Martin Stehling

Induction of Pluripotency in Adult Unipotent Germline Stem Cells

Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.

Direct reprogramming of fibroblasts into epiblast stem cells

Epiblast stem cells (EpiSCs) derived from epiblast tissue of post-implantation embryos are pluripotent and can give rise to all three germ layers in teratoma assays. Introduction of the four transcription factors Oct4, Sox2, Klf4 and c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs) that are very similar to embryonic stem cells (ESCs) in a number of characteristics. However, generation of EpiSCs by the direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here, we show that these transcription factors can be used to directly generate induced EpiSCs (iEpiSCs) under EpiSC culture conditions. iEpiSCs resemble EpiSCs in morphology, gene expression pattern, epigenetic status and chimaera-forming capability. This study demonstrates that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We therefore hypothesize that it will eventually be possible to shape the identity of a directly reprogrammed cell simply by modulating culture conditions.

Pluripotential Reprogramming of the Somatic Genome in Hybrid Cells Occurs with the First Cell Cycle

The fusion of pluripotent embryonic cells with somatic cells results in reprogramming of the somatic cell genome. Oct4‐green fluorescent protein (GFP) transgenes that do not contain the proximal enhancer (PE) region are widely used to visualize reprogramming of the somatic to the pluripotent cell state. The temporal onset of Oct4‐GFP activation has been found to occur 40–48 hours postfusion. We asked whether activation of the transgene actually reflects activation of the endogenous Oct4 gene. In the current study, we show that activation of an Oct4‐GFP transgene that contains the PE region occurs within 22 hours of fusion. In addition, demethylation of the Oct4‐GFP transgene and that of the endogenous Oct4 and Nanog genes was found to occur within 24 hours of fusion. As this timing corresponds with the timing of cell cycle completion in embryonic stem cells and fusion hybrids (∼22 hours), we postulate that pluripotential reprogramming of the somatic cell genome begins during the first cell cycle after the fusion of a somatic cell with a pluripotent cell and has been completed by day 2 postfusion.

BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL(+) Lin(-)Sca-1(+)c-kit(+) (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin(-)Sca-1(-)c-kit(+)), nor mature granulocytes (CD11b(+)Gr-1(+)), nor potential stem cell niche cells (CD45(-)Ter119(-)) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL(+) LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

Regulation of Developmental Lymphangiogenesis by Syk+ Leukocytes

Lymphatic vessels are essential for tissue homeostasis and immune surveillance and contribute to pathological conditions. Lymphatic endothelium differentiates from veins and forms an independent vascular tree with only few connections to the venous circulation. Failure of blood and lymphatic vessel separation results in hemorrhage and edema. VEGF-C and -D are strong inducers of lymphangiogenesis and have essential (VEGF-C) and modulatory (VEGF-D) roles during developmental lymphangiogenesis. We describe here a myeloid population that is defined by expression of the tyrosine kinase Syk, comprises largely M2-polarized mononuclear cells, and robustly expresses angiogenic factors, including VEGF-C/-D and chemokines. These cells stimulate lymphangiogenesis in vivo. Deletion of Syk causes increased chemotractant production, enhanced transmigration, and accumulation in the skin. Ensuing lymphatic hyperplasia and vessel dilation cause the formation of blood-lymphatic shunts. This mechanism does not involve circulating endothelial progenitor cells and demonstrates the potential of hematopoietic cells to control developmental lymphangiogenesis.

Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile

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.

Identification of a clonally expanding haematopoietic compartment in bone marrow.

In mammals, postnatal haematopoiesis occurs in the bone marrow (BM) and involves specialized microenvironments controlling haematopoietic stem cell (HSC) behaviour and, in particular, stem cell dormancy and self‐renewal. While these processes have been linked to a number of different stromal cell types and signalling pathways, it is currently unclear whether BM has a homogenous architecture devoid of structural and functional partitions. Here, we show with genetic labelling techniques, high‐resolution imaging and functional experiments in mice that the periphery of the adult BM cavity harbours previously unrecognized compartments with distinct properties. These units, which we have termed hemospheres, were composed of endothelial, haematopoietic and mesenchymal cells, were enriched in CD150+ CD48− putative HSCs, and enabled rapid haematopoietic cell proliferation and clonal expansion. Inducible gene targeting of the receptor tyrosine kinase VEGFR2 in endothelial cells disrupted hemospheres and, concomitantly, reduced the number of CD150+ CD48− cells. Our results identify a previously unrecognized, vessel‐associated BM compartment with a specific localization and properties distinct from the marrow cavity.

Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells

Mural cells of the vessel wall, namely pericytes and vascular smooth muscle cells, are essential for vascular integrity. The developmental sources of these cells and molecular mechanisms controlling their progenitors in the heart are only partially understood. Here we show that endocardial endothelial cells are progenitors of pericytes and vascular smooth muscle cells in the murine embryonic heart. Endocardial cells undergo endothelial–mesenchymal transition and convert into primitive mesenchymal progenitors expressing the platelet-derived growth factor receptors, PDGFRα and PDGFRβ. These progenitors migrate into the myocardium, differentiate and assemble the wall of coronary vessels, which requires canonical Wnt signalling involving Frizzled4, β-catenin and endothelial cell-derived Wnt ligands. Our findings identify a novel and unexpected population of progenitors for coronary mural cells with potential relevance for heart function and disease conditions.

Novel imatinib-sensitive PDGFRA-activating point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia-like disease

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients.

Smed-SmB, a member of the LSm protein superfamily, is essential for chromatoid body organization and planarian stem cell proliferation

Planarians are an ideal model system to study in vivo the dynamics of adult pluripotent stem cells. However, our knowledge of the factors necessary for regulating the ‘stemness’ of the neoblasts, the adult stem cells of planarians, is sparse. Here, we report on the characterization of the first planarian member of the LSm protein superfamily, Smed-SmB, which is expressed in stem cells and neurons in Schmidtea mediterranea. LSm proteins are highly conserved key players of the splicing machinery. Our study shows that Smed-SmB protein, which is localized in the nucleus and the chromatoid body of stem cells, is required to safeguard the proliferative ability of the neoblasts. The chromatoid body, a cytoplasmatic ribonucleoprotein complex, is an essential regulator of the RNA metabolism required for the maintenance of metazoan germ cells. However, planarian neoblasts and neurons also rely on its functions. Remarkably, Smed-SmB dsRNA-mediated knockdown results in a rapid loss of organization of the chromatoid body, an impairment of the ability to post-transcriptionally process the transcripts of Smed-CycB, and a severe proliferative failure of the neoblasts. This chain of events leads to a quick depletion of the neoblast pool, resulting in a lethal phenotype for both regenerating and intact animals. In summary, our results suggest that Smed-SmB is an essential component of the chromatoid body, crucial to ensure a proper RNA metabolism and essential for stem cell proliferation.