Hans R Schöler

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Saxony, Germany

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Publications (218)1918.04 Total impact

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    ABSTRACT: Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3-6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.
    The EMBO Journal 03/2015; DOI:10.15252/embj.201488049 · 10.75 Impact Factor
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    ABSTRACT: Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes for translational purposes. Multiple cardiomyocyte differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high-efficiency procedure for generating cardiomyocytes both in 2D and 3D culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue was used to monitor culture-induced maturation of the resulting cardiomyocytes (CMs). This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately four weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time-frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation. This article is protected by copyright. All rights reserved.
    Stem Cells 01/2015; DOI:10.1002/stem.1964 · 7.70 Impact Factor
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    ABSTRACT: Several mouse pluripotent stem cell types have been established either from mouse blastocysts and epiblasts. Among these, embryonic stem cells (ESCs) are considered to represent a "naïve", epiblast stem cells (EpiSCs) a "primed" pluripotent state. Although EpiSCs form derivatives of all three germ layers during in vitro differentiation, they rarely incorporate into the inner cell mass of blastocysts and rarely contribute to chimera formation following blastocyst injection. Here we successfully established homogeneous population of EpiSC lines with efficient chimera-forming capability using a medium containing fibroblast growth factor (FGF)-4. The expression levels of Rex1 and Nanog was very low although Oct4 level is comparable to ESCs. EpiSCs also expressed higher levels of epiblast markers, such as Cer1, Eomes, Fgf5, Sox17, and T, and further showed complete DNA methylation of Stella and Dppa5 promoters. However, the EpiSCs were clustered separately from E3 and T9 EpiSC lines and showed a completely different global gene expression pattern to ESCs. Furthermore, the EpiSCs were able to differentiate into all three germ layers in vitro and efficiently formed teratomas and chimeric embryos (21.4%) without germ-line contribution.
    Scientific Reports 12/2014; 4:7477. DOI:10.1038/srep07477 · 5.08 Impact Factor
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    ABSTRACT: Direct reprogramming of somatic cells to pluripotent stem cells entails the obliteration of somatic cell memory and the reestablishment of epigenetic events. Induced pluripotent stem (iPS) cells have been created by reprogramming somatic cells through the transduction of reprogramming factors. During cell reprogramming, female somatic cells must overcome at least one more barrier than male somatic cells in order to enter a pluripotent state, as they must reactivate an inactive X chromosome (Xi). In this study, we investigated whether the sex of somatic cells affects reprogramming efficiency, differentiation potential, and the post-transcriptional processing of Xist RNA after reprogramming. There were no differences between male and female iPS cells with respect to reprogramming efficiency or their differentiation potential in vivo. However, reactivating Xi took longer than reactivating pluripotency-related genes. We also found that direct reprogramming leads to gender appropriate posttranscriptional reprogramming: like male embryonic stem (ES) cells, male iPS cells expressed only the long Xist isoform, whereas female iPS cells, like female ES cells, expressed both the long and short isoforms.
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    ABSTRACT: Aims: To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). Results: We found 11 genes ³1.5-fold overexpressed in EGCs. None of the genes identified were Yamanaka´s genes, but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. Hypoxia induced a glycolytic program in PGCs in detriment of mitochondrial oxidative phosphorylation. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGL), which differentiate into cells of the three germ layers in vitro, and contribute to the ICM in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1α stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signalling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. Innovation and Conclusion: Our data suggests that the properties of stemness, pluripotency and self-renewal, are differentially regulated in primordial germ cell reprogramming induced by hypoxia.
    Antioxidants and Redox Signaling 10/2014; 22(3):205-223. DOI:10.1089/ars.2014.5871 · 7.67 Impact Factor
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    ABSTRACT: Epigenetic memory in induced pluripotent stem cells, with regards to their somatic cell type of origin, might lead to variations in their differentiation capacities. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34+ hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates an epigenetic memory of induced pluripotent stem cells with regards to their somatic cell type of origin, we found a similar hematopoietic induction potential and erythroid differentiation pattern. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the CD34+ hematopoietic stem cell-derived induced pluripotent stem cells. More detailed methylation analysis of the hematopoietic and erythrocyte promoters identified similar CpG methylation levels in the CD34+-derived and neural stem cell-derived induced pluripotent stem cell lines, which confirms their comparable erythroid differentiation potential.
    Haematologica 10/2014; DOI:10.3324/haematol.2014.108068 · 5.94 Impact Factor
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    ABSTRACT: The spinal cord does not spontaneously regenerate and treatment that ensures functional recovery after spinal cord injury (SCI) is still not available. Recently, fibroblasts have been directly converted into induced neural stem cells (iNSCs) by the forced expression defined transcription factors. Although directly converted iNSCs have been considered to be a cell source for clinical applications, their therapeutic potential has not yet been investigated. Here we show that iNSCs directly converted from mouse fibroblasts enhance the functional recovery of SCI animals. Engrafted iNSCs could differentiate into all neuronal lineages, including different subtypes of mature neurons. Furthermore, iNSC-derived neurons could form synapses with host neurons, thus enhancing the locomotor function recovery. A time-course analysis of iNSC-treated SCI animals revealed that engrafted iNSCs effectively reduced the inflammatory response and apoptosis in the injured area. iNSC transplantation also promoted the active regeneration of the endogenous recipient environment in the absence of tumor formation. Therefore, our data suggest that directly converted iNSCs hold therapeutic potential for treatment of SCI and may thus represent a promising cell source for transplantation therapy in patients with SCI.
    Journal of Biological Chemistry 10/2014; DOI:10.1074/jbc.M114.588871 · 4.60 Impact Factor
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    ABSTRACT: Owing to the inherent disconnect between drug pharmacology in heterologous cellular models and drug efficacy in vivo, the quest for more predictive in vitro systems is one of the most urgent challenges of modern drug discovery. An improved pharmacological in vitro profiling would employ primary samples of the proper drug-targeted human tissue or the bona fide human disease-relevant cells. With the advent of induced pluripotent stem (iPS) cell technology the facilitated access to a variety of disease-relevant target cells is now held out in prospect. In this review, we focus on the use of human iPS cell derived neurons for high throughput pharmaceutical drug screening, employing detection technologies that are sufficiently sensitive to measure signaling in cells with physiological target protein expression levels.
    Trends in Pharmacological Sciences 10/2014; 35(10). DOI:10.1016/j.tips.2014.07.003 · 9.99 Impact Factor
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    ABSTRACT: The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34(+) hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.
    Cell Reports 09/2014; DOI:10.1016/j.celrep.2014.08.014 · 7.21 Impact Factor
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    ABSTRACT: Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications.
    09/2014; 3(3):423-31. DOI:10.1016/j.stemcr.2014.06.017
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    ABSTRACT: Though expression of the homeobox transcription factor Nanog is generally restricted to pluripotent cells and early germ cells, many contradictory reports about Nanog’s involvement in tumorigenesis exist. To address this, a modified Tet-On system was utilized to generate Nanog-inducible mice. Following prolonged Nanog expression, phenotypic alterations were found to be restricted to the intestinal tract, leaving other major organs unaffected. Intestinal and colonic epithelium hyperplasia was observed—intestinal villi had doubled in length and hyperplastic epithelium outgrowths were seen after 7 days. Increased proliferation of crypt cells and downregulation of the tumor suppressors Cdx2 and Klf4 was detected. ChIP analysis showed physical interaction of Nanog with the Cdx2 and Klf4 promoters, indicating a regulatory conservation from embryonic development. Despite downregulation of tumor suppressors and increased proliferation, ectopic Nanog expression did not lead to tumor formation. We conclude that unlike other pluripotency-related transcription factors, Nanog cannot be considered an oncogene.
    Stem Cell Research 09/2014; DOI:10.1016/j.scr.2014.08.001 · 3.91 Impact Factor
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    ABSTRACT: Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.
    PLoS ONE 08/2014; 9(8):e103985. DOI:10.1371/journal.pone.0103985 · 3.53 Impact Factor
  • Kee-Pyo Kim, Hans R Schöler
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    ABSTRACT: The manufacturing of clinically relevant cells is a widely used strategy in regenerative medicine. Cahan et al. develop a network biology platform named CellNet to accurately assess the fidelity of such cells and spot aberrant regulatory networks, and Morris et al. apply this platform to improve cell manufacturing.
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    ABSTRACT: In pluripotent cells, OCT4 associates with SOX2 to maintain pluripotency or with SOX17 to induce primitive endoderm commitment. The OCT4-SOX2 and OCT4-SOX17 combinations bind mutually exclusive to two distinct composite DNA elements, known as the "canonical" and "compressed" motifs, respectively. The structural basis for the OCT4-SOX17 cooperativity is unknown. Whereas SOX17 has been engineered to replace SOX2 in the pluripotency circuitry, all generated SOX2 mutants have failed to act like SOX17. From molecular simulations, we revealed the OCT4-SOX17 interaction interface and elucidated the SOX-dependent motif preference of OCT4. Moreover, we designed a SOX2 mutant that we predicted and confirmed experimentally to bind cooperatively with OCT4 to the compressed motif. Ultimately, we found a strong correlation between the experimental and calculated relative cooperative-binding free energies of 12 OCT4-SOX-DNA complexes. Therefore, we validated the OCT4-SOX interfaces and demonstrated that in silico design of DNA-binding cooperativity is suitable for altering transcriptional circuitries.
    Structure 08/2014; 22(9):1274-1286. DOI:10.1016/j.str.2014.06.014 · 6.79 Impact Factor
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    ABSTRACT: The pluripotency factor POU5F1 (OCT4) is well known as a key regulator of stem cell fate. Homologues of POU5F1 exist throughout vertebrates, but the evolutionary and functional relationships between the various family members have been unclear. The level to which function has been conserved within this family provides insight into the evolution of early embryonic potency. Here, we seek to clarify the relationship between POU5F1 homologues in the vertebrate lineage, both phylogenetically and functionally. We resolve the confusion over the identity of the zebrafish gene, which was originally named pou2, then changed to pou5f1 and again, more recently, to pou5f3. We argue that the use of correct nomenclature is crucial when discussing the degree to which the networks regulating early embryonic differentiation are conserved.
    Development 08/2014; 141(15):2921-3. DOI:10.1242/dev.108407 · 6.27 Impact Factor
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    ABSTRACT: Tractable and accurate disease models are essential for understanding disease pathogenesis and for developing new therapeutics. As stem cells are capable of self-renewal and differentiation, they are ideally suited both for generating these models and for obtaining the large quantities of cells required for drug development and transplantation therapies. Although proof of principle for the use of adult stem cells and embryonic stem cells in disease modelling has been established, induced pluripotent stem cells (iPSCs) have demonstrated the greatest utility for modelling human diseases. Furthermore, combining gene editing with iPSCs enables the generation of models of genetically complex disorders.
    Nature Reviews Genetics 07/2014; DOI:10.1038/nrg3764 · 39.79 Impact Factor
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    ABSTRACT: Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for longterm periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feederfree culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.
    Molecules and Cells 05/2014; DOI:10.14348/molcells.2014.0080 · 2.24 Impact Factor
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    ABSTRACT: Terminally differentiated cells can be directly converted into different types of somatic cells by using defined factors, thus circumventing the pluripotent state. However, low reprogramming efficiency, along with the absence of proliferation of some somatic cell types, makes it difficult to generate large numbers of cells with this method. Here we describe a protocol to directly convert mouse fibroblasts into self-renewing induced neural stem cells (iNSCs) that can be expanded in vitro, thereby overcoming the limitations associated with low reprogramming efficiency. The four transcription factors required for direct conversion into iNSCs (Sox2, Klf4, Myc (also known as c-Myc) and Pou3f4 (also known as Brn4)) do not generate a pluripotent cell state, and thus the risk for tumor formation after transplantation is reduced. By following the current protocol, iNSCs are observed 4-5 weeks after transduction. Two additional months are required to establish clonal iNSC cell lines that exhibit retroviral transgene silencing and that differentiate into neurons, astrocytes and oligodendrocytes.
    Nature Protocol 04/2014; 9(4):871-81. DOI:10.1038/nprot.2014.056 · 8.36 Impact Factor
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    ABSTRACT: Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.
    Nature 03/2014; DOI:10.1038/nature13134 · 42.35 Impact Factor
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    ABSTRACT: Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs.
    03/2014; 2(3):351-65. DOI:10.1016/j.stemcr.2014.01.005

Publication Stats

13k Citations
1,918.04 Total Impact Points

Institutions

  • 1990–2015
    • Max Planck Institute of Molecular Cell Biology and Genetics
      Dresden, Saxony, Germany
  • 2013–2014
    • University of Münster
      • Medical Faculty
      Muenster, North Rhine-Westphalia, Germany
    • Konkuk University
      • School of Medicine
      Sŏul, Seoul, South Korea
  • 2005–2014
    • Max Planck Institute for Molecular Biomedicine
      • Department of Cell and Developmental Biology
      Muenster, North Rhine-Westphalia, Germany
  • 2012
    • Seoul National University
      • College of Veterinary Medicine
      Sŏul, Seoul, South Korea
  • 2011
    • Hannover Medical School
      • Department of Gastroenterology, Hepatology and Endocrinology
      Hanover, Lower Saxony, Germany
  • 2010–2011
    • University of Rostock
      Rostock, Mecklenburg-Vorpommern, Germany
  • 2009–2011
    • CHA University
      • Department of Biomedical Science
      Seoul, Seoul, South Korea
  • 2006–2010
    • The Scripps Research Institute
      • • Department of Chemistry
      • • Skaggs Institute for Chemical Biology
      La Jolla, CA, United States
  • 2000–2006
    • University of Pennsylvania
      • • School of Veterinary Medicine
      • • Center for Animal Transgenesis and Germ Cell Research
      • • Department of Animal Biology
      Philadelphia, PA, United States
  • 2004
    • William Penn University
      Worcester, Massachusetts, United States
  • 1994–2004
    • European Molecular Biology Laboratory
      Heidelburg, Baden-Württemberg, Germany
  • 2001
    • Istituto Dermopatico dell'Immacolata
      Roma, Latium, Italy