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Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel Na V 1.7, which is a validated pain target. Na V 1.7 deficiency leads to pain insensitivity, whereas Na V 1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag Na V 1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface Na V 1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust Na V 1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes Na V 1.7 surface expression. A clinically effective Na V 1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling., (© 2024. The Author(s).)

TMEM43 (LUMA) is a ubiquitously expressed protein with unknown function. The protein is phylogenetically highly conserved and also found in Drosophila melanogaster (Klinke et al., 2022). TMEM43-p.S358L is a rare, fully penetrant mutation that leads to arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5). To understand the function of the ARVC5-associated mutation it is first important to understand the function of the TMEM43 protein. Therefore, a TMEM43 knockout induced pluripotent stem cell (iPSC) line was generated using the CRISPR/Cas9 genome editing system. The resulting cell line had a deficiency of TMEM43 and showed normal morphology and a stable karyotype. The colonies were positive for pluripotency markers and could be differentiated into the three germ layers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Induced pluripotent stem cells (iPSCs) and their derivatives have been described to display epigenetic memory of their founder cells, as well as de novo reprogramming-associated alterations. In order to selectively explore changes due to the reprogramming process and not to heterologous somatic memory, we devised a circular reprogramming approach where somatic stem cells are used to generate iPSCs, which are subsequently re-differentiated into their original fate. As somatic founder cells, we employed human embryonic stem cell-derived neural stem cells (NSCs) and compared them to iPSC-derived NSCs derived thereof. Global transcription profiling of this isogenic circular system revealed remarkably similar transcriptomes of both NSC populations, with the exception of 36 transcripts. Amongst these we detected a disproportionately large fraction of X chromosomal genes, all of which were upregulated in iPSC-NSCs. Concurrently, we detected differential methylation of X chromosomal sites spatially coinciding with regions harboring differentially expressed genes. While our data point to a pronounced overall reinstallation of autosomal transcriptomic and methylation signatures when a defined somatic lineage is propagated through pluripotency, they also indicate that X chromosomal genes may partially escape this reinstallation process. Considering the broad application of iPSCs in disease modeling and regenerative approaches, such reprogramming-associated alterations in X chromosomal gene expression and DNA methylation deserve particular attention.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery., Competing Interests: Declaration of interests W.W. and V.T. are involved in Cygenia GmbH (www.cygenia.com), which provides services for DNA methylation analysis to other scientists. S.K. reports funding from Novartis, Bristol-Myers Squibb, and Janssen/Geron; advisory board honoraria from Pfizer, Incyte, Ariad, Novartis, AOP Pharma, BMS, Celgene, Geron, Janssen, CTI, Roche, Baxalta, GSK, Sierra Oncology, and Sanofi; a patent for Bromodomain and Extra-Terminal (BET) inhibitor at RWTH Aachen University; honoraria from Novartis, Bristol Myers Squibb, Celgene, Geron, Janssen, Pfizer, Incyte, Ariad, Shire, Roche, and AOP Pharma; and other financial support (e.g., travel support) from Alexion, Novartis, Bristol Myers Squibb, Incyte, Ariad, AOP Pharma, Baxalta, CTI, Pfizer, Sanofi, Celgene, Shire, Janssen, Geron, Abbvie, Imago Biosciences, Sierra Oncology, GSK, and Karthos. T.H.B. served as a consultant for Janssen, Merck, Novartis, and Pfizer; received research funding form Novartis and Pfizer; and received honoraria from Janssen, Merck, Novartis, and Pfizer., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.

Novel and exciting avenues allow generating dendritic cells (DC) by reprogramming of somatic cells. DC are obtained from induced pluripotent stem cells (iPS cells), referred to as ipDC, and by direct reprogramming of cells toward DC, referred to as induced DC (iDC). iPS cells represent pluripotent stem cells generated by reprogramming of somatic cells and can differentiate into all cell types of the body, including DC. This makes iPS cells and ipDC derived thereof useful for studying various DC subsets, acquiring high cell numbers for research and clinical use, or applying genome editing to generate DC with wanted properties. Thereby, ipDC overcome limitations in specific DC subsets, which are only found in low abundance in blood or lymphoid organs. iDC are generated by direct reprogramming of somatic cells with a specific set of transcription factors and offer an avenue to obtain DC without a pluripotent cell intermediate. ipDC and iDC retain patient and disease-specific mutations and this opens new perspectives for studying DC in disease. This review summarizes the current techniques used to generate ipDC and iDC, and the types and functionality of the DC generated., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Background: DNA methylation is involved in the epigenetic regulation of gene expression during developmental processes and is primarily established by the DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B). DNMT3A is one of the most frequently mutated genes in clonal hematopoiesis and leukemia, indicating that it plays a crucial role for hematopoietic differentiation. However, the functional relevance of Dnmt3a for hematopoietic differentiation and hematological malignancies has mostly been analyzed in mice, with the specific role for human hematopoiesis remaining elusive. In this study, we therefore investigated if DNMT3A is essential for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs)., Results: We generated iPSC lines with knockout of either exon 2, 19, or 23 and analyzed the impact of different DNMT3A exon knockouts on directed differentiation toward mesenchymal and hematopoietic lineages. Exon 19 -/- and 23 -/- lines displayed an almost entire absence of de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation efficiency was only slightly reduced in exon 19 -/- and rather increased in exon 23 -/- lines, while there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A -/- iHPCs recapitulate some DNA methylation patterns of acute myeloid leukemia (AML) with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed growth advantage of exon 23 -/- iHPCs in a syngeneic competitive differentiation assay., Conclusions: Our results demonstrate that iPSCs with homozygous knockout of different exons of DNMT3A remain capable of mesenchymal and hematopoietic differentiation-and exon 23 -/- iHPCs even gained growth advantage-despite loss of almost the entire de novo DNA methylation. Partial recapitulation of DNA methylation patterns of AML with DNMT3A mutations by our DNMT3A knockout iHPCs indicates that our model system can help to elucidate mechanisms of clonal hematopoiesis., (© 2022. The Author(s).)

Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotency. To investigate the dynamics of this process under spatial confinement, we used either polydimethylsiloxane (PDMS) pillars or micro-contact printing of vitronectin. There was a progressive upregulation of OCT4, E-cadherin, and NANOG within 70 μm from the outer rim of iPSC colonies. Single-cell RNA-sequencing and spatial reconstruction of gene expression demonstrated that OCT4 high subsets, residing at the edge of the colony, have pronounced up-regulation of the TGF-β pathway, particularly of NODAL and its inhibitor LEFTY. Interestingly, after 5-7 days, iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size without enzymatic or mechanical treatment. Within the early 3D aggregates, radial organization and differential gene expression continued in analogy to the changes observed during self-organization of iPSC colonies. Early self-detached aggregates revealed up-regulated germline-specific gene expression patterns as compared to conventional EBs. However, there were no marked differences after further directed differentiation toward hematopoietic, mesenchymal, and neuronal lineages. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The chemokine CXCL4/platelet factor 4 (PF4) gene, a key player in myelofibrosis, was knocked out by CRISPR/Cas9 in induced pluripotent stem cells (iPS cells) of a polycythemia vera (PV) patient with JAK2 V617F mutation. Two CXCL4 KO iPS cell lines with and without JAK2 V617F mutation (UKAi002-B-1 and UKAi002-A-1, respectively) were generated. CXCL4 KO iPS cells showed deletion of exon 1 and complete loss of CXCL4 protein. Pluripotency of iPS cells was confirmed by expression of pluripotency markers and trilineage differentiation. CXCL4 KO iPS cells are expected to provide a valuable tool for investigating the role of CXCL4 in human diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC-5) is a dominantly inherited cardiomyopathy caused by the mutation TMEM43-p.S358L. An induced pluripotent stem cell (iPSC) line (HDZi001-A) from an adult male mutation carrier was generated, using the CytoTune Sendai Kit. The resulting iPSCs carried the mutation TMEM43-p.S358L, had a normal morphology, a stable karyotype and were positive for the expression of pluripotency markers. This iPSC line can be differentiated into the three germ layers and might be a useful model for the characterization of ARVC-5 associated pathomechanism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

Background: The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion., Methods: In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays., Results: Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs., Conclusions: Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

The chronic pain syndrome inherited erythromelalgia (IEM) is attributed to mutations in the voltage-gated sodium channel (NaV) 1.7. Still, recent studies targeting NaV1.7 in clinical trials have provided conflicting results. Here, we differentiated induced pluripotent stem cells from IEM patients with the NaV1.7/I848T mutation into sensory nociceptors. Action potentials in these IEM nociceptors displayed a decreased firing threshold, an enhanced upstroke, and afterhyperpolarization, all of which may explain the increased pain experienced by patients. Subsequently, we investigated the voltage dependence of the tetrodotoxin-sensitive NaV activation in these human sensory neurons using a specific prepulse voltage protocol. The IEM mutation induced a hyperpolarizing shift of NaV activation, which leads to activation of NaV1.7 at more negative potentials. Our results indicate that NaV1.7 is not active during subthreshold depolarizations, but that its activity defines the action potential threshold and contributes significantly to the action potential upstroke. Thus, our model system with induced pluripotent stem cell-derived sensory neurons provides a new rationale for NaV1.7 function and promises to be valuable as a translational tool to profile and develop more efficacious clinical analgesics.

Background: Differentiation of induced pluripotent stem cells (iPSCs) toward hematopoietic progenitor cells (HPCs) raises high hopes for disease modeling, drug screening, and cellular therapy. Various differentiation protocols have been established to generate iPSC-derived HPCs (iHPCs) that resemble their primary counterparts in morphology and immunophenotype, whereas a systematic epigenetic comparison was yet elusive., Results: In this study, we compared genome-wide DNA methylation (DNAm) patterns of iHPCs with various different hematopoietic subsets. After 20 days of in vitro differentiation, cells revealed typical hematopoietic morphology, CD45 expression, and colony-forming unit (CFU) potential. DNAm changes were particularly observed in genes that are associated with hematopoietic differentiation. On the other hand, the epigenetic profiles of iHPCs remained overall distinct from natural HPCs. Furthermore, we analyzed if additional co-culture for 2 weeks with syngenic primary mesenchymal stromal cells (MSCs) or iPSC-derived MSCs (iMSCs) further supports epigenetic maturation toward the hematopoietic lineage. Proliferation of iHPCs and maintenance of CFU potential was enhanced upon co-culture. However, DNAm profiles support the notion that additional culture expansion with stromal support did not increase epigenetic maturation of iHPCs toward natural HPCs., Conclusion: Differentiation of iPSCs toward the hematopoietic lineage remains epigenetically incomplete. These results substantiate the need to elaborate advanced differentiation regimen while DNAm profiles provide a suitable measure to track this process.

Induced pluripotent stem cells (iPSCs) can be differentiated toward mesenchymal stromal cells (MSCs), but this transition remains incomplete. It has been suggested that matrix elasticity directs cell-fate decisions. Therefore, we followed the hypothesis that differentiation of primary MSCs and generation of iPSC-derived MSCs (iMSCs) is supported by a soft matrix of human platelet lysate (hPL-gel). We demonstrate that this fibrin-based hydrogel supports growth of primary MSCs with pronounced deposition of extracellular matrix, albeit it hardly impacts on gene expression profiles or in vitro differentiation of MSCs. Furthermore, iPSCs can be effectively differentiated toward MSC-like cells on the hydrogel. Unexpectedly, this complex differentiation process is not affected by the substrate: iMSCs generated on tissue culture plastic (TCP) or hPL-gel have the same morphology, immunophenotype, differentiation potential, and gene expression profiles. Moreover, global DNA methylation patterns are essentially identical in iMSCs generated on TCP or hPL-gel, indicating that they are epigenetically alike. Taken together, hPL-gel provides a powerful matrix that supports growth and differentiation of primary MSCs and iMSCs - but this soft hydrogel does not impact on lineage-specific differentiation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The relevance of topographic cues for commitment of induced pluripotent stem cells (iPSCs) is largely unknown. In this study, we demonstrate that groove-ridge structures with a periodicity in the submicrometer range induce elongation of iPSC colonies, guide the orientation of apical actin fibers, and direct the polarity of cell division. Elongation of iPSC colonies impacts also on their intrinsic molecular patterning, which seems to be orchestrated from the rim of the colonies. BMP4-induced differentiation is enhanced in elongated colonies, and the submicron grooves impact on the spatial modulation of YAP activity upon induction with this morphogen. Interestingly, TAZ, a YAP paralog, shows distinct cytoskeletal localization in iPSCs. These findings demonstrate that topography can guide orientation and organization of iPSC colonies, which may affect the interaction between mechanosensors and mechanotransducers in iPSCs., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8-/- iPS cells and IRF8-/- embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD141+ cross-presenting cDC1 and CD1c+ classical cDC2 and CD303+ plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF8-/- iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF8-/- DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. Stem Cells 2017;35:898-908., (© 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Human pluripotent stem cells represent a powerful tool to study human embryonic development and disease but also open up novel strategies for cell replacement therapies. Their capacity to give rise to every cell type of the human body, meanwhile, enables researchers to generate high yields of mesodermal, ectodermal, but also endodermal-derived tissues such as hepatic, pancreatic, or intestinal cells. Another progress in the field came with the advent of 3-dimensional culture conditions, so-called organoids, which facilitate maturation of stem cells and in turn more faithfully recapitulate human tissue architecture. While several studies reported the derivation of organoid cultures from adult intestinal tissue, the derivation of intestinal organoids derived from plucked human hair of Crohn's disease patients has not been reported. The current research project reports such successful generation and characterization of induced pluripotent stem cells (iPSCs) derived from hair sheet keratinocyte cultures of a patient with Crohn's disease. Stepwise differentiation along the intestinal lineage showed no differences in intermediate stages such as definitive endoderm formation. We also directed the patterned primitive gut tube toward intestinal organoids resembling the cellular architecture of human "miniguts". As expected from current pathophysiological knowledge on Crohn's disease, there were no obvious morphological differences in the "miniguts" derived from healthy control and diseased patient-induced pluripotent stem cells. Taken together, our platform will enable for detailed and complementary phenotyping of the pathophysiology of Crohn's disease in a novel disease-in-a-dish format., (© Georg Thieme Verlag KG Stuttgart · New York.)

Quality control of human induced pluripotent stem cells (iPSCs) can be performed by several methods. These methods are usually relatively labor-intensive, difficult to standardize, or they do not facilitate reliable quantification. Here, we describe a biomarker to distinguish between pluripotent and non-pluripotent cells based on DNA methylation (DNAm) levels at only three specific CpG sites. Two of these CpG sites were selected by their discriminatory power in 258 DNAm profiles - they were either methylated in pluripotent or non-pluripotent cells. The difference between these two β-values provides an Epi-Pluri-Score that was validated on independent DNAm-datasets (264 pluripotent and 1,951 non-pluripotent samples) with 99.9% specificity and 98.9% sensitivity. This score was complemented by a third CpG within the gene POU5F1 (OCT4), which better demarcates early differentiation events. We established pyrosequencing assays for the three relevant CpG sites and thereby correctly classified DNA of 12 pluripotent cell lines and 31 non-pluripotent cell lines. Furthermore, DNAm changes at these three CpGs were tracked in the course of differentiation of iPSCs towards mesenchymal stromal cells. The Epi-Pluri-Score does not give information on lineage-specific differentiation potential, but it provides a simple, reliable, and robust biomarker to support high-throughput classification into either pluripotent or non-pluripotent cells.

Human induced pluripotent stem cells (iPS cells) resemble embryonic stem cells and can differentiate into cell derivatives of all three germ layers. However, frequently the differentiation efficiency of iPS cells into some lineages is rather poor. Here, we found that fusion of iPS cells with human hematopoietic stem cells (HSCs) enhances iPS cell differentiation. Such iPS hybrids showed a prominent differentiation bias toward hematopoietic lineages but also toward other mesendodermal lineages. Additionally, during differentiation of iPS hybrids, expression of early mesendodermal markers-Brachyury (T), MIX1 Homeobox-Like Protein 1 (MIXL1), and Goosecoid (GSC)-appeared with faster kinetics than in parental iPS cells. Following iPS hybrid differentiation there was a prominent induction of NODAL and inhibition of NODAL signaling blunted mesendodermal differentiation. This indicates that NODAL signaling is critically involved in mesendodermal bias of iPS hybrid differentiation. In summary, we demonstrate that iPS cell fusion with HSCs prominently enhances iPS cell differentiation.

Polycomb group (PcG) proteins comprise evolutionary conserved factors with essential functions for embryonic development and adult stem cells. PcG proteins constitute two main multiprotein polycomb repressive complexes (PRC1 and PRC2) that operate in a hierarchical manner to silence gene transcription. Functionally distinct PRC1 complexes are defined by Polycomb group RING finger protein (Pcgf) paralogs. So far, six Pcgf paralogs (Pcgf1-6) have been identified as defining components of different PCR1-type complexes. Paralog-specific functions are not well understood. Here, we show that Pcgf6 is the only Pcgf paralog with high expression in undifferentiated embryonic stem cells (ESCs). Upon differentiation Pcgf6 expression declines. Following Pcgf6 kockdown (KD) in ESCs, the expression of pluripotency genes decreased, while mesodermal- and spermatogenesis-specific genes were derepressed. Concomitantly with the elevated expression of mesodermal lineage markers, Pcgf6 KD ESCs showed increased hemangioblastic and hematopoietic activities upon differentiation suggesting a function of Pcgf6 in repressing mesodermal-specific lineage genes. Consistant with a role in pluripotency, Pcgf6 replaced Sox2 in the generation of germline-competent induced pluripotent stem (iPS) cells. Furthermore, Pcgf6 KD in mouse embryonic fibroblasts reduced the formation of ESC-like colonies in OSKM-driven reprogramming. Together, these analyses indicate that Pcgf6 is nonredundantly involved in maintaining the pluripotent nature of ESCs and it functions in iPS reprogramming., (© 2014 AlphaMed Press.)

Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns-particularly of DNAm patterns associated with tissue type and aging., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Sertoli cells constitute the structural framework in testis and provide an immune-privileged environment for germ cells. Induced pluripotent stem cells (iPS cells) resemble embryonic stem cells (ES cells) and are generated from somatic cells by expression of specific reprogramming transcription factors. Here, we used C57BL/6 (B6) Sertoli cells to generate iPS cells (Ser-iPS cells) and compared the immunogenicity of Ser-iPS cells with iPS cells derived from mouse embryonic fibroblast (MEF-iPS cells). Ser-iPS cells were injected into syngeneic mice to test for their in vivo immunogenicity in teratoma assay. Teratoma assay allows assessing in vivo immunogenicity of iPS cells and of their differentiated progeny simultaneously. We observed that early-passage Ser-iPS cells formed more teratomas with less immune cell infiltration and tissue damage and necrosis than MEF-iPS cells. Differentiating Ser-iPS cells in embryoid bodies (EBs) showed reduced T cell activation potential compared to MEF-iPS cells, which was similar to syngeneic ES cells. However, Ser-iPS cells lost their reduced immunogenicity in vivo after extended passaging in vitro and late-passage Ser-iPS cells exhibited an immunogenicity similar to MEF-iPS cells. These findings indicate that early-passage Ser-iPS cells retain some somatic memory of Sertoli cells that impacts on immunogenicity of iPS cells and iPS cell-derived cells in vivo and in vitro. Our data suggest that immune-privileged Sertoli cells might represent a preferred source for iPS cell generation, if it comes to the use of iPS cell-derived cells for transplantation.

Reprogramming of somatic cells toward pluripotency involves extensive chromatin reorganization and changes in gene expression. Polycomb group (PcG) proteins are key regulators of chromatin structure, cell identity, and development. In this study, we investigated the impact of Ezh2, a core subunit of Polycomb repressive complex 2 (PRC2), on the generation of induced pluripotent stem (iPS) cells. We found that Ezh2 expression is induced during iPS cell generation and iPS cells contain high levels of Ezh2 mRNA and protein. Importantly, shRNA knockdown of Ezh2 during reprogramming severely impairs iPS cell generation. Mechanistically, Ezh2 acts during reprogramming at least in part through repressing the Ink4a/Arf locus, which represents a major roadblock for iPS cell generation. Interestingly, knockdown of Ezh2 in established pluripotent cells leaves pluripotency and self-renewal of embryonic stem cells and iPS cells unaffected. Altogether, our results demonstrate that Ezh2 is critical for efficient iPS cell generation, whereas it is dispensable for maintaining the reprogrammed iPS cell state.

Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs - this is of relevance for standardization and automation of cell culture procedures.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites. Overall, DNA methylation patterns of iP-MSC and ESC were similar whereas some CpG sites revealed highly significant differences, which were not related to parental MSC. Furthermore, hypermethylation in iP-MSC versus ESC occurred preferentially outside of CpG islands and was enriched in genes involved in epidermal differentiation indicating that these differences are not due to random de novo methylation. Subsequently, we searched for CpG sites with donor-specific variation. These "epigenetic fingerprints" were highly enriched in non-promoter regions and outside of CpG islands-and they were maintained upon reprogramming. In conclusion, iP-MSC clones revealed relatively little intraindividual variation but they maintained donor-derived epigenetic differences. In the absence of isogenic controls, it would therefore be more appropriate to compare iPSC from different donors rather than a high number of different clones from the same patient.

Human adult germline stem cells (haGSCs) were established from human testicular biopsies and were claimed to be pluripotent. Recently, the gene expression profile of haGSCs demonstrated that these cells presented with a fibroblast rather than a pluripotent identity. Nevertheless, haGSCs were reported to generate teratomas. In this report, we address this discrepancy. Instead of using haGSCs, which are no longer available for the stem cell community, we used a human testicular fibroblastic cell (hTFC) line that presents with a gene expression profile highly similar to that of haGSCs. Indeed, as shown by microarray analysis, the similarity between hTFCs and haGSCs is comparable to human embryonic stem cell (hESC) lines derived by different laboratories. We argue that the almost identical gene expression profile of hTFCs and haGSCs should result in a very similar if not identical differentiation potential. Strikingly, hTFCs were not able to generate teratomas after injection into nude mice. Instead, they formed a mesenchymal lesion that morphologically resembled the putative haGSC-derived teratomas reported previously. We conclude that haGSCs, which exhibit a profile similar to that of fibroblasts and could not generate teratomas, are not pluripotent. Future work will have to show if pluripotent cells can be derived from human testicular biopsies. Mouse work and certain testicular germ cell tumors indicate that this will be possible., (Copyright © 2011 AlphaMed Press.)

Conrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs). Here we investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues' haGSCs is therefore called into question.