gmail.com; Lee, Hyang-Ae, E-mail: hyangaelee@gmail.com; Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799

2016-04-01

The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effectsmore » of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD{sub 90}) and 50% (APD{sub 50}) repolarization, reduced the maximum upstroke velocity (dV/dt{sub max}) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including I{sub Kr}, I{sub Ks}, I{sub Na}, and I{sub Ca}. Among them, I{sub Kr} and I{sub Na} showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K{sup +} channels. - Highlights: • Nefazodone prolonged APD and decreased upstroke velocity of APs in hiPSC-CMs. • Nefazodone inhibited cardiac ion channels, especially I{sub Kr} and I{sub Na}, in hiPSC-CMs. • Nefazodone-induced AP changes are mainly the result of I{sub Kr} and I{sub Na} inhibition. • hiPSC-CMs are sensitive model to validate nefazodone-induced cardiotoxicity. • hiPSC-CMs provide an integrated approach for evaluating mechanism of drug actions.« less

Human pancreatic islet-derived extracellular vesicles modulate insulin expression in 3D-differentiating iPSC clusters

PubMed Central

Andersson, Eva-Marie; Heath, Nikki; Persson-kry, Anette; Collins, Richard; Hicks, Ryan; Dekker, Niek; Forslöw, Anna

2017-01-01

It has been suggested that extracellular vesicles (EVs) can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs) were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC) clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications. PMID:29117231

Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

PubMed

Jung, Gwanghyun; Fajardo, Giovanni; Ribeiro, Alexandre J S; Kooiker, Kristina Bezold; Coronado, Michael; Zhao, Mingming; Hu, Dong-Qing; Reddy, Sushma; Kodo, Kazuki; Sriram, Krishna; Insel, Paul A; Wu, Joseph C; Pruitt, Beth L; Bernstein, Daniel

2016-04-01

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used β-adrenergic receptor (β-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), β2-ARs are a primary source of cAMP/PKA signaling. With longer culture, β1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic β-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30vs 55% at d 90. Moreover, β-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of β-AR functionalvs remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functionalvs remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation. © FASEB.

Growth Factor-Activated Stem Cell Circuits and Stromal Signals Cooperatively Accelerate Non-Integrated iPSC Reprogramming of Human Myeloid Progenitors

PubMed Central

Park, Tea Soon; Huo, Jeffrey S.; Peters, Ann; Talbot, C. Conover; Verma, Karan; Zimmerlin, Ludovic; Kaplan, Ian M.; Zambidis, Elias T.

2012-01-01

Nonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (∼0.001%–0.5%) of donor cells transfected with non-integrating reprogramming factors. Pluripotency induction of developmentally immature stem-progenitors is generally more efficient than differentiated somatic cell targets. However, the nature of augmented progenitor reprogramming remains obscure, and its potential has not been fully explored for improving the extremely slow pace of non-integrated reprogramming. Here, we report highly optimized four-factor reprogramming of lineage-committed cord blood (CB) myeloid progenitors with bulk efficiencies of ∼50% in purified episome-expressing cells. Lineage-committed CD33+CD45+CD34− myeloid cells and not primitive hematopoietic stem-progenitors were the main targets of a rapid and nearly complete non-integrated reprogramming. The efficient conversion of mature myeloid populations into NANOG+TRA-1-81+ hiPSC was mediated by synergies between hematopoietic growth factor (GF), stromal activation signals, and episomal Yamanaka factor expression. Using a modular bioinformatics approach, we demonstrated that efficient myeloid reprogramming correlated not to increased proliferation or endogenous Core factor expressions, but to poised expression of GF-activated transcriptional circuits that commonly regulate plasticity in both hematopoietic progenitors and embryonic stem cells (ESC). Factor-driven conversion of myeloid progenitors to a high-fidelity pluripotent state was further accelerated by soluble and contact-dependent stromal signals that included an implied and unexpected role for Toll receptor-NFκB signaling. These data provide a paradigm for understanding the augmented reprogramming capacity of somatic progenitors, and reveal that efficient induced pluripotency in other cell types may also require extrinsic activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC. PMID:22905176

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

PubMed

Zanella, Fabian; Sheikh, Farah

2016-01-01

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

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

PubMed

Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.

PubMed

Kang, HyunJun; Minder, Petra; Park, Mi Ae; Mesquitta, Walatta-Tseyon; Torbett, Bruce E; Slukvin, Igor I

2015-12-15

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

Generation of Arbas Cashmere Goat Induced Pluripotent Stem Cells Through Fibroblast Reprogramming.

PubMed

Tai, Dapeng; Liu, Pengxia; Gao, Jing; Jin, Muzi; Xu, Teng; Zuo, Yongchun; Liang, Hao; Liu, Dongjun

2015-08-01

Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.

Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

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

Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu

2014-10-24

Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice.more » Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.« less

Advancing pluripotent stem cell culture: it is a matter of setting the standard.

PubMed

Sartipy, Peter

2013-04-15

Human pluripotent stem cells (hPSCs), defined by their ability to proliferate indefinitely and the capacity to differentiate into all tissue cell types of the adult, represent a platform for the realization of breakthrough technologies for industrial and regenerative medicine applications. We have witnessed tremendous developments over the last decade related to methods for establishment, maintenance, differentiation, and applications of hPSCs and their derivatives. Despite all progress made in the hPSC field, there are still fundamental issues yet to be resolved. For example, our understanding of the pluripotent state remains limited, which in turn may have substantial consequences on how we interpret and communicate scientific data concerning hPSCs. This brief commentary aims to highlight recent important findings that demonstrate additional levels of complexity to the current assessment of pluripotent stem cell cultures. In addition, these data may help to provide some explanations for the challenges in reproducing hPSC differentiation protocols across laboratories.

Inhibition of Apoptosis Blocks Human Motor Neuron Cell Death in a Stem Cell Model of Spinal Muscular Atrophy

PubMed Central

Heins, Brittany M.; McGivern, Jered V.; Ornelas, Loren; Svendsen, Clive N.

2012-01-01

Spinal muscular atrophy (SMA) is a genetic disorder caused by a deletion of the survival motor neuron 1 gene leading to motor neuron loss, muscle atrophy, paralysis, and death. We show here that induced pluripotent stem cell (iPSC) lines generated from two Type I SMA subjects–one produced with lentiviral constructs and the second using a virus-free plasmid–based approach–recapitulate the disease phenotype and generate significantly fewer motor neurons at later developmental time periods in culture compared to two separate control subject iPSC lines. During motor neuron development, both SMA lines showed an increase in Fas ligand-mediated apoptosis and increased caspase-8 and-3 activation. Importantly, this could be mitigated by addition of either a Fas blocking antibody or a caspase-3 inhibitor. Together, these data further validate this human stem cell model of SMA, suggesting that specific inhibitors of apoptotic pathways may be beneficial for patients. PMID:22723941

Comparative Gene Expression Analyses Reveal Distinct Molecular Signatures between Differentially Reprogrammed Cardiomyocytes.

PubMed

Zhou, Yang; Wang, Li; Liu, Ziqing; Alimohamadi, Sahar; Yin, Chaoying; Liu, Jiandong; Qian, Li

2017-09-26

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) or directly reprogrammed from non-myocytes (induced cardiomyocytes [iCMs]) are promising sources for heart regeneration or disease modeling. However, the similarities and differences between iPSC-CMs and iCMs are still unknown. Here, we performed transcriptome analyses of beating iPSC-CMs and iCMs generated from cardiac fibroblasts (CFs) of the same origin. Although both iPSC-CMs and iCMs establish CM-like molecular features globally, iPSC-CMs exhibit a relatively hyperdynamic epigenetic status, whereas iCMs exhibit a maturation status that more closely resembles that of adult CMs. Based on gene expression of metabolic enzymes, iPSC-CMs primarily employ glycolysis, whereas iCMs utilize fatty acid oxidation as the main pathway. Importantly, iPSC-CMs and iCMs exhibit different cell-cycle status, alteration of which influenced their maturation. Therefore, our study provides a foundation for understanding the pros and cons of different reprogramming approaches. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Patient-Specific Induced Pluripotent Stem Cell as a Model for Familial Dilated Cardiomyopathy

PubMed Central

Sun, Ning; Yazawa, Masayuki; Liu, Jianwei; Han, Leng; Sanchez-Freire, Veronica; Abilez, Oscar J.; Navarrete, Enrique G.; Hu, Shijun; Wang, Li; Lee, Andrew; Pavlovic, Aleksandra; Lin, Shin; Chen, Rui; Hajjar, Roger J.; Snyder, Michael P.; Dolmetsch, Ricardo E.; Butte, Manish J.; Ashley, Euan A.; Longaker, Michael T.; Robbins, Robert C.; Wu, Joseph C.

2013-01-01

Dilated cardiomyopathy (DCM) is the most common cardiomyopathy, characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific models, investigating underlying mechanisms, and optimizing therapy. Here we generated cardiomyocytes (CMs) from iPSCs derived from patients of a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to the control healthy individuals in the same family cohort, DCM iPSC-CMs exhibited altered Ca2+ handling, decreased contractility, and abnormal sarcomeric α-actinin distribution. When stimulated with β-adrenergic agonist, DCM iPSC-CMs showed characteristics of failure such as reduced beating rates, compromised contraction, and significantly more cells with abnormal sarcomeric α-actinin distribution. β-adrenergic blocker treatment and over-expression of sarcoplasmic reticulum Ca2+ ATPase (Serca2a) improved DCM iPSC-CMs function. Our study demonstrated that human DCM iPSC-CMs recapitulated to some extent the disease phenotypes morphologically and functionally, and thus can serve as a useful platform for exploring molecular and cellular mechanisms and optimizing treatment of this particular disease. PMID:22517884

Effect of Induced Pluripotent Stem Cell Technology in Blood Banking

PubMed Central

Focosi, Daniele

2016-01-01

Summary Population aging has imposed cost-effective alternatives to blood donations. Artificial blood is still at the preliminary stages of development, and the need for viable cells seems unsurmountable. Because large numbers of viable cells must be promptly available for clinical use, stem cell technologies, expansion, and banking represent ideal tools to ensure a regular supply. Provided key donors can be identified, induced pluripotent stem cell (iPSC) technology could pave the way to a new era in transfusion medicine, just as it is already doing in many other fields of medicine. The present review summarizes the current state of research on iPSC technology in the field of blood banking, highlighting hurdles, and promises. Significance The aging population in Western countries is causing a progressive reduction of blood donors and a constant increase of blood recipients. Because blood is the main therapeutic option to treat acute hemorrhage, cost-effective alternatives to blood donations are being actively investigated. The enormous replication capability of induced pluripotent stem cells and their promising results in many other fields of medicine could be an apt solution to produce the large numbers of viable cells required in transfusion and usher in a new era in transfusion medicine. The present report describes the potentiality, technological hurdles, and promises of induced pluripotent stem cells to generate red blood cells by redifferentiation. PMID:26819256

Induced pluripotent stem cells for the treatment of stroke: the potential and the pitfalls.

PubMed

Yu, Fenggang; Li, Yingying; Morshead, Cindi M

2013-09-01

The extraordinary discovery of induced pluripotent stem cells (iPSCs) has led to the very real possibility that patient-specific cell therapy can be realized. The potential to develop cell replacement therapies outside the ethical and legal limitations, has initiated a new era of hope for regenerative strategies to treat human neurological disease including stroke. In this article, we will review and compare the current approaches to derive iPSCs from different somatic cells, and the induction into neuronal phenotypes, considering the advantages and disadvantages to the methodologies of derivation. We will highlight the work relating to the use of iPSC-based therapies in models of stroke and their potential use in clinical trials. Finally, we will consider future directions and areas of exploration which may promote the realization of iPSC-based cell replacement strategies for the treatment of stroke.

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

PubMed Central

Ramos-Mejía, Verónica; Montes, Rosa; Bueno, Clara; Ayllón, Verónica; Real, Pedro J.; Rodríguez, René; Menendez, Pablo

2012-01-01

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation. PMID:22545141

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture

PubMed Central

Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2017-01-01

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation. PMID:28783133

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture.

PubMed

Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2017-08-07

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase ( p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation.

A comparison of the pro-angiogenic potential of human induced pluripotent stem cell derived endothelial cells and induced endothelial cells in a murine model of peripheral arterial disease.

PubMed

Clayton, Zoe E; Yuen, Gloria S C; Sadeghipour, Sara; Hywood, Jack D; Wong, Jack W T; Huang, Ngan F; Ng, Martin K C; Cooke, John P; Patel, Sanjay

2017-05-01

Endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) promote angiogenesis, and more recently induced endothelial cells (iECs) have been generated via fibroblast trans-differentiation. These cell types have potential as treatments for peripheral arterial disease (PAD). However, it is unknown whether different reprogramming methods produce cells that are equivalent in terms of their pro-angiogenic capabilities. We aimed to directly compare iPSC-ECs and iECs in an animal model of PAD, in order to identify which cell type, if any, displays superior therapeutic potential. IPSC-ECs and iECs were generated from human fibroblasts, and transduced with a reporter construct encoding GFP and firefly luciferase for bioluminescence imaging (BLI). Endothelial phenotype was confirmed using in vitro assays. NOD-SCID mice underwent hindlimb ischaemia surgery and received an intramuscular injection of either 1×10 6 iPSC-ECs, 1×10 6 iECs or control vehicle only. Perfusion recovery was measured by laser Doppler. Hindlimb muscle samples were taken for histological analyses. Perfusion recovery was enhanced in iPSC-EC treated mice on day 14 (Control vs. iPSC-EC; 0.35±0.04 vs. 0.54±0.08, p<0.05) and in iEC treated mice on days 7 (Control vs. iEC; 0.23±0.02 vs. 0.44±0.06, p<0.05), 10 (0.31±0.04 vs. 0.64±0.07, p<0.001) and 14 (0.35±0.04 vs. 0.68±0.07, p<0.001) post-treatment. IEC-treated mice also had greater capillary density in the ischaemic gastrocnemius muscle (Control vs. iEC; 125±10 vs. 179±11 capillaries/image; p<0.05). BLI detected iPSC-EC and iEC presence in vivo for two weeks post-treatment. IPSC-ECs and iECs exhibit similar, but not identical, endothelial functionality and both cell types enhance perfusion recovery after hindlimb ischaemia. Copyright © 2017 Elsevier B.V. All rights reserved.

Reprogramming of blood cells into induced pluripotent stem cells as a new cell source for cartilage repair.

PubMed

Li, Yueying; Liu, Tie; Van Halm-Lutterodt, Nicholas; Chen, JiaYu; Su, Qingjun; Hai, Yong

2016-02-17

An attempt was made to reprogram peripheral blood cells into human induced pluripotent stem cell (hiPSCs) as a new cell source for cartilage repair. We generated chondrogenic lineage from human peripheral blood via hiPSCs using an integration-free method. Peripheral blood cells were either obtained from a human blood bank or freshly collected from volunteers. After transforming peripheral blood cells into iPSCs, the newly derived iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability. iPSCs were differentiated through multiple steps, including embryoid body formation, hiPSC-mesenchymal stem cell (MSC)-like cell expansion, and chondrogenic induction for 21 days. Chondrocyte phenotype was then assessed by morphological, histological and biochemical analysis, as well as the chondrogenic expression. hiPSCs derived from peripheral blood cells were successfully generated, and were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. Flow cytometric analysis showed that MSC markers CD73 and CD105 were present in monolayer cultured hiPSC-MSC-like cells. Both alcian blue and toluidine blue staining of hiPSC-MSC-chondrogenic pellets showed as positive. Immunohistochemistry of collagen II and X staining of the pellets were also positive. The sulfated glycosaminoglycan content was significantly increased, and the expression levels of the chondrogenic markers COL2, COL10, COL9 and AGGRECAN were significantly higher in chondrogenic pellets than in undifferentiated cells. These results indicated that peripheral blood cells could be a potential source for differentiation into chondrogenic lineage in vitro via generation of mesenchymal progenitor cells. This study supports the potential applications of utilizing peripheral blood cells in generating seed cells for cartilage regenerative medicine in a patient-specific and cost-effective approach.

Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy.

PubMed

Turan, Soeren; Farruggio, Alfonso P; Srifa, Waracharee; Day, John W; Calos, Michele P

2016-04-01

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.

Deep phenotyping of human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes.

PubMed

Cyganek, Lukas; Tiburcy, Malte; Sekeres, Karolina; Gerstenberg, Kathleen; Bohnenberger, Hanibal; Lenz, Christof; Henze, Sarah; Stauske, Michael; Salinas, Gabriela; Zimmermann, Wolfram-Hubertus; Hasenfuss, Gerd; Guan, Kaomei

2018-06-21

Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.

Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

PubMed

Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

2014-10-01

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

Establishment of induced pluripotent stem cell (iPSC) line from an 8-year old female patient with ischemic Moyamoya disease.

PubMed

Cardano, Marina; Marsoner, Fabio; Zasso, Jacopo; Marcatili, Matteo; Karnavas, Thodoris; Lanterna, Luigi Andrea; Conti, Luciano

2016-11-01

Peripheral blood mononuclear cells (PBMCs) were collected from an 8-year old female patient affected by ischemic Moyamoya disease (MMD). Patient's PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. The footprint free hiPSC line expressed the major pluripotency markers and exhibited a normal karyotype. Cells were competent to give rise to progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a good tool to in vitro model MMD in order to shed light on the cellular and molecular mechanisms responsible for the occurrence of this syndrome. Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.

Disease-specific induced pluripotent stem cells: a platform for human disease modeling and drug discovery.

PubMed

Jang, Jiho; Yoo, Jeong-Eun; Lee, Jeong-Ah; Lee, Dongjin R; Kim, Ji Young; Huh, Yong Jun; Kim, Dae-Sung; Park, Chul-Yong; Hwang, Dong-Youn; Kim, Han-Soo; Kang, Hoon-Chul; Kim, Dong-Wook

2012-03-31

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.

Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation

PubMed Central

Doi, Daisuke; Samata, Bumpei; Katsukawa, Mitsuko; Kikuchi, Tetsuhiro; Morizane, Asuka; Ono, Yuichi; Sekiguchi, Kiyotoshi; Nakagawa, Masato; Parmar, Malin; Takahashi, Jun

2014-01-01

Summary Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application. PMID:24672756

Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

PubMed

Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

2015-01-01

Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth media supplemented with FBS.

Removal of Transmissible Spongiform Encephalopathy Prion from Large Volumes of Cell Culture Media Supplemented with Fetal Bovine Serum by Using Hollow Fiber Anion-Exchange Membrane Chromatography

PubMed Central

Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

2015-01-01

Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco’s modified Eagle’s medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth media supplemented with FBS. PMID:25874629

A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells

PubMed Central

Burkhardt, Matthew F; Martinez, Fernando J; Wright, Sarah; Ramos, Carla; Volfson, Dmitri; Mason, Michael; Garnes, Jeff; Dang, Vu; Lievers, Jeffery; Shoukat-Mumtaz, Uzma; Martinez, Rita; Gai, Hui; Blake, Robert; Vaisberg, Eugeni; Grskovic, Marica; Johnson, Charles; Irion, Stefan; Bright, Jessica; Cooper, Bonnie; Nguyen, Leane; Griswold-Prenner, Irene; Javaherian, Ashkan

2016-01-01

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients’ fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modelling for drug screening. PMID:23891805

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.

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System.

PubMed

Conway, Michael K; Gerger, Michael J; Balay, Erin E; O'Connell, Rachel; Hanson, Seth; Daily, Neil J; Wakatsuki, Tetsuro

2015-05-14

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

EPA Pesticide Factsheets

Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

Vectors to Increase Production Efficiency of Inducible Pluripotent Stem Cell (iPSC) | NCI Technology Transfer Center | TTC

Cancer.gov

This invention describes the discovery that specific p53 isoform increase the number of inducible pluripotent stem cells (iPS). It is known that the activity of p53 regulates the self-renewal and pluripotency of normal and cancer stem cells, and also affects re-programming efficiency of iPS cells. This p53 isoform-based technology provides a more natural process of increasing iPS cell production than previous methods of decreasing p53. NCI seeks licensees for this technology.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

PubMed

Wu, Jun; Platero-Luengo, Aida; Sakurai, Masahiro; Sugawara, Atsushi; Gil, Maria Antonia; Yamauchi, Takayoshi; Suzuki, Keiichiro; Bogliotti, Yanina Soledad; Cuello, Cristina; Morales Valencia, Mariana; Okumura, Daiji; Luo, Jingping; Vilariño, Marcela; Parrilla, Inmaculada; Soto, Delia Alba; Martinez, Cristina A; Hishida, Tomoaki; Sánchez-Bautista, Sonia; Martinez-Martinez, M Llanos; Wang, Huili; Nohalez, Alicia; Aizawa, Emi; Martinez-Redondo, Paloma; Ocampo, Alejandro; Reddy, Pradeep; Roca, Jordi; Maga, Elizabeth A; Esteban, Concepcion Rodriguez; Berggren, W Travis; Nuñez Delicado, Estrella; Lajara, Jeronimo; Guillen, Isabel; Guillen, Pedro; Campistol, Josep M; Martinez, Emilio A; Ross, Pablo Juan; Izpisua Belmonte, Juan Carlos

2017-01-26

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos. Copyright © 2017 Elsevier Inc. All rights reserved.

Clinical potentials of human pluripotent stem cells.

PubMed

Mora, Cristina; Serzanti, Marialaura; Consiglio, Antonella; Memo, Maurizio; Dell'Era, Patrizia

2017-08-01

Aging, injuries, and diseases can be considered as the result of malfunctioning or damaged cells. Regenerative medicine aims to restore tissue homeostasis by repairing or replacing cells, tissues, or damaged organs, by linking and combining different disciplines including engineering, technology, biology, and medicine. To pursue these goals, the discipline is taking advantage of pluripotent stem cells (PSCs), a peculiar type of cell possessing the ability to differentiate into every cell type of the body. Human PSCs can be isolated from the blastocysts and maintained in culture indefinitely, giving rise to the so-called embryonic stem cells (ESCs). However, since 2006, it is possible to restore in an adult cell a pluripotent ESC-like condition by forcing the expression of four transcription factors with the rejuvenating reprogramming technology invented by Yamanaka. Then the two types of PSC can be differentiated, using standardized protocols, towards the cell type necessary for the regeneration. Although the use of these derivatives for therapeutic transplantation is still in the preliminary phase of safety and efficacy studies, a lot of efforts are presently taking place to discover the biological mechanisms underlying genetic pathologies, by differentiating induced PSCs derived from patients, and new therapies by challenging PSC-derived cells in drug screening.

Pluripotent Stem Cells as a Robust Source of Mesenchymal Stem Cells.

PubMed

Luzzani, Carlos D; Miriuka, Santiago G

2017-02-01

Mesenchymal stem cells (MSC) have been extensively studied over the past years for the treatment of different diseases. Most of the ongoing clinical trials currently involve the use of MSC derived from adult tissues. This source may have some limitations, particularly with therapies that may require extensive and repetitive cell dosage. However, nowadays, there is a staggering growth in literature on a new source of MSC. There is now increasing evidence about the mesenchymal differentiation from pluripotent stem cell (PSC). Here, we summarize the current knowledge of pluripotent-derived mesenchymal stem cells (PD-MSC). We present a historical perspective on the subject, and then discuss some critical questions that remain unanswered.

Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A-Oct4-GFP mice with high efficiency.

PubMed

Lin, Po-Ying; Hsu, Sheng-Chieh; Chen, Hung-Chi; Len, Wen-Bin; Hsiao, Fang-Chi; Liu, Mei-Chun; Pan, Pei-Ling; Lin, Tsai-Chen; Lee, Ying-Hsuan; Meir, Yaa-Jyuhn James

2018-05-01

A reprogrammable transgenic mouse strain, called Col1a1 4F2A-Oct4-GFP, was bred for the present study. Because the somatic cells of this mouse strain contain only two copies of each Yamanaka factor, these animals are inefficient at producing induced pluripotent stem cells (iPSCs; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts (MEFs) of Col1a1 4F2A-Oct4-GFP mice (MEF C ol1a1 4F2A-Oct4- GFP ) was increased to approximately 8%. Further, promotion of cell proliferation by serum supplementation did not enhance iPSC production. Inhibition of transforming growth factor β (TGF-β) in the serum by SB431542 neither affected the growth rate of MEF C ol1a1 4F2A-Oct4- GFP nor promoted iPSC production. However, the use of the gamma-irradiated STO-NEO-LIF (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEF ICR feeders. Interestingly, the use of SB431542 with the γMEF ICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-β expression was 10-fold higher in γMEF ICR than in γSNL cells. Consistent with previous reports, mesenchymal to epithelial transition was found to participate in the initial steps of reprogramming in the specific context of MEF C ol1a1 4F2A-Oct4- GFP . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSCs efficiently generated from our MEF C ol1a1 4F2A-Oct4- GFP resembled mouse embryonic stem cells (mESCs) in aspects of teratoma formation and germline transmission. Depending on the culture conditions, our Col1a1 4F2A-Oct4-GFP mouse system can generate bona fide iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming. © 2018 Federation of European Biochemical Societies.

Utilizing induced pluripotent stem cells (iPSCs) to understand the actions of estrogens in human neurons

PubMed Central

Shum, Carole; Macedo, Sara C.; Warre-Cornish, Katherine; Cocks, Graham; Price, Jack; Srivastava, Deepak P.

2015-01-01

This article is part of a Special Issue “Estradiol and Cognition”. Over recent years tremendous progress has been made towards understanding the molecular and cellular mechanism by which estrogens exert enhancing effects on cognition, and how they act as a neuroprotective or neurotrophic agent in disease. Currently, much of this work has been carried out in animal models with only a limited number of studies using native human tissue or cells. Recent advances in stem cell technology now make it possible to reprogram somatic cells from humans into induced pluripotent stem cells (iPSCs), which can subsequently be differentiated into neurons of specific lineages. Importantly, the reprogramming of cells allows for the generation of iPSCs that retain the genetic “makeup” of the donor. Therefore, it is possible to generate iPSC-derived neurons from patients diagnosed with specific diseases, that harbor the complex genetic background associated with the disorder. Here, we review the iPSC technology and how it's currently being used to model neural development and neurological diseases. Furthermore, we explore whether this cellular system could be used to understand the role of estrogens in human neurons, and present preliminary data in support of this. We further suggest that the use of iPSC technology offers a novel system to not only further understand estrogens' effects in human cells, but also to investigate the mechanism by which estrogens are beneficial in disease. Developing a greater understanding of these mechanisms in native human cells will also aid in the development of safer and more effective estrogen-based therapeutics. PMID:26143621

Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

PubMed

Heslop, James A; Kia, Richard; Pridgeon, Christopher S; Sison-Young, Rowena L; Liloglou, Triantafillos; Elmasry, Mohamed; Fenwick, Stephen W; Mills, John S; Kitteringham, Neil R; Goldring, Chris E; Park, Bong K

2017-05-01

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Geometry-dependent functional changes in iPSC-derived cardiomyocytes probed by functional imaging and RNA sequencing

PubMed Central

Gaublomme, Jellert; Shekhar, Karthik; Butty, Vincent; Yi, B. Alexander; Kralj, Joel M.; Bloxham, William; Boyer, Laurie A.; Regev, Aviv

2017-01-01

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation. PMID:28333933

Low Resting Membrane Potential and Low Inward Rectifier Potassium Currents Are Not Inherent Features of hiPSC-Derived Cardiomyocytes.

PubMed

Horváth, András; Lemoine, Marc D; Löser, Alexandra; Mannhardt, Ingra; Flenner, Frederik; Uzun, Ahmet Umur; Neuber, Christiane; Breckwoldt, Kaja; Hansen, Arne; Girdauskas, Evaldas; Reichenspurner, Hermann; Willems, Stephan; Jost, Norbert; Wettwer, Erich; Eschenhagen, Thomas; Christ, Torsten

2018-03-13

Human induced pluripotent stem cell (hiPSC) cardiomyocytes (CMs) show less negative resting membrane potential (RMP), which is attributed to small inward rectifier currents (I K1 ). Here, I K1 was measured in hiPSC-CMs (proprietary and commercial cell line) cultured as monolayer (ML) or 3D engineered heart tissue (EHT) and, for direct comparison, in CMs from human right atrial (RA) and left ventricular (LV) tissue. RMP was measured in isolated cells and intact tissues. I K1 density in ML- and EHT-CMs from the proprietary line was similar to LV and RA, respectively. I K1 density in EHT-CMs from the commercial line was 2-fold smaller than in the proprietary line. RMP in EHT of both lines was similar to RA and LV. Repolarization fraction and I K,ACh response discriminated best between RA and LV and indicated predominantly ventricular phenotype in hiPSC-CMs/EHT. The data indicate that I K1 is not necessarily low in hiPSC-CMs, and technical issues may underlie low RMP in hiPSC-CMs. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2016-06-06

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

Understanding the Role of TSC1/2 in Cerebellar Purkinje Neurons

DTIC Science & Technology

2017-09-01

patient-specific iPSC lines and rescue the disease phenotypes in patient specific neurons in vitro. We will employ CRISPR -Caspase 9 (Cas9) genome...development from human stem cells. For Aim 2. We at BCH have successfully generated CRISPR -cas9-mediated correction of TSC2- microdeletion in TSC...patient (TSC2+/-) derived hiPSC line (Figure 2). We used puromycin selection for isolation of the CRISPR -cas9 and ssODN transfected cells, and with

Significant differences in genotoxicity induced by retrovirus integration in human T cells and induced pluripotent stem cells.

PubMed

Zheng, Weiyan; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

2013-04-25

Retrovirus is frequently used in the genetic modification of mammalian cells and the establishment of induced pluripotent stem cells (iPSCs) via cell reprogramming. Vector-induced genotoxicity could induce profound effect on the physiology and function of these stem cells and their differentiated progeny. We analyzed retrovirus-induced genotoxicity in somatic cell Jurkat and two iPSC lines. In Jurkat cells, retrovirus frequently activated host gene expression and gene activation was not dependent on the distance between the integration site and the transcription start site of the host gene. In contrast, retrovirus frequently down-regulated host gene expression in iPSCs, possibly due to the action of chromatin silencing that spreads from the provirus to the nearby host gene promoter. Our data raises the issue that some of the phenotypic variability observed among iPSC clones derived from the same parental cell line may be caused by retrovirus-induced gene expression changes rather than by the reprogramming process itself. It also underscores the importance of characterizing retrovirus integration and carrying out risk assessment of iPSCs before they can be applied in basic research and clinics. Copyright © 2013 Elsevier B.V. All rights reserved.

Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation.

PubMed

Chen, Kevin G; Mallon, Barbara S; Johnson, Kory R; Hamilton, Rebecca S; McKay, Ronald D G; Robey, Pamela G

2014-05-01

Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We further highlight core signaling pathways that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyze emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Furthermore, based on the influence of cell-cell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids, which are pivotal steps needed for future clinical applications. Published by Elsevier B.V.

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

PubMed Central

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

2014-01-01

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

PubMed Central

2014-01-01

Induced pluripotent stem cell (iPSC) reprogramming requires sustained expression of multiple reprogramming factors for a limited period of time (10–30 days). Conventional iPSC reprogramming was achieved using lentiviral or simple retroviral vectors. Retroviral reprogramming has flaws of insertional mutagenesis, uncontrolled silencing, residual expression and re-activation of transgenes, and immunogenicity. To overcome these issues, various technologies were explored, including adenoviral vectors, protein transduction, RNA transfection, minicircle DNA, excisable PiggyBac (PB) transposon, Cre-lox excision system, negative-sense RNA replicon, positive-sense RNA replicon, Epstein-Barr virus-based episomal plasmids, and repeated transfections of plasmids. This review provides summaries of the main vectorologies and factor delivery systems used in current reprogramming protocols. PMID:24625220

Generation of Equine-Induced Pluripotent Stem Cells and Analysis of Their Therapeutic Potential for Muscle Injuries.

PubMed

Lee, Eun-Mi; Kim, Ah-Young; Lee, Eun-Joo; Park, Jin-Kyu; Park, Se-Il; Cho, Ssang-Goo; Kim, Hong Kyun; Kim, Shin-Yoon; Jeong, Kyu-Shik

2016-11-01

Horse health has become a major concern with the expansion of horse-related industries and sports; the importance of healthy muscles for horse performance and daily activities is undisputed. Here we generated equine-induced pluripotent stem cells (E-iPSCs) by reprogramming equine adipose-derived stem cells (E-ADSCs) into iPSCs using a polycistronic lentiviral vector encoding four transcription factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and then examined their pluripotent characteristics. Subsequently, established E-iPSCs were transplanted into muscle-injured Rag/ mdx mice. The histopathology results showed that E-iPSC-transplanted mice exhibited enhanced muscle regeneration compared to controls. In addition, E-iPSC-derived myofibers were observed in the injured muscles. In conclusion, we show that E-iPSCs could be successfully generated from equine ADSCs and transplanted into injured muscles and that E-iPSCs have the capacity to induce regeneration of injured muscles.

Induced Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Neurodegenerative Diseases.

PubMed

Cao, Lei; Tan, Lan; Jiang, Teng; Zhu, Xi-Chen; Yu, Jin-Tai

2015-08-01

Although most neurodegenerative diseases have been closely related to aberrant accumulation of aggregation-prone proteins in neurons, understanding their pathogenesis remains incomplete, and there is no treatment to delay the onset or slow the progression of many neurodegenerative diseases. The availability of induced pluripotent stem cells (iPSCs) in recapitulating the phenotypes of several late-onset neurodegenerative diseases marks the new era in in vitro modeling. The iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in these diseases and provides a novel human stem cell platform for screening new candidate therapeutics. Modeling human diseases using iPSCs has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. In this review, we introduce iPSC-based disease modeling in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. In addition, we discuss the implementation of iPSCs in drug discovery associated with some new techniques.

Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

PubMed

Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2011-06-01

Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

ROCK Inhibition Extends Passage of Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium

PubMed Central

Croze, Roxanne H.; Buchholz, David E.; Radeke, Monte J.; Thi, William J.; Hu, Qirui; Coffey, Peter J.

2014-01-01

Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-β pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture. PMID:25069775

Efficient generation of endothelial cells from human pluripotent stem cells and characterization of their functional properties.

PubMed

Song, Wei; Kaufman, Dan S; Shen, Wei

2016-03-01

Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs), large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34, respectively, from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGFβ-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542 + hESC-ECs, SB431542 - hESC-ECs, and HUVECs showed similar permeability to 10,000 Da dextran, but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542 + hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542 - hESC-ECs and HUVECs responded differently to VEGF and bFGF, which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542 - hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 678-687, 2016. © 2015 Wiley Periodicals, Inc.

Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening.

PubMed

Rana, Payal; Anson, Blake; Engle, Sandra; Will, Yvonne

2012-11-01

Cardiotoxicity remains the number one reason for drug withdrawal from the market, and Food and Drug Administration issued black box warnings, thus demonstrating the need for more predictive preclinical safety screening, especially early in the drug discovery process when much chemical substrate is available. Whereas human-ether-a-go-go related gene screening has become routine to mitigate proarrhythmic risk, the development of in vitro assays predicting additional on- and off-target biochemical toxicities will benefit from cellular models exhibiting true cardiomyocyte characteristics such as native tissue-like mitochondrial activity. Human stem cell-derived tissue cells may provide such a model. This hypothesis was tested using a combination of flux analysis, gene and protein expression, and toxicity-profiling techniques to characterize mitochondrial function in induced pluripotent stem cell (iPSC) derived human cardiomyocytes in the presence of differing carbon sources over extended periods in cell culture. Functional analyses demonstrate that iPSC-derived cardiomyocytes are (1) capable of utilizing anaerobic or aerobic respiration depending upon the available carbon substrate and (2) bioenergetically closest to adult heart tissue cells when cultured in galactose or galactose supplemented with fatty acids. We utilized this model to test a variety of kinase inhibitors with known clinical cardiac liabilities for their potential toxicity toward these cells. We found that the kinase inhibitors showed a dose-dependent toxicity to iPSC cardiomyocytes grown in galactose and that oxygen consumption rates were significantly more affected than adenosine triphosphate production. Sorafenib was found to have the most effect, followed by sunitinib, dasatinib, imatinib, lapatinib, and nioltinib.

ROCK Inhibition Extends Passage of Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium.

PubMed

Croze, Roxanne H; Buchholz, David E; Radeke, Monte J; Thi, William J; Hu, Qirui; Coffey, Peter J; Clegg, Dennis O

2014-09-01

Human embryonic stem cells (hESCs) offer a potentially unlimited supply of cells for emerging cell-based therapies. Unfortunately, the process of deriving distinct cell types can be time consuming and expensive. In the developed world, age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with more than 7.2 million people afflicted in the U.S. alone. Both hESC-derived retinal pigmented epithelium (hESC-RPE) and induced pluripotent stem cell-derived RPE (iPSC-RPE) are being developed for AMD therapies by multiple groups, but their potential for expansion in culture is limited. To attempt to overcome this passage limitation, we examined the involvement of Rho-associated, coiled-coil protein kinase (ROCK) in hESC-RPE and iPSC-RPE culture. We report that inhibiting ROCK1/2 with Y-27632 allows extended passage of hESC-RPE and iPSC-RPE. Microarray analysis suggests that ROCK inhibition could be suppressing an epithelial-to-mesenchymal transition through various pathways. These include inhibition of key ligands of the transforming growth factor-β pathway (TGFB1 and GDF6) and Wnt signaling. Two important processes are affected, allowing for an increase in hESC-RPE expansion. First, ROCK inhibition promotes proliferation by inducing multiple components that are involved in cell cycle progression. Second, ROCK inhibition affects many pathways that could be converging to suppress RPE-to-mesenchymal transition. This allows hESC-RPE to remain functional for an extended but finite period in culture. ©AlphaMed Press.

Epigenetics of cell fate reprogramming and its implications for neurological disorders modelling.

PubMed

Grzybek, Maciej; Golonko, Aleksandra; Walczak, Marta; Lisowski, Pawel

2017-03-01

The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies. We conclude that sample heterogeneity masks DNA methylation signatures in subpopulations of cells and thus believe that beside a genetic evaluation, extensive epigenomic screening should become a standard procedure to ensure hiPSCs state before they are used for genome editing and differentiation into neurons of interest. In particular, we suggest that exploitation of the single-cell composition of the epigenome will provide important insights into heterogeneity within hiPSCs subpopulations to fast forward development of reliable hiPSC-based analytical platforms in neurological disorders modelling and before completed hiPSC technology will be implemented in clinical approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression of Pluripotency Genes in Chondrocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells

PubMed Central

Stelcer, Ewelina; Kulcenty, Katarzyna; Rucinski, Marcin; Jopek, Karol; Trzeciak, Tomasz; Richter, Magdalena; Wroblewska, Joanna P.; Suchorska, Wiktoria M.

2018-01-01

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies. PMID:29439516

Pluripotent stem cell-derived organoids: using principles of developmental biology to grow human tissues in a dish.

PubMed

McCauley, Heather A; Wells, James M

2017-03-15

Pluripotent stem cell (PSC)-derived organoids are miniature, three-dimensional human tissues generated by the application of developmental biological principles to PSCs in vitro The approach to generate organoids uses a combination of directed differentiation, morphogenetic processes, and the intrinsically driven self-assembly of cells that mimics organogenesis in the developing embryo. The resulting organoids have remarkable cell type complexity, architecture and function similar to their in vivo counterparts. In the past five years, human PSC-derived organoids with components of all three germ layers have been generated, resulting in the establishment of a new human model system. Here, and in the accompanying poster, we provide an overview of how principles of developmental biology have been essential for generating human organoids in vitro , and how organoids are now being used as a primary research tool to investigate human developmental biology. © 2017. Published by The Company of Biologists Ltd.

Extracellular vesicles from human-induced pluripotent stem cell-derived mesenchymal stromal cells (hiPSC-MSCs) protect against renal ischemia/reperfusion injury via delivering specificity protein (SP1) and transcriptional activating of sphingosine kinase 1 and inhibiting necroptosis.

PubMed

Yuan, Xiaodong; Li, Dawei; Chen, Xiaosong; Han, Conghui; Xu, Longmei; Huang, Tao; Dong, Zhen; Zhang, Ming

2017-12-11

Renal ischemia-reperfusion is a main cause of acute kidney injury (AKI), which is associated with high mortality. Here we show that extracellular vesicles (EVs) secreted from hiPSC-MSCs play a critical role in protection against renal I/R injury. hiPSC-MSCs-EVs can fuse with renal cells and deliver SP1 into target cells, subsequently active SK1 expression and increase S1P formation. Chromatin immunoprecipitation (ChIP) analyses and luciferase assay were used to confirm SP1 binds directly to the SK1 promoter region and promote promoter activity. Moreover, SP1 inhibition (MIT) or SK1 inhibition (SKI-II) completely abolished the renal protective effect of hiPSC-MSCs-EVs in rat I/R injury mode. However, pre-treatment of necroptosis inhibitor Nec-1 showed no difference with the administration of hiPSC-MSCs-EVs only. We then generated an SP1 knockout hiPSC-MSC cell line by CRISPR/Cas9 system and found that SP1 knockout failed to show the protective effect of hiPSC-MSCs-EVs unless restoring the level of SP1 by Ad-SP1 in vitro and in vivo. In conclusion, this study describes an anti-necroptosis effect of hiPSC-MSCs-EVs against renal I/R injury via delivering SP1 into target renal cells and intracellular activating the expression of SK1 and the generation of S1P. These findings suggest a novel mechanism for renal protection against I/R injury, and indicate a potential therapeutic approach for a variety of renal diseases and renal transplantation.

Induction of pluripotent stem cells from a cynomolgus monkey using a polycistronic simian immunodeficiency virus-based vector, differentiation toward functional cardiomyocytes, and generation of stably expressing reporter lines.

PubMed

Wunderlich, Stephanie; Haase, Alexandra; Merkert, Sylvia; Beier, Jennifer; Schwanke, Kristin; Schambach, Axel; Glage, Silke; Göhring, Gudrun; Curnow, Eliza C; Martin, Ulrich

2012-12-01

Induced pluripotent stem cells (iPSCs) represent a novel cell source for regenerative therapies. Many emerging iPSC-based therapeutic concepts will require preclinical evaluation in suitable large animal models. Among the large animal species frequently used in preclinical efficacy and safety studies, macaques show the highest similarities to humans at physiological, cellular, and molecular levels. We have generated iPSCs from cynomolgus monkeys (Macaca fascicularis) as a segue to regenerative therapy model development in this species. Because typical human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors show poor transduction of simian cells, a simian immunodeficiency virus (SIV)-based vector was chosen for efficient transduction of cynomolgus skin fibroblasts. A corresponding polycistronic vector with codon-optimized reprogramming factors was constructed for reprogramming. Growth characteristics as well as cell and colony morphology of the resulting cynomolgus iPSCs (cyiPSCs) were demonstrated to be almost identical to cynomolgus embryonic stem cells (cyESCs), and cyiPSCs expressed typical pluripotency markers including OCT4, SOX2, and NANOG. Furthermore, differentiation in vivo and in vitro into derivatives of all three germ layers, as well as generation of functional cardiomyocytes, could be demonstrated. Finally, a highly efficient technique for generation of transgenic cyiPSC clones with stable reporter expression in undifferentiated cells as well as differentiated transgenic cyiPSC progeny was developed to enable cell tracking in recipient animals. In conclusion, our data indicate that cyiPSCs represent a valuable cell source for establishment of macaque-based allogeneic and autologous preclinical cell transplantation models for various fields of regenerative medicine.

iPSC-derived cardiomyocytes reveal abnormal TGFβ signaling in left ventricular non-compaction cardiomyopathy

PubMed Central

Kodo, Kazuki; Ong, Sang-Ging; Jahanbani, Fereshteh; Termglinchan, Vittavat; Hirono, Keiichi; InanlooRahatloo, Kolsoum; Ebert, Antje D.; Shukla, Praveen; Abilez, Oscar J.; Churko, Jared M.; Karakikes, Ioannis; Jung, Gwanghyun; Ichida, Fukiko; Wu, Sean M.; Snyder, Michael P.; Bernstein, Daniel; Wu, Joseph C.

2016-01-01

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and was associated with perturbed transforming growth factor beta (TGFβ) signaling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGFβ signaling. TBX20 regulates the expression of TGFβ signaling modifiers including a known genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGFβ signaling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC. PMID:27642787

The assessment of electrophysiological activity in human-induced pluripotent stem cell-derived cardiomyocytes exposed to dimethyl sulfoxide and ethanol by manual patch clamp and multi-electrode array system.

PubMed

Hyun, Soo-Wang; Kim, Bo-Ram; Hyun, Sung-Ae; Seo, Joung-Wook

2017-09-01

Recently, electrophysiological activity has been effectively measured in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to predict drug-induced arrhythmia. Dimethyl sulfoxide (DMSO) and ethanol have been used as diluting agents in many experiments. However, the maximum DMSO and ethanol concentrations that can be effectively used in the measurement of electrophysiological parameters in hiPSC-CMs-based patch clamp and multi-electrode array (MEA) have not been fully elucidated. We investigated the effects of varying concentrations of DMSO and ethanol used as diluting agents on several electrophysiological parameters in hiPSC-CMs using patch clamp and MEA. Both DMSO and ethanol at concentrations>1% in external solution resulted in osmolality >400mOsmol/kg, but pH was not affected by either agent. Neither DMSO nor ethanol led to cell death at the concentrations examined. However, resting membrane potential, action potential amplitude, action potential duration at 90% and 40%, and corrected field potential duration were decreased significantly at 1% ethanol concentration. DMSO at 1% also significantly decreased the sodium spike amplitude. In addition, the waveform of action potential and field potential was recorded as irregular at 3% concentrations of both DMSO and ethanol. Concentrations of up to 0.3% of either agent did not affect osmolality, pH, cell death, or electrophysiological parameters in hiPSC-CMs. Our findings suggest that 0.3% is the maximum concentration at which DMSO or ethanol should be used for dilution purposes in hiPSC-CMs-based patch clamp and MEA. Copyright © 2017 Elsevier Inc. All rights reserved.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.

PubMed

Schweizer, Patrick A; Darche, Fabrice F; Ullrich, Nina D; Geschwill, Pascal; Greber, Boris; Rivinius, Rasmus; Seyler, Claudia; Müller-Decker, Karin; Draguhn, Andreas; Utikal, Jochen; Koenen, Michael; Katus, Hugo A; Thomas, Dierk

2017-10-16

Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca 2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

Energy utilization of induced pluripotent stem cell-derived cardiomyocyte in Fabry disease.

PubMed

Chou, Shih-Jie; Yu, Wen-Chung; Chang, Yuh-Lih; Chen, Wen-Yeh; Chang, Wei-Chao; Chien, Yueh; Yen, Jiin-Cherng; Liu, Yung-Yang; Chen, Shih-Jen; Wang, Chien-Ying; Chen, Yu-Han; Niu, Dau-Ming; Lin, Shing-Jong; Chen, Jaw-Wen; Chiou, Shih-Hwa; Leu, Hsin-Bang

2017-04-01

Fabry disease (FD) is a lysosomal storage disease in which glycosphingolipids (GB3) accumulate in organs of the human body, leading to idiopathic hypertrophic cardiomyopathy and target organ damage. Its pathophysiology is still poorly understood. We aimed to generate patient-specific induced pluripotent stem cells (iPSC) from FD patients presenting cardiomyopathy to determine whether the model could recapitulate key features of the disease phenotype and to investigate the energy metabolism in Fabry disease. Peripheral blood mononuclear cells from a 30-year-old Chinese man with a diagnosis of Fabry disease, GLA gene (IVS4+919G>A) mutation were reprogrammed into iPSCs and differentiated into iPSC-CMs and energy metabolism was analyzed in iPSC-CMs. The FD-iPSC-CMs recapitulated numerous aspects of the FD phenotype including reduced GLA activity, cellular hypertrophy, GB3 accumulation and impaired contractility. Decreased energy metabolism with energy utilization shift to glycolysis was observed, but the decreased energy metabolism was not modified by enzyme rescue replacement (ERT) in FD-iPSCs-CMs. This model provided a promising in vitro model for the investigation of the underlying disease mechanism and development of novel therapeutic strategies for FD. This potential remedy for enhancing the energetic network and utility efficiency warrants further study to identify novel therapies for the disease. Copyright © 2017 Elsevier B.V. All rights reserved.

CRISPR correction of the PRKAG2 gene mutation in the patient's induced pluripotent stem cell-derived cardiomyocytes eliminates electrophysiological and structural abnormalities.

PubMed

Ben Jehuda, Ronen; Eisen, Binyamin; Shemer, Yuval; Mekies, Lucy N; Szantai, Agnes; Reiter, Irina; Cui, Huanhuan; Guan, Kaomei; Haron-Khun, Shiraz; Freimark, Dov; Sperling, Silke R; Gherghiceanu, Mihaela; Arad, Michael; Binah, Ofer

2018-02-01

Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial Wolff-Parkinson-White (WPW) syndrome. Patients carrying the R302Q mutation in PRKAG2 present with sinus bradycardia, escape rhythms, ventricular preexcitation, supraventricular tachycardia, and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW syndrome, HCM, and arrhythmias remains unknown. The purpose of this study was to investigate the pathological changes caused by the PRKAG2 mutation. We tested the hypothesis that patient's induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease. Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we corrected the mutation and then generated isogenic iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. Using a microelectrode array setup, we recorded electrograms from iPSC-CMs clusters. Transmission electron microscopy was used to detect ultrastructural abnormalities in the mutated iPSC-CMs. PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations, triggered arrhythmias, and augmented beat rate variability. Importantly, CRISPR correction eliminated the electrophysiological abnormalities, the augmented glycogen, storage, and cardiomyocyte hypertrophy. PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using CRISPR technology. Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Generating trunk neural crest from human pluripotent stem cells

PubMed Central

Huang, Miller; Miller, Matthew L.; McHenry, Lauren K.; Zheng, Tina; Zhen, Qiqi; Ilkhanizadeh, Shirin; Conklin, Bruce R.; Bronner, Marianne E.; Weiss, William A.

2016-01-01

Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior “cranial” NCC form craniofacial bone, whereas solely posterior “trunk” NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages. PMID:26812940

X Chromosome of female cells shows dynamic changes in status during human somatic cell reprogramming.

PubMed

Kim, Kun-Yong; Hysolli, Eriona; Tanaka, Yoshiaki; Wang, Brandon; Jung, Yong-Wook; Pan, Xinghua; Weissman, Sherman Morton; Park, In-Hyun

2014-06-03

Induced pluripotent stem cells (iPSCs) acquire embryonic stem cell (ESC)-like epigenetic states, including the X chromosome. Previous studies reported that human iPSCs retain the inactive X chromosome of parental cells, or acquire two active X chromosomes through reprogramming. Most studies investigated the X chromosome states in established human iPSC clones after completion of reprogramming. Thus, it is still not fully understood when and how the X chromosome reactivation occurs during reprogramming. Here, we report a dynamic change in the X chromosome state throughout reprogramming, with an initial robust reactivation of the inactive X chromosome followed by an inactivation upon generation of nascent iPSC clones. iPSCs with two active X chromosomes or an eroded X chromosome arise in passaging iPSCs. These data provide important insights into the plasticity of the X chromosome of human female iPSCs and will be crucial for the future application of such cells in cell therapy and X-linked disease modeling.

Preclinical studies for induced pluripotent stem cell-based therapeutics.

PubMed

Harding, John; Mirochnitchenko, Oleg

2014-02-21

Induced pluripotent stem cells (iPSCs) and their differentiated derivatives can potentially be applied to cell-based therapy for human diseases. The properties of iPSCs are being studied intensively both to understand the basic biology of pluripotency and cellular differentiation and to solve problems associated with therapeutic applications. Examples of specific preclinical applications summarized briefly in this minireview include the use of iPSCs to treat diseases of the liver, nervous system, eye, and heart and metabolic conditions such as diabetes. Early stage studies illustrate the potential of iPSC-derived cells and have identified several challenges that must be addressed before moving to clinical trials. These include rigorous quality control and efficient production of required cell populations, improvement of cell survival and engraftment, and development of technologies to monitor transplanted cell behavior for extended periods of time. Problems related to immune rejection, genetic instability, and tumorigenicity must be solved. Testing the efficacy of iPSC-based therapies requires further improvement of animal models precisely recapitulating human disease conditions.

Characterization of porcine partially reprogrammed iPSCs from adipose-derived stem cells.

PubMed

Wei, Chao; Li, Xia; Zhang, Pengfei; Zhang, Yu; Liu, Tong; Jiang, Shaoshuai; Han, Fei; Zhang, Yunhai

2015-05-01

Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos. © 2015 Society for Reproduction and Fertility.

Reverse engineering human neurodegenerative disease using pluripotent stem cell technology.

PubMed

Liu, Ying; Deng, Wenbin

2016-05-01

With the technology of reprogramming somatic cells by introducing defined transcription factors that enables the generation of "induced pluripotent stem cells (iPSCs)" with pluripotency comparable to that of embryonic stem cells (ESCs), it has become possible to use this technology to produce various cells and tissues that have been difficult to obtain from living bodies. This advancement is bringing forth rapid progress in iPSC-based disease modeling, drug screening, and regenerative medicine. More and more studies have demonstrated that phenotypes of adult-onset neurodegenerative disorders could be rather faithfully recapitulated in iPSC-derived neural cell cultures. Moreover, despite the adult-onset nature of the diseases, pathogenic phenotypes and cellular abnormalities often exist in early developmental stages, providing new "windows of opportunity" for understanding mechanisms underlying neurodegenerative disorders and for discovering new medicines. The cell reprogramming technology enables a reverse engineering approach for modeling the cellular degenerative phenotypes of a wide range of human disorders. An excellent example is the study of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS) using iPSCs. ALS is a progressive neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs), culminating in muscle wasting and death from respiratory failure. The iPSC approach provides innovative cell culture platforms to serve as ALS patient-derived model systems. Researchers have converted iPSCs derived from ALS patients into MNs and various types of glial cells, all of which are involved in ALS, to study the disease. The iPSC technology could be used to determine the role of specific genetic factors to track down what's wrong in the neurodegenerative disease process in the "disease-in-a-dish" model. Meanwhile, parallel experiments of targeting the same specific genes in human ESCs could also be performed to control and to complement the iPSC-based approach for ALS disease modeling studies. Much knowledge has been generated from the study of both ALS iPSCs and ESCs. As these methods have advantages and disadvantages that should be balanced on experimental design in order for them to complement one another, combining the diverse methods would help build an expanded knowledge of ALS pathophysiology. The goals are to reverse engineer the human disease using ESCs and iPSCs, generate lineage reporter lines and in vitro disease models, target disease related genes, in order to better understand the molecular and cellular mechanisms of differentiation regulation along neural (neuronal versus glial) lineages, to unravel the pathogenesis of the neurodegenerative disease, and to provide appropriate cell sources for replacement therapy. This article is part of a Special Issue entitled SI: PSC and the brain. Copyright © 2015 Elsevier B.V. All rights reserved.

Erythropoiesis from Human Embryonic Stem Cells Through Erythropoietin-Independent AKT Signaling

PubMed Central

Kim, William S.; Zhu, Yuhua; Deng, Qiming; Chin, Chee Jia; He, Chong Bin; Grieco, Amanda J.; Dravid, Gautam G.; Parekh, Chintan; Hollis, Roger P.; Lane, Timothy F.; Bouhassira, Eric E.; Kohn, Donald B.; Crooks, Gay M.

2014-01-01

Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homo-dimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of the present study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESC) and to identify the signaling pathways activated by this strategy. We present here evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates the GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC. PMID:24677652

Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases

PubMed Central

Shi, Liang; Cui, Yazhou; Luan, Jing; Zhou, Xiaoyan; Han, Jinxiang

2016-01-01

Summary Rare diseases with a low prevalence are a key public health issue because the causes of those diseases are difficult to determine and those diseases lack a clearly established or curative treatment. Thus, investigating the molecular mechanisms that underlie the pathology of rare diseases and facilitating the development of novel therapies using disease models is crucial. Human induced pluripotent stem cells (iPSCs) are well suited to modeling rare diseases since they have the capacity for self-renewal and pluripotency. In addition, iPSC technology provides a valuable tool to generate patient-specific iPSCs. These cells can be differentiated into cell types that have been affected by a disease. These cells would circumvent ethical concerns and avoid immunological rejection, so they could be used in cell replacement therapy or regenerative medicine. To date, human iPSCs could have been generated from multiple donor sources, such as skin, adipose tissue, and peripheral blood. However, these cells are obtained via invasive procedures. In contrast, several groups of researchers have found that urine may be a better source for producing iPSCs from normal individuals or patients. This review discusses urinary iPSC (UiPSC) as a candidate for modeling rare diseases. Cells obtained from urine have overwhelming advantages compared to other donor sources since they are safely, affordably, and frequently obtained and they are readily obtained from patients. The use of iPSC-based models is also discussed. UiPSCs may prove to be a key means of modeling rare diseases and they may facilitate the treatment of those diseases in the future. PMID:27672542

Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

PubMed

Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

2017-08-01

Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

Human pluripotent stem cell-derived cardiomyocytes for heart regeneration, drug discovery and disease modeling: from the genetic, epigenetic, and tissue modeling perspectives.

PubMed

Chow, Maggie; Boheler, Kenneth R; Li, Ronald A

2013-08-14

Heart diseases remain a major cause of mortality and morbidity worldwide. However, terminally differentiated human adult cardiomyocytes (CMs) possess a very limited innate ability to regenerate. Directed differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into CMs has enabled clinicians and researchers to pursue the novel therapeutic paradigm of cell-based cardiac regeneration. In addition to tissue engineering and transplantation studies, the need for functional CMs has also prompted researchers to explore molecular pathways and develop strategies to improve the quality, purity and quantity of hESC-derived and iPSC-derived CMs. In this review, we describe various approaches in directed CM differentiation and driven maturation, and discuss potential limitations associated with hESCs and iPSCs, with an emphasis on the role of epigenetic regulation and chromatin remodeling, in the context of the potential and challenges of using hESC-CMs and iPSC-CMs for drug discovery and toxicity screening, disease modeling, and clinical applications.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

PubMed

Romero-Moya, Damià; Santos-Ocaña, Carlos; Castaño, Julio; Garrabou, Gloria; Rodríguez-Gómez, José A; Ruiz-Bonilla, Vanesa; Bueno, Clara; González-Rodríguez, Patricia; Giorgetti, Alessandra; Perdiguero, Eusebio; Prieto, Cristina; Moren-Nuñez, Constanza; Fernández-Ayala, Daniel J; Victoria Cascajo, Maria; Velasco, Iván; Canals, Josep Maria; Montero, Raquel; Yubero, Delia; Jou, Cristina; López-Barneo, José; Cardellach, Francesc; Muñoz-Cánoves, Pura; Artuch, Rafael; Navas, Plácido; Menendez, Pablo

2017-07-01

Coenzyme Q 10 (CoQ 10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ 10 biosynthesis (COQ genes) cause primary CoQ 10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ 10 ], CoQ 10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4 ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4 ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ 10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ 10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703. © 2017 AlphaMed Press.

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

Complex aberrant splicing in the induced pluripotent stem cell-derived cardiomyocytes from a patient with long-QT syndrome carrying KCNQ1-A344Aspl mutation.

PubMed

Wuriyanghai, Yimin; Makiyama, Takeru; Sasaki, Kenichi; Kamakura, Tsukasa; Yamamoto, Yuta; Hayano, Mamoru; Harita, Takeshi; Nishiuchi, Suguru; Chen, Jiarong; Kohjitani, Hirohiko; Hirose, Sayako; Yokoi, Fumika; Gao, Jingshan; Chonabayashi, Kazuhisa; Watanabe, Ken; Ohno, Seiko; Yoshida, Yoshinori; Kimura, Takeshi; Horie, Minoru

2018-05-29

Long-QT syndrome type 1 (LQT1) is caused by mutations in KCNQ1, which encodes the α-subunit of the slow delayed rectifier potassium current (I Ks ) channel. We previously reported that a synonymous mutation, c.1032G>A, p.A344Aspl in KCNQ1 is most commonly identified in the genotyped LQT1 Japanese patients, and the aberrant splicing was analyzed in the lymphocytes isolated from patients' blood samples. However, the mechanisms underlying the observed processes in human cardiomyocytes remains unclear. To establish and analyze patient-specific human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model carrying KCNQ1-A344Aspl. We generated hiPSCs from the peripheral blood mononuclear cells obtained from an LQT1 patient carrying KCNQ1-A344Aspl. Using the differentiated cardiomyocytes, we analyzed splicing variants and performed electrophysiological studies. We identified seven aberrant RNA variants in A344Aspl-hiPSC-CMs, more complex compared with those in the peripheral lymphocytes. Multi-electrode array analysis revealed that 1 μM isoproterenol significantly prolonged the duration of corrected field potential in A344Aspl-hiPSC-CMs, compared with that in the controls. Additionally, 100 nM E-4031, I Kr blocker, was shown to induce early afterdepolarization-like waveforms in A344Aspl-hiPSC-CMs. Action potential durations (APDs) did not significantly differ between the hiPSC-CM groups. After administrating 500 nM isoproterenol, APDs of A344Aspl-hiPSC-CMs were significantly longer than those of the controls. ML277 and phenylboronic acid, I Ks activators, ameliorated the APDs of hiPSC-CMs. We identified complex aberrant mRNA variants in the A344Aspl-hiPSC-CM model, and successfully recapitulated the clinical phenotypes of the patient with concealed LQT1. This model allows the investigation of the underlying mechanisms and development of novel therapies. Copyright © 2018. Published by Elsevier Inc.

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.

Advanced feeder-free generation of induced pluripotent stem cells directly from blood cells.

PubMed

Trokovic, Ras; Weltner, Jere; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Salomaa, Veikko; Jalanko, Anu; Otonkoski, Timo; Kyttälä, Aija

2014-12-01

Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids. However, most of the published protocols require expansion and/or activation of a specific cell population from PBMCs. We have recently collected a PBMC cohort from the Finnish population containing more than 2,000 subjects. Here we report efficient generation of iPSCs directly from PBMCs in feeder-free conditions in approximately 2 weeks. The produced iPSC clones are pluripotent and transgene-free. Together, these properties make this novel method a powerful tool for large-scale reprogramming of PBMCs and for iPSC biobanking. ©AlphaMed Press.

Human-Induced Pluripotent Stem Cell Technology and Cardiomyocyte Generation: Progress and Clinical Applications.

PubMed

Di Baldassarre, Angela; Cimetta, Elisa; Bollini, Sveva; Gaggi, Giulia; Ghinassi, Barbara

2018-05-25

Human-induced pluripotent stem cells (hiPSCs) are reprogrammed cells that have hallmarks similar to embryonic stem cells including the capacity of self-renewal and differentiation into cardiac myocytes. The improvements in reprogramming and differentiating methods achieved in the past 10 years widened the use of hiPSCs, especially in cardiac research. hiPSC-derived cardiac myocytes (CMs) recapitulate phenotypic differences caused by genetic variations, making them attractive human disease models and useful tools for drug discovery and toxicology testing. In addition, hiPSCs can be used as sources of cells for cardiac regeneration in animal models. Here, we review the advances in the genetic and epigenetic control of cardiomyogenesis that underlies the significant improvement of the induced reprogramming of somatic cells to CMs; the methods used to improve scalability of throughput assays for functional screening and drug testing in vitro; the phenotypic characteristics of hiPSCs-derived CMs and their ability to rescue injured CMs through paracrine effects; we also cover the novel approaches in tissue engineering for hiPSC-derived cardiac tissue generation, and finally, their immunological features and the potential use in biomedical applications.

An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives.

PubMed

Randolph, Lauren N; Bao, Xiaoping; Zhou, Chikai; Lian, Xiaojun

2017-05-08

Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies due to their unique combination of two properties: pluripotency and unlimited proliferative capacity. However, directed differentiation of hPSCs to clinically relevant cell lineages is needed to achieve the goal of hPSC-based therapies. This requires a deep understanding of how cell signaling pathways converge on the nucleus to control differentiation and the ability to dissect gene function in a temporal manner. Here, we report the use of the PiggyBac transposon and a Tet-On 3G drug-inducible gene expression system to achieve versatile inducible gene expression in hPSC lines. Our new system, XLone, offers improvement over previous Tet-On systems with significantly reduced background expression and increased sensitivity to doxycycline. Transgene expression in hPSCs is tightly regulated in response to doxycycline treatment. In addition, the PiggyBac elements in our XLone construct provide a rapid and efficient strategy for generating stable transgenic hPSCs. Our inducible gene expression PiggyBac transposon system should facilitate the study of gene function and directed differentiation in human stem cells.

Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

PubMed Central

Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J.; Iannaccone, Philip M.; Hendrix, Mary J.C.

2016-01-01

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique

NASA Astrophysics Data System (ADS)

Chen, Wanjuan; Liu, Jingxin; Zhang, Longmei; Xu, Huijuan; Guo, Xiaogang; Deng, Sihao; Liu, Lipeng; Yu, Daiguan; Chen, Yonglong; Li, Zhiyuan

2014-06-01

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.

An Atypical Human Induced Pluripotent Stem Cell Line With a Complex, Stable, and Balanced Genomic Rearrangement Including a Large De Novo 1q Uniparental Disomy

PubMed Central

Steichen, Clara; Maluenda, Jérôme; Tosca, Lucie; Luce, Eléanor; Pineau, Dominique; Dianat, Noushin; Hannoun, Zara; Tachdjian, Gérard; Melki, Judith

2015-01-01

Human induced pluripotent stem cells (hiPSCs) hold great promise for cell therapy through their use as vital tools for regenerative and personalized medicine. However, the genomic integrity of hiPSCs still raises some concern and is one of the barriers limiting their use in clinical applications. Numerous articles have reported the occurrence of aneuploidies, copy number variations, or single point mutations in hiPSCs, and nonintegrative reprogramming strategies have been developed to minimize the impact of the reprogramming process on the hiPSC genome. Here, we report the characterization of an hiPSC line generated by daily transfections of modified messenger RNAs, displaying several genomic abnormalities. Karyotype analysis showed a complex genomic rearrangement, which remained stable during long-term culture. Fluorescent in situ hybridization analyses were performed on the hiPSC line showing that this karyotype is balanced. Interestingly, single-nucleotide polymorphism analysis revealed the presence of a large 1q region of uniparental disomy (UPD), demonstrating for the first time that UPD can occur in a noncompensatory context during nonintegrative reprogramming of normal fibroblasts. PMID:25650439

Exon skipping and gene transfer restore dystrophin expression in human induced pluripotent stem cells-cardiomyocytes harboring DMD mutations.

PubMed

Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns; Denning, Chris

2013-10-15

With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47-50 or 48-50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart.

Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations

PubMed Central

Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H.; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns

2013-01-01

With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47–50 or 48–50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart. PMID:23829870

A dangerous method? The use of induced pluripotent stem cells as a model for schizophrenia.

PubMed

Jacobs, Benjamin Meir

2015-10-01

Schizophrenia is a devastating and prevalent psychiatric illness. Progress in understanding the basic pathophysiological processes underlying this disorder has been hindered by the lack of appropriate models. With the advent of induced pluripotent stem cell (iPSC) technology, it is now possible to generate live neurons in vitro from somatic tissue of schizophrenia patients. Despite its several limitations, this revolutionary technology has already helped to advance our understanding of schizophrenia. The phenotypic insights garnered with iPSC models of schizophrenia include transcriptional dysregulation, oxidative stress synaptic dysregulation, and neurodevelopmental abnormalities. Potential pitfalls of this work include the possibility of introducing random genetic mutations during the reprogramming process, the inadequacy of using neurons from other patients as controls, the inability to capture the complex environmental contribution to schizophrenia pathogenesis, the difficulty in modelling neurodevelopment, and the difficulty in modelling the interaction of multiple neuronal and non-neuronal cell types. However, with the increasing sophistication of available reprogramming techniques, co-culture technology, and gene correction strategies, iPSC-derived neurons will continue to elucidate how neuronal function is disrupted in schizophrenia. Copyright © 2015 Elsevier B.V. All rights reserved.

Ajmaline blocks INa and IKr without eliciting differences between Brugada syndrome patient and control human pluripotent stem cell-derived cardiac clusters.

PubMed

Miller, Duncan C; Harmer, Stephen C; Poliandri, Ariel; Nobles, Muriel; Edwards, Elizabeth C; Ware, James S; Sharp, Tyson V; McKay, Tristan R; Dunkel, Leo; Lambiase, Pier D; Tinker, Andrew

2017-12-01

The class Ia anti-arrhythmic drug ajmaline is used clinically to unmask latent type I ECG in Brugada syndrome (BrS) patients, although its mode of action is poorly characterised. Our aims were to identify ajmaline's mode of action in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), and establish a simple BrS hiPSC platform to test whether differences in ajmaline response could be determined between BrS patients and controls. Control hiPSCs were differentiated into spontaneously contracting cardiac clusters. It was found using multi electrode array (MEA) that ajmaline treatment significantly lengthened cluster activation-recovery interval. Patch clamping of single CMs isolated from clusters revealed that ajmaline can block both I Na and I Kr . Following generation of hiPSC lines from BrS patients (absent of pathogenic SCN5A sodium channel mutations), analysis of hiPSC-CMs from patients and controls revealed that differentiation and action potential parameters were similar. Comparison of cardiac clusters by MEA showed that ajmaline lengthened activation-recovery interval consistently across all lines. We conclude that ajmaline can block both depolarisation and repolarisation of hiPSC-CMs at the cellular level, but that a more refined integrated tissue model may be necessary to elicit differences in its effect between BrS patients and controls. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease

PubMed Central

Sánchez-Danés, Adriana; Richaud-Patin, Yvonne; Carballo-Carbajal, Iria; Jiménez-Delgado, Senda; Caig, Carles; Mora, Sergio; Di Guglielmo, Claudia; Ezquerra, Mario; Patel, Bindiben; Giralt, Albert; Canals, Josep M; Memo, Maurizio; Alberch, Jordi; López-Barneo, José; Vila, Miquel; Cuervo, Ana Maria; Tolosa, Eduard; Consiglio, Antonella; Raya, Angel

2012-01-01

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type. PMID:22407749

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.

Generation of human induced pluripotent stem cells from urinary cells of a healthy donor using a non-integration system.

PubMed

Uhm, Kyung-Ok; Jo, Eun Hee; Go, Gue Youn; Kim, So-Jung; Choi, Hye Young; Im, Young Sam; Ha, Hye-Yeong; Jung, Ji-Won; Koo, Soo Kyung

2017-05-01

Urinary cells can be an ideal source for generating hiPSCs and progenitors, as they are easily accessible, non-invasive, and universally available. We generated human induced pluripotent stem cells (hiPSCs) from the urinary cells of a healthy donor using a Sendai virus-based gene delivery method. The generated hiPSC line, KSCBi001-A, has a normal karyotype (46,XY). The pluripotency and capacity of multilineage differentiation were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from National Stem Cell Bank, Korea National Institute of Health. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

PubMed

Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

2017-10-10

Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

Engineering the human pluripotent stem cell microenvironment to direct cell fate

PubMed Central

Hazeltine, Laurie B.; Selekman, Joshua A.; Palecek, Sean P.

2013-01-01

Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell-cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystems technologies for high-throughput screening of spatial-temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types. PMID:23510904

Engineering the human pluripotent stem cell microenvironment to direct cell fate.

PubMed

Hazeltine, Laurie B; Selekman, Joshua A; Palecek, Sean P

2013-11-15

Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell-cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystem technologies for high-throughput screening of spatial-temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types. Copyright © 2013 Elsevier Inc. All rights reserved.

Cellular trajectories and molecular mechanisms of iPSC reprogramming.

PubMed

Apostolou, Effie; Stadtfeld, Matthias

2018-06-16

The discovery of induced pluripotent stem cells (iPSCs) has solidified the concept of transcription factors as major players in controlling cell identity and provided a tractable tool to study how somatic cell identity can be dismantled and pluripotency established. A number of landmark studies have established hallmarks and roadmaps of iPSC formation by describing relative kinetics of transcriptional, protein and epigenetic changes, including alterations in DNA methylation and histone modifications. Recently, technological advancements such as single-cell analyses, high-resolution genome-wide chromatin assays and more efficient reprogramming systems have been used to challenge and refine our understanding of the reprogramming process. Here, we will outline novel insights into the molecular mechanisms underlying iPSC formation, focusing on how the core reprogramming factors OCT4, KLF4, SOX2 and MYC (OKSM) drive changes in gene expression, chromatin state and 3D genome topology. In addition, we will discuss unexpected consequences of reprogramming factor expression in in vitro and in vivo systems that may point towards new applications of iPSC technology. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Alternative Splicing of MBD2 Supports Self-Renewal in Human Pluripotent Stem Cells

PubMed Central

Lu, Yu; Loh, Yuin-Han; Li, Hu; Cesana, Marcella; Ficarro, Scott B.; Parikh, Jignesh R.; Salomonis, Nathan; Toh, Cheng-Xu Delon; Andreadis, Stelios T.; Luckey, C. John; Collins, James J.; Daley, George Q.; Marto, Jarrod A.

2014-01-01

Summary Alternative RNA splicing (AS) regulates proteome diversity, including isoform-specific expression of several pluripotency genes. Here, we integrated global gene expression and proteomic analyses and identified a molecular signature suggesting a central role for AS in maintaining human pluripotent stem cell (hPSC) self-renewal. We demonstrate the splicing factor SFRS2 is an OCT4 target gene required for pluripotency. SFRS2 regulates AS of the methyl-CpG-binding protein MBD2, whose isoforms play opposing roles in maintenance of, and reprogramming to, pluripotency. While both MDB2a and MBD2c are enriched at the OCT4 and NANOG promoters, MBD2a preferentially interacts with repressive NuRD chromatin remodeling factors and promotes hPSC differentiation, whereas overexpression of MBD2c enhances reprogramming of fibroblasts to pluripotency. The miR-301 and miR-302 families provide additional regulation by targeting SFRS2 and MDB2a. These data suggest that OCT4, SFRS2, and MBD2 participate in a positive feedback loop, regulating proteome diversity complexity in support of hPSC self-renewal and reprogramming. PMID:24813856

Zika virus crosses an in vitro human blood brain barrier model.

PubMed

Alimonti, Judie B; Ribecco-Lutkiewicz, Maria; Sodja, Caroline; Jezierski, Anna; Stanimirovic, Danica B; Liu, Qing; Haqqani, Arsalan S; Conlan, Wayne; Bani-Yaghoub, Mahmud

2018-05-15

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.

Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.

PubMed

Liu, Chun; Oikonomopoulos, Angelos; Sayed, Nazish; Wu, Joseph C

2018-03-08

The advent of human induced pluripotent stem cells (iPSCs) presents unprecedented opportunities to model human diseases. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers have proven to be a relatively simple tool for exploring disease pathogenesis and underlying mechanisms. In this Spotlight article, we discuss the progress and limitations of the current 2D iPSC disease-modeling platform, as well as recent advancements in the development of human iPSC models that mimic in vivo tissues and organs at the three-dimensional (3D) level. Recent bioengineering approaches have begun to combine different 3D organoid types into a single '4D multi-organ system'. We summarize the advantages of this approach and speculate on the future role of 4D multi-organ systems in human disease modeling. © 2018. Published by The Company of Biologists Ltd.

TALEN- and CRISPR/Cas9-Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid-Based Transfection.

PubMed

Hendriks, William T; Jiang, Xin; Daheron, Laurence; Cowan, Chad A

2015-08-03

Using custom-engineered nuclease-mediated genome editing, such as Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can be generated and differentiated into various cell types. This strategy of genome engineering in hPSCs will prove invaluable for studying human biology and disease. Here, we provide a detailed protocol for design and construction of TALEN and CRISPR vectors, testing of their nuclease activity, and delivery of TALEN or CRISPR vectors into hPSCs. In addition, we describe the use of single-stranded oligodeoxynucleotides (ssODNs) to introduce or repair point mutations. Next, we describe the identification of edited hPSC clones without antibiotic selection, including their clonal selection, genotyping, and expansion for downstream applications. Copyright © 2015 John Wiley & Sons, Inc.

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.

PubMed

Yumlu, Saniye; Stumm, Jürgen; Bashir, Sanum; Dreyer, Anne-Kathrin; Lisowski, Pawel; Danner, Eric; Kühn, Ralf

2017-05-15

Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones. Copyright © 2017 Elsevier Inc. All rights reserved.

Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.

PubMed

Kim, Han-Seop; Lee, Jungwoon; Lee, Da Yong; Kim, Young-Dae; Kim, Jae Yun; Lim, Hyung Jin; Lim, Sungmin; Cho, Yee Sook

2017-06-06

Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation

PubMed Central

Song, Wei; Lu, Yen-Chun; Frankel, Angela S.; An, Duo; Schwartz, Robert E.; Ma, Minglin

2015-01-01

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies. PMID:26592180

A single-cell and feeder-free culture system for monkey embryonic stem cells.

PubMed

Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

2014-01-01

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

PubMed

Rodrigues, Gonçalo M C; Fernandes, Tiago G; Rodrigues, Carlos A V; Cabral, Joaquim M S; Diogo, Maria Margarida

2015-01-01

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

Binary colloidal crystals (BCCs) as a feeder-free system to generate human induced pluripotent stem cells (hiPSCs)

PubMed Central

Wang, Peng-Yuan; Hung, Sandy Shen-Chi; Thissen, Helmut; Kingshott, Peter; Wong, Raymond Ching-Bong

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of differentiating into any cell type and provide significant advances to cell therapy and regenerative medicine. However, the current protocol for hiPSC generation is relatively inefficient and often results in many partially reprogrammed colonies, which increases the cost and reduces the applicability of hiPSCs. Biophysical stimulation, in particular from tuning cell-surface interactions, can trigger specific cellular responses that could in turn promote the reprogramming process. In this study, human fibroblasts were reprogrammed into hiPSCs using a feeder-free system and episomal vectors using novel substrates based on binary colloidal crystals (BCCs). BCCs are made from two different spherical particle materials (Si and PMMA) ranging in size from nanometers to micrometers that self-assemble into hexagonal close-packed arrays. Our results show that the BCCs, particularly those made from a crystal of 2 μm Si and 0.11 μm PMMA particles (2SiPM) facilitate the reprogramming process and increase the proportion of fully reprogrammed hiPSC colonies, even without a vitronectin coating. Subsequent isolation of clonal hiPSC lines demonstrates that they express pluripotent markers (OCT4 and TRA-1-60). This proof-of-concept study demonstrates that cell reprogramming can be improved on substrates where surface properties are tailored to the application. PMID:27833126

Modulation of Human Allogeneic and Syngeneic Pluripotent Stem Cells and Immunological Implications for Transplantation

PubMed Central

Sackett, S.D.; Brown, M.E.; Tremmel, D.M.; Ellis, T.; Burlingham, W.J.; Odorico, J.S.

2016-01-01

Tissues derived from induced pluripotent stem cells (iPSCs) are a promising source of cells for building various regenerative medicine therapies; from simply transplanting cells to reseeding decellularized organs to reconstructing multicellular tissues. Although reprogramming strategies for producing iPSCs have improved, the clinical use of iPSCs is limited by the presence of unique human leukocyte antigen (HLA) genes, the main immunologic barrier to transplantation. In order to overcome the immunological hurdles associated with allogeneic tissues and organs, the generation of patient-histocompatible iPSCs (autologous or HLA-matched cells) provides an attractive platform for personalized medicine. However, concerns have been raised as to the fitness, safety and immunogenicity of iPSC derivatives because of variable differentiation potential of different lines and the identification of genetic and epigenetic aberrations that can occur during the reprogramming process. In addition, significant cost and regulatory barriers may deter commercialization of patient specific therapies in the short-term. Nonetheless, recent studies provide some evidence of immunological benefit for using autologous iPSCs. Yet, more studies are needed to evaluate the immunogenicity of various autologous and allogeneic human iPSC-derived cell types as well as test various methods to abrogate rejection. Here, we present perspectives of using allogeneic vs autologous iPSCs for transplantation therapies and the advantages and disadvantages of each related to differentiation potential, immunogenicity, genetic stability and tumorigenicity. We also review the current literature on the immunogenicity of syngeneic iPSCs and discuss evidence that questions the feasibility of HLA-matched iPSC banks. Finally, we will discuss emerging methods of abrogating or reducing host immune responses to PSC derivatives. PMID:26970668

Modulation of human allogeneic and syngeneic pluripotent stem cells and immunological implications for transplantation.

PubMed

Sackett, S D; Brown, M E; Tremmel, D M; Ellis, T; Burlingham, W J; Odorico, J S

2016-04-01

Tissues derived from induced pluripotent stem cells (iPSCs) are a promising source of cells for building various regenerative medicine therapies; from simply transplanting cells to reseeding decellularized organs to reconstructing multicellular tissues. Although reprogramming strategies for producing iPSCs have improved, the clinical use of iPSCs is limited by the presence of unique human leukocyte antigen (HLA) genes, the main immunologic barrier to transplantation. In order to overcome the immunological hurdles associated with allogeneic tissues and organs, the generation of patient-histocompatible iPSCs (autologous or HLA-matched cells) provides an attractive platform for personalized medicine. However, concerns have been raised as to the fitness, safety and immunogenicity of iPSC derivatives because of variable differentiation potential of different lines and the identification of genetic and epigenetic aberrations that can occur during the reprogramming process. In addition, significant cost and regulatory barriers may deter commercialization of patient specific therapies in the short-term. Nonetheless, recent studies provide some evidence of immunological benefit for using autologous iPSCs. Yet, more studies are needed to evaluate the immunogenicity of various autologous and allogeneic human iPSC-derived cell types as well as test various methods to abrogate rejection. Here, we present perspectives of using allogeneic vs. autologous iPSCs for transplantation therapies and the advantages and disadvantages of each related to differentiation potential, immunogenicity, genetic stability and tumorigenicity. We also review the current literature on the immunogenicity of syngeneic iPSCs and discuss evidence that questions the feasibility of HLA-matched iPSC banks. Finally, we will discuss emerging methods of abrogating or reducing host immune responses to PSC derivatives. Copyright © 2016 Elsevier Inc. All rights reserved.

Divergent effects of adrenaline in human induced pluripotent stem cell-derived cardiomyocytes obtained from hypertrophic cardiomyopathy

PubMed Central

Prajapati, Chandra

2018-01-01

ABSTRACT Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disease that affects the heart muscle with diverse clinical outcomes. HCM can cause sudden cardiac death (SCD) during or immediately after mild to rigorous physical activity in young patients. However, the mechanism causing SCD as a result of exercise remains unknown, but exercise-induced ventricular arrhythmias are thought to be responsible for this fatal consequence. To understand the disease mechanism behind HCM in a better way, we generated patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from HCM patients carrying either the MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. We extensively investigated the effects of low to high concentrations of adrenaline on action potential characteristics, and the occurrence of arrhythmias in the presence of various concentrations of adrenaline and in wash-out condition. We classified and quantified different types of arrhythmias observed in hiPSC-CMs, and found that the occurrence of arrhythmias was dependent on concentrations of adrenaline and positions of mutations in genes causing HCM. In addition, we observed ventricular tachycardia types of arrhythmias in hiPSC-CMs carrying the TPM1-Asp175Asn mutation. We additionally examined the antiarrhythmic potency of bisoprolol in HCM-specific hiPSC-CMs. However, bisoprolol could not reduce the occurrence of arrhythmias during administration or during the wash-out condition of adrenaline in HCM-specific hiPSC-CMs. Our study demonstrates hiPSC-CMs as a promising tool for studying HCM. The experimental design used in this study could be suitable and beneficial for studying other components and drugs related to cardiac disease in general. PMID:29361520

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

PubMed Central

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A.; de Boer, Jan; Watt, Fiona M.

2016-01-01

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation. PMID:26757610

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells.

PubMed

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A; de Boer, Jan; Watt, Fiona M

2016-01-13

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation.

A High Proliferation Rate is Critical for Reproducible and Standardized Embryoid Body Formation from Laminin-521-Based Human Pluripotent Stem Cell Cultures.

PubMed

Dziedzicka, Dominika; Markouli, Christina; Barbé, Lise; Spits, Claudia; Sermon, Karen; Geens, Mieke

2016-12-01

When aiming for homogenous embryoid body (EB) differentiation, the use of equal-sized EBs is required to avoid a size-induced differentiation bias. In this study we developed an efficient and standardized EB formation protocol for human pluripotent stem cells (hPSC) cultured in a laminin-521-based xeno-free system. As the cell proliferation rate of the cells growing on laminin-521 strongly affected the efficiency of aggregate formation, we found that recently passaged cells, as well as the addition of ROCK inhibitor, were essential for reproducible EB formation from hPSC single-cell suspensions. EBs could be obtained in a variety of differentiation media, in 96-well round-bottom plates and in hanging drops. Gene expression studies on differentially sized EBs from three individual human embryonic stem cell lines demonstrated that the medium used for differentiation influenced the differentiation outcome to a much greater extent than the number of cells used for the initial EB formation. Our findings give a new insight into factors that influence the EB formation and differentiation process. This optimized method allows us to easily manipulate EB formation and provide an excellent starting point for downstream EB-based differentiation protocols.

Induced pluripotent stem cell-based therapy for age-related macular degeneration.

PubMed

Bracha, Peter; Moore, Nicholas A; Ciulla, Thomas A

2017-09-01

In age-related macular degeneration (AMD), stem cells could possibly replace or regenerate disrupted pathologic retinal pigment epithelium (RPE), and produce supportive growth factors and cytokines such as brain-derived neurotrophic factor.  Induced pluripotent stem cells (iPSCs)-derived RPE was first subretinally transplanted in a neovascular AMD patient in 2014. Areas covered: Induced PSCs are derived from the introduction of transcription factors to adult cells under specific cell culture conditions, followed by differentiation into RPE cells. Induced PSC-derived RPE cells exhibit ion transport, membrane potential, polarized VEGF secretion and gene expression that is similar to native RPE. Despite having similar in vitro function, morphology, immunostaining and microscopic analysis, it remains to be seen if iPSC-derived RPE can replicate the myriad of in vivo functions, including immunomodulatory effects, of native RPE cells.  Historically, adjuvant RPE transplantation during CNV resections were technically difficult and complicated by immune rejection. Autologous iPSCs are hypothesized to reduce the risk of immune rejection, but their production is time-consuming and expensive.  Alternatively, allogenic transplantation using human leukocyte antigen (HLA)-matched iPSCs, similar to HLA-matched organ transplantation, is currently being investigated. Expert opinion: Challenges to successful transplantation with iPSCs include surgical technique, a pathologic subretinal microenvironment, possible immune rejection, and complications of immunosuppression.

Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system.

PubMed

Song, Bing; Fan, Yong; He, Wenyin; Zhu, Detu; Niu, Xiaohua; Wang, Ding; Ou, Zhanhui; Luo, Min; Sun, Xiaofang

2015-05-01

The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.

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

A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells.

PubMed

Gunhanlar, N; Shpak, G; van der Kroeg, M; Gouty-Colomer, L A; Munshi, S T; Lendemeijer, B; Ghazvini, M; Dupont, C; Hoogendijk, W J G; Gribnau, J; de Vrij, F M S; Kushner, S A

2018-05-01

Progress in elucidating the molecular and cellular pathophysiology of neuropsychiatric disorders has been hindered by the limited availability of living human brain tissue. The emergence of induced pluripotent stem cells (iPSCs) has offered a unique alternative strategy using patient-derived functional neuronal networks. However, methods for reliably generating iPSC-derived neurons with mature electrophysiological characteristics have been difficult to develop. Here, we report a simplified differentiation protocol that yields electrophysiologically mature iPSC-derived cortical lineage neuronal networks without the need for astrocyte co-culture or specialized media. This protocol generates a consistent 60:40 ratio of neurons and astrocytes that arise from a common forebrain neural progenitor. Whole-cell patch-clamp recordings of 114 neurons derived from three independent iPSC lines confirmed their electrophysiological maturity, including resting membrane potential (-58.2±1.0 mV), capacitance (49.1±2.9 pF), action potential (AP) threshold (-50.9±0.5 mV) and AP amplitude (66.5±1.3 mV). Nearly 100% of neurons were capable of firing APs, of which 79% had sustained trains of mature APs with minimal accommodation (peak AP frequency: 11.9±0.5 Hz) and 74% exhibited spontaneous synaptic activity (amplitude, 16.03±0.82 pA; frequency, 1.09±0.17 Hz). We expect this protocol to be of broad applicability for implementing iPSC-based neuronal network models of neuropsychiatric disorders.

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.

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.

PubMed

Yang, Xiulan; Rodriguez, Marita; Pabon, Lil; Fischer, Karin A; Reinecke, Hans; Regnier, Michael; Sniadecki, Nathan J; Ruohola-Baker, Hannele; Murry, Charles E

2014-07-01

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens. Copyright © 2014 Elsevier Ltd. All rights reserved.

Human pluripotent stem cells: an emerging model in developmental biology.

PubMed

Zhu, Zengrong; Huangfu, Danwei

2013-02-01

Developmental biology has long benefited from studies of classic model organisms. Recently, human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, have emerged as a new model system that offers unique advantages for developmental studies. Here, we discuss how studies of hPSCs can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development 'in a dish'. We also summarize some of the recently developed genetic tools that greatly facilitate the interrogation of gene function during hPSC differentiation. With the development of high-throughput screening technologies, hPSCs have the potential to revolutionize gene discovery in mammalian development.

The Therapeutic Potential of Induced Pluripotent Stem Cells After Stroke: Evidence from Rodent Models.

PubMed

Zents, Karlijn; Copray, Sjef

2016-01-01

Stroke is the second most common cause of death and the leading cause of disability in the world. About 30% of the people that are affected by stroke die within a year; 25% of the patients that survive stroke remain in need of care after a year. Therefore, stroke is a major burden for health care costs. The most common subtype is ischemic stroke. This type is characterized by a reduced and insufficient blood supply to a certain part of the brain. Despite the high prevalence of stroke, the currently used therapeutic interventions are limited. No therapies that aim to restore damaged neuronal tissue or to promote recovery are available nowadays. Transplantation of stem cell-derived cells has been investigated as a potential regenerative and protective treatment. Embryonic stem cell (ESC)-based cell therapy in rodent models of stroke has been shown to improve functional outcome. However, the clinical use of ESCs still raises ethical questions and implantation of ESC-derived cells requires continuous immunosuppression. The groundbreaking detection of induced pluripotent stem cells (iPSCs) has provided a most promising alternative. This mini-review summarizes current literature in which the potential use of iPSC-derived cells has been tested in rodent models of stroke. iPSC-based cell therapy has been demonstrated to improve motor function, decrease stroke volume, promote neurogenesis and angiogenesis and to exert immunomodulatory, anti-inflammatory effects in the brain of stroke-affected rodents.

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening

PubMed Central

Smith, Alec S.T.; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A.; Kim, Deok-Ho

2016-01-01

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. PMID:28007615

Process-Based Expansion and Neural Differentiation of Human Pluripotent Stem Cells for Transplantation and Disease Modeling

PubMed Central

Stover, Alexander E.; Brick, David J.; Nethercott, Hubert E.; Banuelos, Maria G.; Sun, Lei; O’Dowd, Diane K.; Schwartz, Philip H.

2014-01-01

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials. PMID:23893392

Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications.

PubMed

Baghbaderani, Behnam Ahmadian; Syama, Adhikarla; Sivapatham, Renuka; Pei, Ying; Mukherjee, Odity; Fellner, Thomas; Zeng, Xianmin; Rao, Mahendra S

2016-08-01

We have recently described manufacturing of human induced pluripotent stem cells (iPSC) master cell banks (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Reports, 5(4), 647-659, 2015). In this manuscript, we describe the detailed characterization of the two iPSC clones generated using this process, including whole genome sequencing (WGS), microarray, and comparative genomic hybridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibration material and with a reporter subclone and lines made by a similar process from different donors. We believe that iPSCs are likely to be used to make multiple clinical products. We further believe that the lines used as input material will be used at different sites and, given their immortal status, will be used for many years or even decades. Therefore, it will be important to develop assays to monitor the state of the cells and their drift in culture. We suggest that a detailed characterization of the initial status of the cells, a comparison with some calibration material and the development of reporter sublcones will help determine which set of tests will be most useful in monitoring the cells and establishing criteria for discarding a line.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

PubMed

Meraviglia, Viviana; Zanon, Alessandra; Lavdas, Alexandros A; Schwienbacher, Christine; Silipigni, Rosamaria; Di Segni, Marina; Chen, Huei-Sheng Vincent; Pramstaller, Peter P; Hicks, Andrew A; Rossini, Alessandra

2015-06-05

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease.

PubMed

Fox, Ira J; Daley, George Q; Goldman, Steven A; Huard, Johnny; Kamp, Timothy J; Trucco, Massimo

2014-08-22

Pluripotent stem cells (PSCs) directed to various cell fates holds promise as source material for treating numerous disorders. The availability of precisely differentiated PSC-derived cells will dramatically affect blood component and hematopoietic stem cell therapies and should facilitate treatment of diabetes, some forms of liver disease and neurologic disorders, retinal diseases, and possibly heart disease. Although an unlimited supply of specific cell types is needed, other barriers must be overcome. This review of the state of cell therapies highlights important challenges. Successful cell transplantation will require optimizing the best cell type and site for engraftment, overcoming limitations to cell migration and tissue integration, and occasionally needing to control immunologic reactivity, as well as a number of other challenges. Collaboration among scientists, clinicians, and industry is critical for generating new stem cell-based therapies. Copyright © 2014, American Association for the Advancement of Science.

Corneal repair by human corneal keratocyte-reprogrammed iPSCs and amphiphatic carboxymethyl-hexanoyl chitosan hydrogel.

PubMed

Chien, Yueh; Liao, Yi-Wen; Liu, Dean-Mo; Lin, Heng-Liang; Chen, Shih-Jen; Chen, Hen-Li; Peng, Chi-Hsien; Liang, Chang-Min; Mou, Chung-Yuan; Chiou, Shih-Hwa

2012-11-01

Induced pluripotent stem cells (iPSCs) have promising potential in regenerative medicine, but whether iPSCs can promote corneal reconstruction remains undetermined. In this study, we successfully reprogrammed human corneal keratocytes into iPSCs. To prevent feeder cell contamination, these iPSCs were cultured onto a serum- and feeder-free system in which they remained stable through 30 passages and showed ESC-like pluripotent property. To investigate the availability of iPSCs as bioengineered substitutes in corneal repair, we developed a thermo-gelling injectable amphiphatic carboxymethyl-hexanoyl chitosan (CHC) nanoscale hydrogel and found that such gel increased the viability and CD44+proportion of iPSCs, and maintained their stem-cell like gene expression, in the presence of culture media. Combined treatment of iPSC with CHC hydrogel (iPSC/CHC hydrogel) facilitated wound healing in surgical abrasion-injured corneas. In severe corneal damage induced by alkaline, iPSC/CHC hydrogel enhanced corneal reconstruction by downregulating oxidative stress and recruiting endogenous epithelial cells to restore corneal epithelial thickness. Therefore, we demonstrated that these human keratocyte-reprogrammed iPSCs, when combined with CHC hydrogel, can be used as a rapid delivery system to efficiently enhance corneal wound healing. In addition, iPSCs reprogrammed from corneal surgical residues may serve as an alternative cell source for personalized therapies for human corneal damage. Copyright © 2012 Elsevier Ltd. All rights reserved.

Combinatorial Polymer Matrices Enhance In Vitro Maturation of Human Induced Pluripotent Cell Cell-Derived Cardiomyocytes

PubMed Central

Chun, Young Wook; Balikov, Daniel A.; Feaster, Tromondae K.; Williams, Charles H.; Sheng, Calvin C.; Lee, Jung-Bok; Boire, Timothy C.; Neely, M. Diana; Bellan, Leon M.; Ess, Kevin C.; Bowman, Aaron B.; Sung, Hak-Joon; Hong, Charles C.

2015-01-01

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-95%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions. PMID:26204225

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.

PubMed

Lai, Frank Pui-Ling; Lau, Sin-Ting; Wong, John Kwong-Leong; Gui, Hongsheng; Wang, Reeson Xu; Zhou, Tingwen; Lai, Wing Hon; Tse, Hung-Fat; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Ngan, Elly Sau-Wai

2017-07-01

Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET +/- and RET -/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells.

PubMed

Stebbins, Matthew J; Wilson, Hannah K; Canfield, Scott G; Qian, Tongcheng; Palecek, Sean P; Shusta, Eric V

2016-05-15

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties, the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease, yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently, in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here, we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R; Weissman, Amir; Binah, Ofer

2016-12-01

Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Induced pluripotent stem cell-derived gamete-associated proteins incite rejection of induced pluripotent stem cells in syngeneic mice.

PubMed

Kim, Eun-Mi; Manzar, Gohar; Zavazava, Nicholas

2017-06-01

The safety of induced pluripotent stem cells (iPSCs) in autologous recipients has been questioned after iPSCs, but not embryonic stem cells (ESCs), were reported to be rejected in syngeneic mice. This important topic has remained controversial because there has not been a mechanistic explanation for this phenomenon. Here, we hypothesize that iPSCs, but not ESCs, readily differentiate into gamete-forming cells that express meiotic antigens normally found in immune-privileged gonads. Because peripheral blood T cells are not tolerized to these antigens in the thymus, gamete-associated-proteins (GAPs) sensitize T cells leading to rejection. Here, we provide evidence that GAPs expressed in iPSC teratomas, but not in ESC teratomas, are responsible for the immunological rejection of iPSCs. Furthermore, silencing the expression of Stra8, 'the master regulator of meiosis', in iPSCs, using short hairpin RNA led to significant abrogation of the rejection of iPSCs, supporting our central hypothesis that GAPs expressed after initiation of meiosis in iPSCs were responsible for rejection. In contrast to iPSCs, iPSC-derivatives, such as haematopoietic progenitor cells, are able to engraft long-term into syngeneic recipients because they no longer express GAPs. Our findings, for the first time, provide a unifying explanation of why iPSCs, but not ESCs, are rejected in syngeneic recipients, ending the current controversy on the safety of iPSCs and their derivatives. © 2017 John Wiley & Sons Ltd.

Large-scale production of human pluripotent stem cell derived cardiomyocytes.

PubMed

Kempf, Henning; Andree, Birgit; Zweigerdt, Robert

2016-01-15

Regenerative medicine, including preclinical studies in large animal models and tissue engineering approaches as well as innovative assays for drug discovery, will require the constant supply of hPSC-derived cardiomyocytes and other functional progenies. Respective cell production processes must be robust, economically viable and ultimately GMP-compliant. Recent research has enabled transition of lab scale protocols for hPSC expansion and cardiomyogenic differentiation towards more controlled processing in industry-compatible culture platforms. Here, advanced strategies for the cultivation and differentiation of hPSCs will be reviewed by focusing on stirred bioreactor-based techniques for process upscaling. We will discuss how cardiomyocyte mass production might benefit from recent findings such as cell expansion at the cardiovascular progenitor state. Finally, remaining challenges will be highlighted, specifically regarding three dimensional (3D) hPSC suspension culture and critical safety issues ahead of clinical translation. Copyright © 2015 Elsevier B.V. All rights reserved.

A single cell high content assay detects mitochondrial dysfunction in iPSC-derived neurons with mutations in SNCA.

PubMed

Little, Daniel; Luft, Christin; Mosaku, Olukunbi; Lorvellec, Maëlle; Yao, Zhi; Paillusson, Sébastien; Kriston-Vizi, Janos; Gandhi, Sonia; Abramov, Andrey Y; Ketteler, Robin; Devine, Michael J; Gissen, Paul

2018-06-13

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson's disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Patient-derived iPSCs show premature neural differentiation and neuron-type specific phenotypes relevant to neurodevelopment

PubMed Central

Yeh, Erika; Dao, Dang Q.; Wu, Zhi Y.; Kandalam, Santoshi M.; Camacho, Federico M.; Tom, Curtis; Zhang, Wandong; Krencik, Robert; Rauen, Katherine A.; Ullian, Erik M.; Weiss, Lauren A.

2017-01-01

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events. PMID:29158583

hiPSC-derived neural stem cells from patients with schizophrenia induce an impaired angiogenesis.

PubMed

Casas, Bárbara S; Vitória, Gabriela; do Costa, Marcelo N; Madeiro da Costa, Rodrigo; Trindade, Pablo; Maciel, Renata; Navarrete, Nelson; Rehen, Stevens K; Palma, Verónica

2018-02-22

Schizophrenia is a neurodevelopmental disease characterized by cerebral connectivity impairment and loss of gray matter. It was described in adult schizophrenia patients (SZP) that concentration of VEGFA, a master angiogenic factor, is decreased. Recent evidence suggests cerebral hypoperfusion related to a dysfunctional Blood Brain Barrier (BBB) in SZP. Since neurogenesis and blood-vessel formation occur in a coincident and coordinated fashion, a defect in neurovascular development could result in increased vascular permeability and, therefore, in poor functionality of the SZP's neurons. Here, we characterized the conditioned media (CM) of human induced Pluripotent Stem Cells (hiPSC)-derived Neural Stem Cells of SZP (SZP NSC) versus healthy subjects (Ctrl NSC), and its impact on angiogenesis. Our results reveal that SZP NSC have an imbalance in the secretion and expression of several angiogenic factors, among them non-canonical neuro-angiogenic guidance factors. SZP NSC migrated less and their CM was less effective in inducing migration and angiogenesis both in vitro and in vivo. Since SZP originates during embryonic brain development, our findings suggest a defective crosstalk between NSC and endothelial cells (EC) during the formation of the neuro-angiogenic niche.

Disease Modeling Using 3D Organoids Derived from Human Induced Pluripotent Stem Cells.

PubMed

Ho, Beatrice Xuan; Pek, Nicole Min Qian; Soh, Boon-Seng

2018-03-21

The rising interest in human induced pluripotent stem cell (hiPSC)-derived organoid culture has stemmed from the manipulation of various combinations of directed multi-lineage differentiation and morphogenetic processes that mimic organogenesis. Organoids are three-dimensional (3D) structures that are comprised of multiple cell types, self-organized to recapitulate embryonic and tissue development in vitro. This model has been shown to be superior to conventional two-dimensional (2D) cell culture methods in mirroring functionality, architecture, and geometric features of tissues seen in vivo. This review serves to highlight recent advances in the 3D organoid technology for use in modeling complex hereditary diseases, cancer, host-microbe interactions, and possible use in translational and personalized medicine where organoid cultures were used to uncover diagnostic biomarkers for early disease detection via high throughput pharmaceutical screening. In addition, this review also aims to discuss the advantages and shortcomings of utilizing organoids in disease modeling. In summary, studying human diseases using hiPSC-derived organoids may better illustrate the processes involved due to similarities in the architecture and microenvironment present in an organoid, which also allows drug responses to be properly recapitulated in vitro.

Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain.

PubMed

Emborg, Marina E; Liu, Yan; Xi, Jiajie; Zhang, Xiaoqing; Yin, Yingnan; Lu, Jianfeng; Joers, Valerie; Swanson, Christine; Holden, James E; Zhang, Su-Chun

2013-03-28

The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Cryopreservation of embryonic stem cell-derived multicellular neural aggregates labeled with micron-sized particles of iron oxide for magnetic resonance imaging.

PubMed

Yan, Yuanwei; Sart, Sébastien; Calixto Bejarano, Fabian; Muroski, Megan E; Strouse, Geoffrey F; Grant, Samuel C; Li, Yan

2015-01-01

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)-derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre-labeled neural cells, especially in three-dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC-derived multicellular NPC aggregates labeled with micron-sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70-80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post-cryopreservation. MRI analysis showed comparable detectability for the MPIO-labeled cells before and after cryopreservation indicated by T2 and T2* relaxation rates. Cryopreserving MPIO-labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers.

Development and function of human cerebral cortex neural networks from pluripotent stem cells in vitro

PubMed Central

Kirwan, Peter; Turner-Bridger, Benita; Peter, Manuel; Momoh, Ayiba; Arambepola, Devika; Robinson, Hugh P. C.; Livesey, Frederick J.

2015-01-01

A key aspect of nervous system development, including that of the cerebral cortex, is the formation of higher-order neural networks. Developing neural networks undergo several phases with distinct activity patterns in vivo, which are thought to prune and fine-tune network connectivity. We report here that human pluripotent stem cell (hPSC)-derived cerebral cortex neurons form large-scale networks that reflect those found in the developing cerebral cortex in vivo. Synchronised oscillatory networks develop in a highly stereotyped pattern over several weeks in culture. An initial phase of increasing frequency of oscillations is followed by a phase of decreasing frequency, before giving rise to non-synchronous, ordered activity patterns. hPSC-derived cortical neural networks are excitatory, driven by activation of AMPA- and NMDA-type glutamate receptors, and can undergo NMDA-receptor-mediated plasticity. Investigating single neuron connectivity within PSC-derived cultures, using rabies-based trans-synaptic tracing, we found two broad classes of neuronal connectivity: most neurons have small numbers (<10) of presynaptic inputs, whereas a small set of hub-like neurons have large numbers of synaptic connections (>40). These data demonstrate that the formation of hPSC-derived cortical networks mimics in vivo cortical network development and function, demonstrating the utility of in vitro systems for mechanistic studies of human forebrain neural network biology. PMID:26395144

Development and function of human cerebral cortex neural networks from pluripotent stem cells in vitro.

PubMed

Kirwan, Peter; Turner-Bridger, Benita; Peter, Manuel; Momoh, Ayiba; Arambepola, Devika; Robinson, Hugh P C; Livesey, Frederick J

2015-09-15

A key aspect of nervous system development, including that of the cerebral cortex, is the formation of higher-order neural networks. Developing neural networks undergo several phases with distinct activity patterns in vivo, which are thought to prune and fine-tune network connectivity. We report here that human pluripotent stem cell (hPSC)-derived cerebral cortex neurons form large-scale networks that reflect those found in the developing cerebral cortex in vivo. Synchronised oscillatory networks develop in a highly stereotyped pattern over several weeks in culture. An initial phase of increasing frequency of oscillations is followed by a phase of decreasing frequency, before giving rise to non-synchronous, ordered activity patterns. hPSC-derived cortical neural networks are excitatory, driven by activation of AMPA- and NMDA-type glutamate receptors, and can undergo NMDA-receptor-mediated plasticity. Investigating single neuron connectivity within PSC-derived cultures, using rabies-based trans-synaptic tracing, we found two broad classes of neuronal connectivity: most neurons have small numbers (<10) of presynaptic inputs, whereas a small set of hub-like neurons have large numbers of synaptic connections (>40). These data demonstrate that the formation of hPSC-derived cortical networks mimics in vivo cortical network development and function, demonstrating the utility of in vitro systems for mechanistic studies of human forebrain neural network biology. © 2015. Published by The Company of Biologists Ltd.

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

PubMed

Mosqueira, Diogo; Mannhardt, Ingra; Bhagwan, Jamie R; Lis-Slimak, Katarzyna; Katili, Puspita; Scott, Elizabeth; Hassan, Mustafa; Prondzynski, Maksymilian; Harmer, Stephen C; Tinker, Andrew; Smith, James G W; Carrier, Lucie; Williams, Philip M; Gaffney, Daniel; Eschenhagen, Thomas; Hansen, Arne; Denning, Chris

2018-05-08

Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-β-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-βMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient βMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.

Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses.

PubMed

Sima, Ni; Li, Rong; Huang, Wei; Xu, Miao; Beers, Jeanette; Zou, Jizhong; Titus, Steven; Ottinger, Elizabeth A; Marugan, Juan J; Xie, Xing; Zheng, Wei

2018-04-10

Infantile and late infantile neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases affecting the central nervous system (CNS). The infantile NCL (INCL) is caused by mutations in the PPT1 gene and late-infantile NCL (LINCL) is due to mutations in the TPP1 gene. Deficiency in PPT1 or TPP1 enzyme function results in lysosomal accumulation of pathological lipofuscin-like material in the patient cells. There is currently no small-molecular drug treatment for NCLs. We have generated induced pluripotent stem cells (iPSC) from three patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). Using these new disease models, we evaluated the effect of δ-tocopherol (DT) and hydroxypropyl-β-cyclodextrin (HPBCD) with the enzyme replacement therapy as the control. Treatment with the relevant recombinant enzyme or DT significantly ameliorated the lipid accumulation and lysosomal enlargement in the disease cells. A combination therapy of δ-tocopherol and HPBCD further improved the effect compared to that of either drug used as a single therapy. The results demonstrate that these patient iPSC derived NCL NSCs are valid cell- based disease models with characteristic disease phenotypes that can be used for study of disease pathophysiology and drug development.

Pluripotent Stem Cells and Gene Therapy

PubMed Central

Simara, Pavel; Motl, Jason A.; Kaufman, Dan S.

2013-01-01

Human pluripotent stem cells represent an accessible cell source for novel cell-based clinical research and therapies. With the realization of induced pluripotent stem cells (iPSCs), it is possible to produce almost any desired cell type from any patient's cells. Current developments in gene modification methods have opened the possibility for creating genetically corrected human iPSCs for certain genetic diseases that could be used later in autologous transplantation. Promising preclinical studies have demonstrated correction of disease-causing mutations in a number of hematological, neuronal and muscular disorders. This review aims to summarize these recent advances with a focus on iPSC generation techniques, as well as gene modification methods. We will then further discuss some of the main obstacles remaining to be overcome before successful application of human pluripotent stem cell-based therapy arrives in the clinic and what the future of stem cell research may look like. PMID:23353080

The Autism Spectrum Disorders Stem Cell Resource at Children’s Hospital of Orange County: Implications for Disease Modeling and Drug Discovery

PubMed Central

Brick, David J.; Nethercott, Hubert E.; Montesano, Samantha; Banuelos, Maria G.; Stover, Alexander E.; Schutte, Soleil Sun; O’Dowd, Diane K.; Hagerman, Randi J.; Ono, Michele; Hessl, David R.; Tassone, Flora

2014-01-01

The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD-specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action-potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC-based models of ASD-associated diseases. The ASD Stem Cell Resource at the Children’s Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community. PMID:25273538

The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.

PubMed

Brick, David J; Nethercott, Hubert E; Montesano, Samantha; Banuelos, Maria G; Stover, Alexander E; Schutte, Soleil Sun; O'Dowd, Diane K; Hagerman, Randi J; Ono, Michele; Hessl, David R; Tassone, Flora; Schwartz, Philip H

2014-11-01

The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD-specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action-potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC-based models of ASD-associated diseases. The ASD Stem Cell Resource at the Children's Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community. ©AlphaMed Press.

New lessons learned from disease modeling with induced Pluripotent Stem Cells

PubMed Central

Onder, Tamer T.; Daley, George Q.

2012-01-01

Cellular reprogramming and generation of induced pluripotent stem cells (iPSCs) from adult cell types has enabled the creation of patient-specific stem cells for use in disease modeling. To date, many iPSC lines have been generated from a variety of disorders, which have then been differentiated into disease-relevant cell types. When a disease-specific phenotype is detectable in such differentiated cells, the reprogramming technology provides a new opportunity to identify aberrant disease-associated pathways and drugs that can block them. Here, we highlight recent progress as well as limitations in the use of iPSCs to recapitulate disease phenotypes and to screen for therapeutics in vitro. PMID:22749051

Rapid Electrical Stimulation Increased Cardiac Apoptosis Through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

PubMed

Geng, Le; Wang, Zidun; Cui, Chang; Zhu, Yue; Shi, Jiaojiao; Wang, Jiaxian; Chen, Minglong

2018-06-15

Heart failure induced by tachycardia, the most common arrhythmia, is frequently observed in clinical practice. This study was designed to investigate the underlying mechanisms. Rapid electrical stimulation (RES) at a frequency of 3 Hz was applied on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for 7 days, with 8 h/day and 24 h/day set to represent short-term and long-term tachycardia, respectively. Age-matched hiPSC-CMs without electrical stimulation or with slow electrical stimulation (1 Hz) were set as no electrical stimulation (NES) control or low-frequency electrical stimulation (LES) control. Following stimulation, JC-1 staining flow cytometry analysis was performed to examine mitochondrial conditions. Apoptosis in hiPSC-CMs was evaluated using Hoechst staining and Annexin V/propidium iodide (AV/PI) staining flow cytometry analysis. Calcium transients and L-type calcium currents were recorded to evaluate calcium homeostasis. Western blotting and qPCR were performed to evaluate the protein and mRNA expression levels of apoptosis-related genes and calcium homeostasis-regulated genes. Compared to the controls, hiPSC-CMs following RES presented mitochondrial dysfunction and an increased apoptotic percentage. Amplitudes of calcium transients and L-type calcium currents were significantly decreased in hiPSC-CMs with RES. Molecular analysis demonstrated upregulated expression of Caspase3 and increased Bax/Bcl-2 ratio. Genes related to calcium re-sequence were downregulated, while phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) was significantly upregulated following RES. There was no significant difference between the NES control and LES control groups in these aspects. Inhibition of CaMKII with 1 µM KN93 partly reversed these adverse effects of RES. RES on hiPSC-CMs disturbed calcium homeostasis, which led to mitochondrial stress, promoted cell apoptosis and caused electrophysiological remodeling in a time-dependent manner. CaMKII played a central role in the damages induced by RES, pharmacological inhibition of CaMKII activity partly reversed the adverse effects of RES on both structural and electrophysiological properties of cells. © 2018 The Author(s). Published by S. Karger AG, Basel.

Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.

PubMed

Han, Xiaoping; Chen, Haide; Huang, Daosheng; Chen, Huidong; Fei, Lijiang; Cheng, Chen; Huang, He; Yuan, Guo-Cheng; Guo, Guoji

2018-04-05

Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved. We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells. Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming

PubMed Central

Huang, Delun; Wang, Ling; Duan, Jingyue; Huang, Chang; Tian, Xiuchun (Cindy); Zhang, Ming

2018-01-01

ABSTRACT The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation. PMID:29212799

The Combination of CRISPR/Cas9 and iPSC Technologies in the Gene Therapy of Human β-thalassemia in Mice.

PubMed

Ou, Zhanhui; Niu, Xiaohua; He, Wenyin; Chen, Yuchang; Song, Bing; Xian, Yexing; Fan, Di; Tang, Daolin; Sun, Xiaofang

2016-09-01

β-thalassemia results from point mutations or small deletions in the β-globin (HBB) gene that ultimately cause anemia. The generation of induced pluripotent stem cells (iPSCs) from the somatic cells of patients in combination with subsequent homologous recombination-based gene correction provides new approaches to cure this disease. CRISPR/Cas9 is a genome editing tool that is creating a buzz in the scientific community for treating human diseases, especially genetic disorders. Here, we reported that correction of β-thalassemia mutations in patient-specific iPSCs using the CRISPR/Cas9 tool promotes hematopoietic differentiation in vivo. CRISPR/Cas9-corrected iPSC-derived hematopoietic stem cells (HSCs) were injected into sublethally-irradiated NOD-scid-IL2Rg-/- (NSI) mice. HBB expression was observed in these HSCs after hematopoietic differentiation in the NSI mice. Importantly, no tumor was found in the livers, lungs, kidneys, or bone marrow at 10 weeks in the NSI mice after implantation with these HSCs. Collectively, our findings demonstrated that CRISPR/Cas9 successfully corrects β-thalassemia mutations in patient-specific iPSCs. These CRISPR/Cas9-corrected iPSC-derived HSCs express normal HBB in mice without tumorigenic potential, suggesting a safe strategy for personalized treatment of β-thalassemia.

Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells

PubMed Central

Takayama, Naoya; Nishimura, Satoshi; Nakamura, Sou; Shimizu, Takafumi; Ohnishi, Ryoko; Endo, Hiroshi; Yamaguchi, Tomoyuki; Otsu, Makoto; Nishimura, Ken; Nakanishi, Mahito; Sawaguchi, Akira; Nagai, Ryozo; Takahashi, Kazutoshi; Yamanaka, Shinya; Nakauchi, Hiromitsu

2010-01-01

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a+CD42b+ platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b+ platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones. PMID:21098095

High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

PubMed

Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

2016-07-30

Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

Examining FKBP5 mRNA expression in human iPSC-derived neural cells

PubMed Central

Lieberman, Richard; Kranzler, Henry R.; Levine, Eric S.; Covault, Jonathan

2016-01-01

In peripheral blood leukocytes, FKBP5 mRNA expression is upregulated following glucocorticoid receptor activation. The single nucleotide polymorphism rs1360780 in FKBP5 is associated with psychiatric illness and has functional molecular effects. However, examination of FKBP5 regulation has largely been limited to peripheral cells, which may not reflect regulation in neural cells. We used 27 human induced pluripotent stem cell lines (iPSCs) derived from 20 subjects to examine FKBP5 mRNA expression following GR activation. Following differentiation into forebrain-lineage neural cultures, cells were exposed to 1μM dexamethasone and mRNA expression of FKBP5 and NR3C1 analyzed. Results from the iPSC-derived neural cells were compared with those from 15 donor matched fibroblast lines. Following dexamethasone treatment, there was a 670% increase in FKBP5 expression in fibroblasts, mimicking findings in peripheral blood-derived cells, but only a 23% increase in iPSC-derived neural cultures. FKBP5 rs1360780 genotype did not affect the induction of FKBP5 mRNA in either fibroblasts or neural cells. These results suggest that iPSC-derived forebrain-lineage neurons may not be an optimal neural cell type in which to examine relationships between GR activation, FKBP5 expression, and genetic variation in human subjects. Further, FKBP5 induction following GR activation may differ between cell types derived from the same individual. PMID:27915167

Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

PubMed

Sakai-Takemura, Fusako; Narita, Asako; Masuda, Satoru; Wakamatsu, Toshifumi; Watanabe, Nobuharu; Nishiyama, Takashi; Nogami, Ken'ichiro; Blanc, Matthias; Takeda, Shin'ichi; Miyagoe-Suzuki, Yuko

2018-04-26

Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(-) CD108(-) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-β inhibitors. TGF-β inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx 4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

PubMed Central

Hämäläinen, Riikka H.; Ahlqvist, Kati J.; Ellonen, Pekka; Lepistö, Maija; Logan, Angela; Otonkoski, Timo; Murphy, Michael P.; Suomalainen, Anu

2015-01-01

Summary mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS)/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs) and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function. PMID:26027936

Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers

NASA Astrophysics Data System (ADS)

Tang, Yadong; Liu, Li; Li, Junjun; Yu, Leqian; Wang, Li; Shi, Jian; Chen, Yong

2016-07-01

Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape of the hiPSC colony could be controlled from a flat layer to a hemisphere. Afterwards, the induction and differentiation of hiPSCs were achieved on the same patch, leading to a uniform cardiac layer with homogeneous contraction. This cardiac layer could then be used for extracellular recording with a commercial multi-electrode array, showing representative field potential waveforms of matured cardiac tissues with appropriate drug responses.Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape of the hiPSC colony could be controlled from a flat layer to a hemisphere. Afterwards, the induction and differentiation of hiPSCs were achieved on the same patch, leading to a uniform cardiac layer with homogeneous contraction. This cardiac layer could then be used for extracellular recording with a commercial multi-electrode array, showing representative field potential waveforms of matured cardiac tissues with appropriate drug responses. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr04545f

Alternative functional in vitro models of human intestinal epithelia

PubMed Central

Kauffman, Amanda L.; Gyurdieva, Alexandra V.; Mabus, John R.; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J.

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport. PMID:23847534

Alternative functional in vitro models of human intestinal epithelia.

PubMed

Kauffman, Amanda L; Gyurdieva, Alexandra V; Mabus, John R; Ferguson, Chrissa; Yan, Zhengyin; Hornby, Pamela J

2013-01-01

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

Human pluripotent stem cells: an emerging model in developmental biology

PubMed Central

Zhu, Zengrong; Huangfu, Danwei

2013-01-01

Developmental biology has long benefited from studies of classic model organisms. Recently, human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, have emerged as a new model system that offers unique advantages for developmental studies. Here, we discuss how studies of hPSCs can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development ‘in a dish’. We also summarize some of the recently developed genetic tools that greatly facilitate the interrogation of gene function during hPSC differentiation. With the development of high-throughput screening technologies, hPSCs have the potential to revolutionize gene discovery in mammalian development. PMID:23362344

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.

GABA and Gap Junctions in the Development of Synchronized Activity in Human Pluripotent Stem Cell-Derived Neural Networks

PubMed Central

Mäkinen, Meeri Eeva-Liisa; Ylä-Outinen, Laura; Narkilahti, Susanna

2018-01-01

The electrical activity of the brain arises from single neurons communicating with each other. However, how single neurons interact during early development to give rise to neural network activity remains poorly understood. We studied the emergence of synchronous neural activity in human pluripotent stem cell (hPSC)-derived neural networks simultaneously on a single-neuron level and network level. The contribution of gamma-aminobutyric acid (GABA) and gap junctions to the development of synchronous activity in hPSC-derived neural networks was studied with GABA agonist and antagonist and by blocking gap junctional communication, respectively. We characterized the dynamics of the network-wide synchrony in hPSC-derived neural networks with high spatial resolution (calcium imaging) and temporal resolution microelectrode array (MEA). We found that the emergence of synchrony correlates with a decrease in very strong GABA excitation. However, the synchronous network was found to consist of a heterogeneous mixture of synchronously active cells with variable responses to GABA, GABA agonists and gap junction blockers. Furthermore, we show how single-cell distributions give rise to the network effect of GABA, GABA agonists and gap junction blockers. Finally, based on our observations, we suggest that the earliest form of synchronous neuronal activity depends on gap junctions and a decrease in GABA induced depolarization but not on GABAA mediated signaling. PMID:29559893

Adoptive transfer of CD8+ T cells generated from induced pluripotent stem cells triggers regressions of large tumors along with immunological memory

PubMed Central

Saito, Hidehito; Okita, Keisuke; Chang, Alfred E.; Ito, Fumito

2016-01-01

Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with useful in vivo characteristics. Theoretically, this limitation could be overcome by using induced pluripotent stem cells (iPSCs) that could provide an unlimited source of autologous T cells. However, the therapeutic efficacy of iPSC-derived regenerated T cells remains to be demonstrated. Here we report the first successful reprogramming of T-cell receptor (TCR) transgenic CD8+ T cells into pluripotency. As part of the work, we established a syngeneic mouse model for evaluating in vitro and in vivo antitumor reactivity of regenerated T cells from iPSCs bearing a rearranged TCR of known antigen specificity. Stably TCR retained T cell-derived iPSCs differentiated into CD4+CD8+ T cells that expressed CD3 and the desired TCR in vitro. Stimulation of iPSC-derived CD4+CD8+ T cells with the cognate antigen in the presence of IL-7 and IL-15 followed by expansion with IL-2, IL-7 and IL-15 generated large numbers of less-differentiated CD8+ T cells with antigen-specific potent cytokine production and cytolytic capacity. Furthermore, adoptively transferred iPSC-derived CD8+ T cells escaped immune rejection, mediated effective regression of large tumors, improved survival, and established antigen-specific immunological memory. Our findings illustrate the translational potential of iPSCs to provide an unlimited number of phenotypically defined, functional, and expandable autologous antigen-specific T cells with the characteristics needed to enable in vivo effectiveness. PMID:27197199

Isolation and characterization of ventricular-like cells derived from NKX2-5eGFP/w and MLC2vmCherry/w double knock-in human pluripotent stem cells.

PubMed

Yamauchi, Kaori; Li, Junjun; Morikawa, Kumi; Liu, Li; Shirayoshi, Yasuaki; Nakatsuji, Norio; Elliott, David A; Hisatome, Ichiro; Suemori, Hirofumi

2018-01-01

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5 eGFP/w and MLC2v mCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5 eGFP/w and MLC2v mCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources. Copyright © 2017 Elsevier Inc. All rights reserved.

Reactive oxygen species-dependent Toll/NF-κB activation in the Drosophila hematopoietic niche confers resistance to wasp parasitism.

PubMed

Louradour, Isabelle; Sharma, Anurag; Morin-Poulard, Ismael; Letourneau, Manon; Vincent, Alain; Crozatier, Michèle; Vanzo, Nathalie

2017-11-01

Hematopoietic stem/progenitor cells in the adult mammalian bone marrow ensure blood cell renewal. Their cellular microenvironment, called 'niche', regulates hematopoiesis both under homeostatic and immune stress conditions. In the Drosophila hematopoietic organ, the lymph gland, the posterior signaling center (PSC) acts as a niche to regulate the hematopoietic response to immune stress such as wasp parasitism. This response relies on the differentiation of lamellocytes, a cryptic cell type, dedicated to pathogen encapsulation and killing. Here, we establish that Toll/NF-κB pathway activation in the PSC in response to wasp parasitism non-cell autonomously induces the lymph gland immune response. Our data further establish a regulatory network where co-activation of Toll/NF-κB and EGFR signaling by ROS levels in the PSC/niche controls lymph gland hematopoiesis under parasitism. Whether a similar regulatory network operates in mammals to control emergency hematopoiesis is an open question.

Integration-free induced pluripotent stem cells derived from a patient with autosomal recessive Alport syndrome (ARAS).

PubMed

Kuebler, Bernd; Aran, Begoña; Miquel-Serra, Laia; Muñoz, Yolanda; Ars, Elisabet; Bullich, Gemma; Furlano, Monica; Torra, Roser; Marti, Merce; Veiga, Anna; Raya, Angel

2017-12-01

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Generation of integration-free induced pluripotent stem cell lines derived from two patients with X-linked Alport syndrome (XLAS).

PubMed

Kuebler, Bernd; Aran, Begoña; Miquel-Serra, Laia; Muñoz, Yolanda; Ars, Elisabet; Bullich, Gemma; Furlano, Monica; Torra, Roser; Marti, Merce; Veiga, Anna; Raya, Angel

2017-12-01

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Developmental changes in electrophysiological characteristics of human induced Pluripotent Stem Cell-derived cardiomyocytes

PubMed Central

Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R.; Weissman, Amir; Binah, Ofer

2016-01-01

Background Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) only 3 types of action potentials (AP) exist: nodal, atrial and ventricular-like. Objective To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) during culture development a cardiac precursor cell is present that - depending on age - can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of nodal phenotype, transient appearance of atrial phenotype, evolution to ventricular phenotype, and persistence of transitional phenotypes. Methods To test these hypotheses we: (1) performed FACS analysis of nodal, atrial and ventricular markers; (2) recorded AP from 280 7-to-95 day old iPSC-CMs; (3) analyzed AP characteristics. Results The major findings were: (1) FACS analysis of 30 and 60-day old cultures showed that an iPSC-CMs population shifts from nodal into atrial/ventricular phenotype, while including significant transitional populations.(2) The AP population did not consist of 3 distinct phenotypes; (3) Culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57–70 days) appearance of atrial phenotype; (4) Beat Rate Variability was more prominent in nodal than ventricular cardiomyocytes while If density increased in older cultures. Conclusions From the onset of development the iPSC-CMs population includes nodal, atrial and ventricular AP and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial which appears only transiently, yet dominates at 57–70 days of evolution. PMID:27639456

Drusen in patient-derived hiPSC-RPE models of macular dystrophies

PubMed Central

Galloway, Chad A.; Dalvi, Sonal; Hung, Sandy S. C.; MacDonald, Leslie A.; Latchney, Lisa R.; Wong, Raymond C. B.; Guymer, Robyn H.; Williams, David S.; Chung, Mina M.; Gamm, David M.; Pébay, Alice; Hewitt, Alex W.; Singh, Ruchira

2017-01-01

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby’s fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid- and protein-rich “drusen-like” composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE–ECM interface. PMID:28878022

A panel of induced pluripotent stem cells from chimpanzees: a resource for comparative functional genomics

PubMed Central

Gallego Romero, Irene; Pavlovic, Bryan J; Hernando-Herraez, Irene; Zhou, Xiang; Ward, Michelle C; Banovich, Nicholas E; Kagan, Courtney L; Burnett, Jonathan E; Huang, Constance H; Mitrano, Amy; Chavarria, Claudia I; Friedrich Ben-Nun, Inbar; Li, Yingchun; Sabatini, Karen; Leonardo, Trevor R; Parast, Mana; Marques-Bonet, Tomas; Laurent, Louise C; Loring, Jeanne F; Gilad, Yoav

2015-01-01

Comparative genomics studies in primates are restricted due to our limited access to samples. In order to gain better insight into the genetic processes that underlie variation in complex phenotypes in primates, we must have access to faithful model systems for a wide range of cell types. To facilitate this, we generated a panel of 7 fully characterized chimpanzee induced pluripotent stem cell (iPSC) lines derived from healthy donors. To demonstrate the utility of comparative iPSC panels, we collected RNA-sequencing and DNA methylation data from the chimpanzee iPSCs and the corresponding fibroblast lines, as well as from 7 human iPSCs and their source lines, which encompass multiple populations and cell types. We observe much less within-species variation in iPSCs than in somatic cells, indicating the reprogramming process erases many inter-individual differences. The low within-species regulatory variation in iPSCs allowed us to identify many novel inter-species regulatory differences of small magnitude. DOI: http://dx.doi.org/10.7554/eLife.07103.001 PMID:26102527

Comment on “Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells”

PubMed Central

Bilican, Bilada; Serio, Andrea; Barmada, Sami J.; Nishimura, Agnes Lumi; Sullivan, Gareth J.; Carrasco, Monica; Phatnani, Hemali P.; Puddifoot, Clare A.; Story, David; Fletcher, Judy; Park, In-Hyun; Friedman, Brad A.; Daley, George Q.; Wyllie, David J. A.; Hardingham, Giles E.; Wilmut, Ian; Finkbeiner, Steven; Maniatis, Tom; Shaw, Christopher E.; Chandran, Siddharthan

2014-01-01

Egawa et al. recently showed the value of patient-specific induced pluripotent stem cells (iPSCs) for modeling amyotrophic lateral sclerosis in vitro. Their study and our work highlight the need for complementary assays to detect small, but potentially important, phenotypic differences between control iPSC lines and those carrying disease mutations. PMID:23740897

Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

PubMed Central

Feng, Qiang; Shabrani, Namrata; Thon, Jonathan N.; Huo, Hongguang; Thiel, Austin; Machlus, Kellie R.; Kim, Kyungho; Brooks, Julie; Li, Feng; Luo, Chenmei; Kimbrel, Erin A.; Wang, Jiwu; Kim, Kwang-Soo; Italiano, Joseph; Cho, Jaehyung; Lu, Shi-Jiang; Lanza, Robert

2014-01-01

Summary Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness. PMID:25418726

Scalable generation of universal platelets from human induced pluripotent stem cells.

PubMed

Feng, Qiang; Shabrani, Namrata; Thon, Jonathan N; Huo, Hongguang; Thiel, Austin; Machlus, Kellie R; Kim, Kyungho; Brooks, Julie; Li, Feng; Luo, Chenmei; Kimbrel, Erin A; Wang, Jiwu; Kim, Kwang-Soo; Italiano, Joseph; Cho, Jaehyung; Lu, Shi-Jiang; Lanza, Robert

2014-11-11

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid "surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Genetic correction of tauopathy phenotypes in neurons derived from human induced pluripotent stem cells.

PubMed

Fong, Helen; Wang, Chengzhong; Knoferle, Johanna; Walker, David; Balestra, Maureen E; Tong, Leslie M; Leung, Laura; Ring, Karen L; Seeley, William W; Karydas, Anna; Kshirsagar, Mihir A; Boxer, Adam L; Kosik, Kenneth S; Miller, Bruce L; Huang, Yadong

2013-01-01

Tauopathies represent a group of neurodegenerative disorders characterized by the accumulation of pathological TAU protein in brains. We report a human neuronal model of tauopathy derived from induced pluripotent stem cells (iPSCs) carrying a TAU-A152T mutation. Using zinc-finger nuclease-mediated gene editing, we generated two isogenic iPSC lines: one with the mutation corrected, and another with the homozygous mutation engineered. The A152T mutation increased TAU fragmentation and phosphorylation, leading to neurodegeneration and especially axonal degeneration. These cellular phenotypes were consistent with those observed in a patient with TAU-A152T. Upon mutation correction, normal neuronal and axonal morphologies were restored, accompanied by decreases in TAU fragmentation and phosphorylation, whereas the severity of tauopathy was intensified in neurons with the homozygous mutation. These isogenic TAU-iPSC lines represent a critical advancement toward the accurate modeling and mechanistic study of tauopathies with human neurons and will be invaluable for drug-screening efforts and future cell-based therapies.

Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

PubMed

Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

2013-09-01

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.

Autologous Pluripotent Stem Cell-Derived β-Like Cells for Diabetes Cellular Therapy.

PubMed

Millman, Jeffrey R; Pagliuca, Felicia W

2017-05-01

Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into β-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach. © 2017 by the American Diabetes Association.

Induced pluripotent stem cells meet genome editing

PubMed Central

Hockemeyer, Dirk; Jaenisch, Rudolf

2016-01-01

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments --the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-- have fundamentally reshaped our approach to biomedical research, stem cell biology and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided, and have set the stage for their success. PMID:27152442

Induced Pluripotent Stem Cells Meet Genome Editing.

PubMed

Hockemeyer, Dirk; Jaenisch, Rudolf

2016-05-05

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success. Copyright © 2016 Elsevier Inc. All rights reserved.

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells

PubMed Central

Bhutani, Nidhi; Decker, Matthew N.; Brady, Jennifer J.; Bussat, Rose T.; Burns, David M.; Corbel, Stephane Y.; Blau, Helen M.

2013-01-01

Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.—Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells. PMID:23212122

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening.

PubMed

Smith, Alec S T; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A; Kim, Deok-Ho

Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. Published by Elsevier Inc.

Fabrication of macroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors.

PubMed

Sladkova, Martina; Palmer, Michael; Öhman, Caroline; Alhaddad, Rawan Jaragh; Esmael, Asmaa; Engqvist, Håkan; de Peppo, Giuseppe Maria

2016-12-01

Calcium phosphate cements (CPCs) have been extensively used in reconstructive dentistry and orthopedics, but it is only recently that CPCs have been combined with stem cells to engineer biological substitutes with enhanced healing potential. In the present study, macroporous CPC scaffolds with defined composition were fabricated using an easily reproduced synthesis method, with minimal fabrication and processing steps. Scaffold pore size and porosity, essential for cell infiltration and tissue ingrowth, were tuned by varying the content and size of polyethylene glycol (PEG) particles, resulting in 9 groups with different architectural features. The scaffolds were characterized for chemical composition, porosity and mechanical properties, then tested in vitro with human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MPs). Biomimetic decellularized bone scaffolds were used as reference material in this study. Our manufacturing process resulted in the formation of macroporous monetite scaffolds with no residual traces of PEG. The size and content of PEG particles was found to affect scaffold porosity, and thus mechanical properties. Irrespective of pore size and porosity, the CPC scaffolds fabricated in this study supported adhesion and viability of human iPSC-MPs similarly to decellularized bone scaffolds. However, the architectural features of the scaffolds were found to affect the expression of bone specific genes, suggesting that specific scaffold groups could be more suitable to direct human iPSC-MPs in vitro toward an osteoblastic phenotype. Our simplistic fabrication method allows rapid, inexpensive and reproducible construction of macroporous CPC scaffolds with tunable architecture for potential use in dental and orthopedic applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Induced pluripotent stem cells (iPSC)-derived retinal cells in disease modeling and regenerative medicine.

PubMed

Rathod, Reena; Surendran, Harshini; Battu, Rajani; Desai, Jogin; Pal, Rajarshi

2018-02-12

Retinal degenerative disorders are a leading cause of the inherited, irreversible and incurable vision loss. While various rodent model systems have provided crucial information in this direction, lack of disease-relevant tissue availability and species-specific differences have proven to be a major roadblock. Human induced pluripotent stem cells (iPSC) have opened up a whole new avenue of possibilities not just in understanding the disease mechanism but also potential therapeutic approaches towards a cure. In this review, we have summarized recent advances in the methods of deriving retinal cell types from iPSCs which can serve as a renewable source of disease-relevant cell population for basic as well as translational studies. We also provide an overview of the ongoing efforts towards developing a suitable in vitro model for modeling retinal degenerative diseases. This basic understanding in turn has contributed to advances in translational goals such as drug screening and cell-replacement therapies. Furthermore we discuss gene editing approaches for autologous repair of genetic disorders and allogeneic transplantation of stem cell-based retinal derivatives for degenerative disorders with an ultimate goal to restore vision. It is pertinent to note however, that these exciting new developments throw up several challenges that need to be overcome before their full clinical potential can be realized. Copyright © 2018 Elsevier B.V. All rights reserved.

Cryopreservation of pluripotent stem cell aggregates in defined protein-free formulation.

PubMed

Sart, Sébastien; Ma, Teng; Li, Yan

2013-01-01

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate-based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size-dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein-free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three-germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein-free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes.

PubMed

Chai, Sam; Wan, Xiaoping; Nassal, Drew M; Liu, Haiyan; Moravec, Christine S; Ramirez-Navarro, Angelina; Deschênes, Isabelle

2017-06-01

Two-pore K + (K 2p ) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K 2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K 2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K 2p channels in the heart. Comparing quantitative PCR expression of K 2p channels between human heart tissue and iPSC-CMs revealed K 2p 1.1, K 2p 2.1, K 2p 5.1, and K 2p 17.1 to be higher expressed in cHVT, whereas K 2p 3.1 and K 2p 13.1 were higher in iPSC-CMs. Notably, K 2p 17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K 2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K 2p 2.1, K 2p 3.1, K 2p 6.1, and K 2p 17.1. Here, we report the expression level of 10 human K 2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K 2p 17.1 as significantly reduced in niHF tissues and K 2p 4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K 2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K + (K 2p ) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K 2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K 2p channels. Copyright © 2017 the American Physiological Society.

Large field of view quantitative phase imaging of induced pluripotent stem cells and optical pathlength reference materials

NASA Astrophysics Data System (ADS)

Kwee, Edward; Peterson, Alexander; Stinson, Jeffrey; Halter, Michael; Yu, Liya; Majurski, Michael; Chalfoun, Joe; Bajcsy, Peter; Elliott, John

2018-02-01

Induced pluripotent stem cells (iPSCs) are reprogrammed cells that can have heterogeneous biological potential. Quality assurance metrics of reprogrammed iPSCs will be critical to ensure reliable use in cell therapies and personalized diagnostic tests. We present a quantitative phase imaging (QPI) workflow which includes acquisition, processing, and stitching multiple adjacent image tiles across a large field of view (LFOV) of a culture vessel. Low magnification image tiles (10x) were acquired with a Phasics SID4BIO camera on a Zeiss microscope. iPSC cultures were maintained using a custom stage incubator on an automated stage. We implement an image acquisition strategy that compensates for non-flat illumination wavefronts to enable imaging of an entire well plate, including the meniscus region normally obscured in Zernike phase contrast imaging. Polynomial fitting and background mode correction was implemented to enable comparability and stitching between multiple tiles. LFOV imaging of reference materials indicated that image acquisition and processing strategies did not affect quantitative phase measurements across the LFOV. Analysis of iPSC colony images demonstrated mass doubling time was significantly different than area doubling time. These measurements were benchmarked with prototype microsphere beads and etched-glass gratings with specified spatial dimensions designed to be QPI reference materials with optical pathlength shifts suitable for cell microscopy. This QPI workflow and the use of reference materials can provide non-destructive traceable imaging method for novel iPSC heterogeneity characterization.

Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome.

PubMed

Ikemoto, Yu; Takayama, Yoshinaga; Fujii, Katsunori; Masuda, Mokuri; Kato, Chise; Hatsuse, Hiromi; Fujitani, Kazuko; Nagao, Kazuaki; Kameyama, Kohzoh; Ikehara, Hajime; Toyoda, Masashi; Umezawa, Akihiro; Miyashita, Toshiyuki

2017-08-01

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 ( PTCH1 ) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%-15.6% depending on the tissue) identical to the one found in iPSC clones. This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Electrotaxis of cardiac progenitor cells, cardiac fibroblasts, and induced pluripotent stem cell-derived cardiac progenitor cells requires serum and is directed via PI3'K pathways.

PubMed

Frederich, Bert J; Timofeyev, Valeriy; Thai, Phung N; Haddad, Michael J; Poe, Adam J; Lau, Victor C; Moshref, Maryam; Knowlton, Anne A; Sirish, Padmini; Chiamvimonvat, Nipavan

2017-11-01

The limited regenerative capacity of cardiac tissue has long been an obstacle to treating damaged myocardium. Cell-based therapy offers an enormous potential to the current treatment paradigms. However, the efficacy of regenerative therapies remains limited by inefficient delivery and engraftment. Electrotaxis (electrically guided cell movement) has been clinically used to improve recovery in a number of tissues but has not been investigated for treating myocardial damage. The purpose of this study was to test the electrotactic behaviors of several types of cardiac cells. Cardiac progenitor cells (CPCs), cardiac fibroblasts (CFs), and human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) were used. CPCs and CFs electrotax toward the anode of a direct current electric field, whereas hiPSC-CPCs electrotax toward the cathode. The voltage-dependent electrotaxis of CPCs and CFs requires the presence of serum in the media. Addition of soluble vascular cell adhesion molecule to serum-free media restores directed migration. We provide evidence that CPC and CF electrotaxis is mediated through phosphatidylinositide 3-kinase signaling. In addition, very late antigen-4, an integrin and growth factor receptor, is required for electrotaxis and localizes to the anodal edge of CPCs in response to direct current electric field. The hiPSC-derived CPCs do not express very late antigen-4, migrate toward the cathode in a voltage-dependent manner, and, similar to CPCs and CFs, require media serum and phosphatidylinositide 3-kinase activity for electrotaxis. The electrotactic behaviors of these therapeutic cardiac cells may be used to improve cell-based therapy for recovering function in damaged myocardium. Published by Elsevier Inc.

Altered neurite morphology and cholinergic function of induced pluripotent stem cell-derived neurons from a patient with Kleefstra syndrome and autism

PubMed Central

Nagy, J; Kobolák, J; Berzsenyi, S; Ábrahám, Z; Avci, H X; Bock, I; Bekes, Z; Hodoscsek, B; Chandrasekaran, A; Téglási, A; Dezső, P; Koványi, B; Vörös, E T; Fodor, L; Szél, T; Németh, K; Balázs, A; Dinnyés, A; Lendvai, B; Lévay, G; Román, V

2017-01-01

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD. PMID:28742076

Systematic review of induced pluripotent stem cell technology as a potential clinical therapy for spinal cord injury.

PubMed

Kramer, Anne S; Harvey, Alan R; Plant, Giles W; Hodgetts, Stuart I

2013-01-01

Transplantation therapies aimed at repairing neurodegenerative and neuropathological conditions of the central nervous system (CNS) have utilized and tested a variety of cell candidates, each with its own unique set of advantages and disadvantages. The use and popularity of each cell type is guided by a number of factors including the nature of the experimental model, neuroprotection capacity, the ability to promote plasticity and guided axonal growth, and the cells' myelination capability. The promise of stem cells, with their reported ability to give rise to neuronal lineages to replace lost endogenous cells and myelin, integrate into host tissue, restore functional connectivity, and provide trophic support to enhance and direct intrinsic regenerative ability, has been seen as a most encouraging step forward. The advent of the induced pluripotent stem cell (iPSC), which represents the ability to "reprogram" somatic cells into a pluripotent state, hails the arrival of a new cell transplantation candidate for potential clinical application in therapies designed to promote repair and/or regeneration of the CNS. Since the initial development of iPSC technology, these cells have been extensively characterized in vitro and in a number of pathological conditions and were originally reported to be equivalent to embryonic stem cells (ESCs). This review highlights emerging evidence that suggests iPSCs are not necessarily indistinguishable from ESCs and may occupy a different "state" of pluripotency with differences in gene expression, methylation patterns, and genomic aberrations, which may reflect incomplete reprogramming and may therefore impact on the regenerative potential of these donor cells in therapies. It also highlights the limitations of current technologies used to generate these cells. Moreover, we provide a systematic review of the state of play with regard to the use of iPSCs in the treatment of neurodegenerative and neuropathological conditions. The importance of balancing the promise of this transplantation candidate in the light of these emerging properties is crucial as the potential application in the clinical setting approaches. The first of three sections in this review discusses (A) the pathophysiology of spinal cord injury (SCI) and how stem cell therapies can positively alter the pathology in experimental SCI. Part B summarizes (i) the available technologies to deliver transgenes to generate iPSCs and (ii) recent data comparing iPSCs to ESCs in terms of characteristics and molecular composition. Lastly, in (C) we evaluate iPSC-based therapies as a candidate to treat SCI on the basis of their neurite induction capability compared to embryonic stem cells and provide a summary of available in vivo data of iPSCs used in SCI and other disease models.