Critical role of Lin28‐TNFR2 signalling in cardiac stem cell activation and differentiation

Tumour necrotic factor receptor‐2 (TNFR2) has been to be cardiac‐protective and is expressed in cardiac progenitor cells. Our goal is to define the mechanism for TNFR2‐mediated cardiac stem cell activation and differentiation. By employing a protocol of in vitro cardiac stem cell (CSC) differentiation from human inducible pluripotent stem cell (hiPSC), we show that expression of TNFR2 precedes expression of CSC markers followed by expression of mature cardiomyocyte proteins. Activation of TNFR2 by a specific agonist promotes whereas inhibition of TNFR2 by neutralizing antibody diminishes hiPSC‐based CSC differentiation. Interestingly, pluripotent cell factor RNA‐binding protein Lin28 enhances TNFR2 protein expression in early CSC activation by directly binding to a conserved Lin28‐motif within the 3'UTR of Tnfr2 mRNA. Furthermore, inhibition of Lin28 blunts TNFR2 expression and TNFR2‐dependent CSC activation and differentiation. Our study demonstrates a critical role of Lin28‐TNFR2 axis in CSC activation and survival, providing a novel strategy to enhance stem cell‐based therapy for the ischaemic heart diseases.     

Sialyl-lactotetra,a Novel Cell Surface Marker of Undifferentiated Human Pluripotent Stem Cells

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 × 10⁹ 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.     

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.     

A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability

Background

The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34⁺ cord blood using non-viral, non-integrating methods.

Methodology/Principal Findings

We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34⁺ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I⁺ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.

Conclusion/Significance

This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.     

Two new protocols to enhance the production and isolation of human induced pluripotent stem cell lines

There are two critical stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. One is the production of high titer virus required to reprogram somatic cells; the other is identification of true hiPSC colonies from heterogeneous cell populations, and their isolation and expansion to generate a sustainable, pluripotent stem cell line. Here we describe simple, time-saving methods to address the current difficulties at these two critical junctures. First, we have developed a method to increase the number of infectious viral units 600-fold. Second, we have developed a TRA-1-81-based positive selection column method for isolating “true” hiPSCs from the heterogeneous cell populations, which overcomes the labor-intensive and highly subjective method of manual selection of hiPSC colonies. We have used these techniques to produce 8 hiPSC lines from human fibroblasts and we believe that they are of considerable utility to researchers in the hiPSC field.     

基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

Structural characterization of the human fast skeletal muscle troponin I gene (TNNI2)

Three troponin I genes have been identified in vertebrates that encode the isoforms expressed in adult cardiac muscle (TNNI3), slow skeletal muscle (TNNI1) and fast skeletal muscle (TNNI2), respectively. While the organization and regulation of human cardiac and slow skeletal muscle genes have been investigated in detail, the fast skeletal troponin I gene has to date only been examined in birds. Here, we describe the structure and complete sequence of the human fast skeletal muscle troponin I gene (TNNI2) and identify putative regulatory elements within both the 5' flanking region and the first intron. In particular, a region containing MEF-2, E-box, CCAC and CAGG elements was identified in intron 1 that closely resembles the fast internal regulatory element (FIRE) of the quail intronic enhancer. We have previously shown that the fast skeletal muscle troponin I gene is located at 11p15.5 and noted potential close linkage with the fast skeletal muscle troponin T gene (TNNT3). Here, we have isolated two independent human PAC genomic clones that contain either TNNI2 or TNNT3 and demonstrate by interphase FISH mapping that they are less than 100 kb apart in the genome. The results demonstrate that the human TNNI2 gene is closely related to its avian counterparts with conserved elements within both the putative promoter and first intron. Our data further confirm close physical linkage of TNNI2 and TNNI3 on 11p15.5.     

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.     

综述:基于人多潜能干细胞的人类疾病研究与治疗

人多潜能干细胞(hPSC)包括人胚胎干细胞(hESC)和诱导性多潜能干细胞(hiPSC),理论上具有分化成为人类所有细胞类型的能力.基于hPSC的基因打靶技术,不但可以纠正人基因组中的遗传突变用于细胞治疗,还可以通过反向遗传学的方式向hPSC引入疾病特异的突变.将携带人类疾病遗传基因的hPSC分化为特定的细胞类型,在理论上可以在体外模拟人类疾病的发生,研究人类疾病发生的机理,并建立体外筛选平台寻找治疗性药物.基因编辑和干细胞技术的结合将为人类疾病的机制研究和再生医学治疗带来革命性的突破.     

Serum supplemented culture medium masks hypertrophic phenotypes in human pluripotent stem cell derived cardiomyocytes

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here, we determined how serum affected cardiomyocytes from human embryonic‐ (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC‐ and hiPSC‐derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine, which normally induces cardiac hypertrophy, had no additional effects under serum conditions. Likewise, hiPSC‐derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype, did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum.     

Human Induced Pluripotent Stem Cells Are Targets for Allogeneic and Autologous Natural Killer (NK) Cells and Killing Is Partly Mediated by the Activating NK Receptor DNAM-1

Human induced pluripotent stem cells (hiPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, iPSC-derived grafts are at risk of giving rise to teratomas in the host, if residuals of tumorigenic cells are not rejected by the recipient. We have analyzed the susceptibility of hiPSC lines to allogeneic and autologous natural killer (NK) cells. IL-2-activated, in contrast to resting NK cells killed hiPSC lines efficiently (P=1.69x10^-39). Notably, the specific lysis of the individual hiPSC lines by IL-2-activated NK cells was significantly different (P=1.72x10^-6) and ranged between 46 % and 64 % in ⁵¹Cr-release assays when compared to K562 cells. The hiPSC lines were killed by both allogeneic and autologous NK cells although autologous NK cells were less efficient (P=8.63x10^-6). Killing was partly dependent on the activating NK receptor DNAM-1 (P=8.22x10^-7). The DNAM-1 ligands CD112 and CD155 as well as the NKG2D ligands MICA and MICB were expressed on the hiPSC lines. Low amounts of human leukocyte antigen (HLA) class I proteins, which serve as ligands for inhibitory and activating NK receptors were also detected. Thus, the susceptibility to NK cell killing appears to constitute a common feature of hiPSCs. Therefore, NK cells might reduce the risk of teratoma formation even after autologous transplantations of pluripotent stem cell-derived grafts that contain traces of pluripotent cells.     

A Novel Feeder-Free Culture System for Human Pluripotent Stem Cell Culture and Induced Pluripotent Stem Cell Derivation

Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC) to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC) lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.     

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

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

Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression

Rationale

Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.

Method and Result

We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30–70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7–8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95–98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5−10×10⁵ VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.

Conclusion

We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.     

miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells

The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.     

Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.