Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.     

Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.     

Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.     

One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming. H9 human embryonic stem cells (hESCs) served as control. CMs and MSCs were co-generated from hiPSCs and hESCs via embryoid body-dependent cardiac differentiation protocol involving a serum-free and insulin-depleted medium containing a p38 MAPK inhibitor, SB 203580. Comparing to bone marrow and umbilical cord blood-derived MSCs, hiPSC-derived MSCs (iMSCs) expressed common MSC markers and were capable of adipogenesis, osteogenesis and chondrogenesis. Moreover, iMSCs continuously proliferated for more than 32 population doublings without cellular senescence and showed superior pro-angiogenic and wound healing properties. In summary, we generated a large number of homogenous MSCs in conjunction with CMs in a low-cost and efficient one step manner. Functionally competent CMs and MSCs co-generated from hiPSCs may be useful for autologous cardiac repair.     

Effective Cardiac Myocyte Differentiation of Human Induced Pluripotent Stem Cells Requires VEGF

Perhaps one of the most significant achievements in modern science is the discovery of human induced pluripotent stem cells (hiPSCs), which have paved the way for regeneration therapy using patients’ own cells. Cardiomyocytes differentiated from hiPSCs (hiPSC-CMs) could be used for modelling patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, the present cardiomyocyte differentiation protocols exhibit variable differentiation efficiency across different hiPSC lines, which inhibit the application of this technology significantly. Here, we demonstrate a novel myocyte differentiation protocol that can yield a significant, high percentage of cardiac myocyte differentiation (>85%) in 2 hiPSC lines, which makes the fabrication of a human cardiac muscle patch possible. The established hiPSCs cell lines being examined include the transgene integrated UCBiPS7 derived from cord blood cells and non-integrated PCBC16iPS from skin fibroblasts. The results indicate that hiPSC-CMs derived from established hiPSC lines respond to adrenergic or acetylcholine stimulation and beat regularly for greater than 60 days. This data also demonstrates that this novel differentiation protocol can efficiently generate hiPSC-CMs from iPSC lines that are derived not only from fibroblasts, but also from blood mononuclear cells.     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

Derivation of Induced Pluripotent Stem Cells from Human Peripheral Blood T Lymphocytes

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (“TiPS”) retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR) gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages.     

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

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.     

Residual expression of the reprogramming factors prevents differentiation of iPSC generated from human fibroblasts and cord blood CD34+ progenitors

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.     

Cancer hallmarks in induced pluripotent cells: New insights

Studies are beginning to emerge that demonstrate intriguing differences between human‐induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Here, we investigated the expression of key members of the Nodal embryonic signaling pathway, critical to the maintenance of pluripotency in hESCs. Western blot and real‐time RT‐PCR analyses reveal slightly lower levels of Nodal (a TGF‐β family member) and Cripto‐1 (Nodal's co‐receptor) and a dramatic decrease in Lefty (Nodal's inhibitor and TGF‐β family member) in hiPSCs compared with hESCs. The noteworthy drop in hiPSC's Lefty expression correlated with an increase in the methylation of Lefty B CpG island. Based on these findings, we addressed a more fundamental question related to the consequences of epigenetically reprogramming hiPSCs, especially with respect to maintaining a stable ESC phenotype. A global comparative analysis of 365 microRNAs (miRs) in two hiPSC versus four hESC lines ultimately identified 10 highly expressed miRs in hiPCSs with >10‐fold difference, which have been shown to be cancer related. These data demonstrate cancer hallmarks expressed by hiPSCs, which will require further assessment for their impact on future therapies. J. Cell. Physiol. 225: 390–393, 2010. © 2010 Wiley‐Liss, Inc.     

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate ^1-3. We used this method to reprogram late passage (>p10) human adult fibroblasts derived from Friedreich''s ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged ^4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich''s ataxia patients and control individuals ⁶, human newborn fibroblasts, as well as human keratinocytes.     

Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage,blood outgrowth endothelial cells and cells from milk teeth

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative.     

Telomere Dynamics in Human Cells Reprogrammed to Pluripotency

Background

Human induced pluripotent stem cells (IPSCs) have enormous potential in the development of cellular models of human disease and represent a potential source of autologous cells and tissues for therapeutic use. A question remains as to the biological age of IPSCs, in particular when isolated from older subjects. Studies of cloned animals indicate that somatic cells reprogrammed to pluripotency variably display telomere elongation, a common indicator of cell “rejuvenation.”

Methodology/Principal Findings

We examined telomere lengths in human skin fibroblasts isolated from younger and older subjects, fibroblasts converted to IPSCs, and IPSCs redifferentiated through teratoma formation and explant culture. In IPSCs analyzed at passage five (P5), telomeres were significantly elongated in 6/7 lines by >40% and approximated telomere lengths in human embryonic stem cells (hESCs). In cell lines derived from three IPSC-teratoma explants cultured to P5, two displayed telomeres shortened to lengths similar to input fibroblasts while the third line retained elongated telomeres.

Conclusions/Significance

While these results reveal some heterogeneity in the reprogramming process with respect to telomere length, human somatic cells reprogrammed to pluripotency generally displayed elongated telomeres that suggest that they will not age prematurely when isolated from subjects of essentially any age.     

Gene targeting in human pluripotent stem cells with adeno-associated virus vectors

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.     

人胚胎干细胞和诱导型人多能干细胞分化的心肌细胞在药物心脏毒性筛选中的应用

心脏毒性是药物研发失败的主要原因之一,也是临床前安全评价研究的难题之一。人胚胎干细胞和诱导型人多能干细胞均具有无限增殖、自我更新和多向分化的特性,为体外心脏毒性筛选实验提供了细胞资源。人胚胎干细胞和诱导型人多能干细胞诱导分化的心肌细胞相似,具有相同的形态结构,且随着培养时间的推移,功能性心、Na^＋、Ca^2＋通道密度逐渐增加、心肌特异性基因ANF、α—MHC、MLC-2α的表达量增加,具有相似的动作电位时程和收缩性等特点,相当于幼稚型心肌细胞。将它们应用于已知作用药物的心脏毒性筛选,检测心肌细胞离子通道、动作电位、心脏损伤标志物、收缩功能的变化,获得与临床相似的结果。因此,建立人胚胎干细胞和诱导型人多能干细胞诱导分化心肌细胞的体外评价模型,大大减少了药物研发的时间和成本,克服了种属间的差异,推动了心脏毒性体外评价方法的发展。     

Fourier transform infrared microspectroscopy reveals unique phenotypes for human embryonic and induced pluripotent stem cell lines and their progeny

Fourier transform infrared (FTIR) microspectroscopy was employed to elucidate the macromolecular phenotype of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and their differentiated progeny. Undifferentiated hESCs and hiPSC lines were found to be not clearly distinguishable from each other. However, although both hESC and hiPSC variants appeared to undergo similar changes during differentiation in terms of cell surface antigens, the derived cell types from all cell lines could be discriminated using FTIR spectroscopy. We foresee a possible future role for FTIR microspectroscopy as a powerful and objective investigative and quality control tool in regenerative medicine. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)