Reinier van der Linden

Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

Gata2 is required for HSC generation and survival

GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition and thereafter for HSC survival

Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation.

Using highly sensitive RNAseq to examine the whole transcriptome of enriched aortic hematopoietic stem cells and endothelial cells, the authors find G-protein–coupled receptor, Gpr56, is required to generate the first HSCs during endothelial to hematopoietic cell transition.

Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells

Summary Therapeutic application of human embryonic stem cells (hESCs) requires precise control over their differentiation. However, spontaneous differentiation is prevalent, and growth factors induce multiple cell types; e.g., the mesoderm inducer BMP4 generates both mesoderm and trophoblast. Here we identify endogenous WNT signals as BMP targets that are required and sufficient for mesoderm induction, while trophoblast induction is WNT independent, enabling the exclusive differentiation toward either lineage. Furthermore, endogenous WNT signals induce loss of pluripotency in hESCs and their murine counterparts, epiblast stem cells (EpiSCs). WNT inhibition obviates the need to manually remove differentiated cells to maintain cultures and improves the efficiency of directed differentiation. In EpiSCs, WNT inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras. Our findings show that endogenous WNT signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs.

HIF1α is a regulator of hematopoietic progenitor and stem cell development in hypoxic sites of the mouse embryo

Hypoxia affects many physiologic processes during early stages of mammalian ontogeny, particularly placental and vascular development. In the adult, the hypoxic bone marrow microenvironment plays a role in regulating hematopoietic stem cell (HSC) function. HSCs are generated from the major vasculature of the embryo, but whether the hypoxic response affects the generation of these HSCs is as yet unknown. Here we examined whether Hypoxia Inducible Factor1-alpha (HIF1α), a key modulator of the response to hypoxia, is essential for HSC development. We found hypoxic cells in embryonic tissues that generate and expand hematopoietic cells (aorta, placenta and fetal liver), and specifically aortic endothelial and hematopoietic cluster cells. A Cre/loxP conditional knockout (cKO) approach was taken to delete HIF1α in Vascular Endothelial-Cadherin expressing endothelial cells, the precursors to definitive hematopoietic cells. Functional assays show that HSC and hematopoietic progenitor cells (HPCs) are significantly reduced in cKO aorta and placenta. Moreover, decreases in phenotypic aortic hematopoietic cluster cells in cKO embryos indicate that HIF1α is necessary for generation and/or expansion of HPCs and HSCs. cKO adult BM HSCs are also affected under transplantation conditions. Thus, HIF1α is a regulator of HSC generation and function beginning at the earliest embryonic stages.

BMP signalling differentially regulates distinct haematopoietic stem cell types

Adult haematopoiesis is the outcome of distinct haematopoietic stem cell (HSC) subtypes with self-renewable repopulating ability, but with different haematopoietic cell lineage outputs. The molecular basis for this heterogeneity is largely unknown. BMP signalling regulates HSCs as they are first generated in the aorta-gonad-mesonephros region, but at later developmental stages, its role in HSCs is controversial. Here we show that HSCs in murine fetal liver and the bone marrow are of two types that can be prospectively isolated—BMP activated and non-BMP activated. Clonal transplantation demonstrates that they have distinct haematopoietic lineage outputs. Moreover, the two HSC types differ in intrinsic genetic programs, thus supporting a role for the BMP signalling axis in the regulation of HSC heterogeneity and lineage output. Our findings provide insight into the molecular control mechanisms that define HSC types and have important implications for reprogramming cells to HSC fate and treatments targeting distinct HSC types.

Stable X chromosome reactivation in female human induced pluripotent stem cells

Summary In placental mammals, balanced expression of X-linked genes is accomplished by X chromosome inactivation (XCI) in female cells. In humans, random XCI is initiated early during embryonic development. To investigate whether reprogramming of female human fibroblasts into induced pluripotent stem cells (iPSCs) leads to reactivation of the inactive X chromosome (Xi), we have generated iPSC lines from fibroblasts heterozygous for large X-chromosomal deletions. These fibroblasts show completely skewed XCI of the mutated X chromosome, enabling monitoring of X chromosome reactivation (XCR) and XCI using allele-specific single-cell expression analysis. This approach revealed that XCR is robust under standard culture conditions, but does not prevent reinitiation of XCI, resulting in a mixed population of cells with either two active X chromosomes (Xas) or one Xa and one Xi. This mixed population of XaXa and XaXi cells is stabilized in naive human stem cell medium, allowing expansion of clones with two Xas.

Integrin αIIb (CD41) plays a role in the maintenance of hematopoietic stem cell activity in the mouse embryonic aorta

Summary Integrins are transmembrane receptors that play important roles as modulators of cell behaviour through their adhesion properties and the initiation of signaling cascades. The &agr;IIb integrin subunit (CD41) is one of the first cell surface markers indicative of hematopoietic commitment. &agr;IIb pairs exclusively with &bgr;3 to form the &agr;IIb&bgr;3 integrin. &bgr;3 (CD61) also pairs with &agr;v (CD51) to form the &agr;v&bgr;3 integrin. The expression and putative role of these integrins during mouse hematopoietic development is as yet unknown. We show here that hematopoietic stem cells (HSCs) differentially express &agr;IIb&bgr;3 and &agr;v&bgr;3 integrins throughout development. Whereas the first HSCs generated in the aorta at mid-gestation express both integrins, HSCs from the placenta only express &agr;v&bgr;3, and most fetal liver HSCs do not express either integrin. By using &agr;IIb deficient embryos, we show that &agr;IIb is not only a reliable HSC marker but it also plays an important and specific function in maintaining the HSC activity in the mouse embryonic aorta.

Functional and molecular characterization of mouse Gata2-independent hematopoietic progenitors.

The Gata2 transcription factor is a pivotal regulator of hematopoietic cell development and maintenance, highlighted by the fact that Gata2 haploinsufficiency has been identified as the cause of some familial cases of acute myelogenous leukemia/myelodysplastic syndrome and in MonoMac syndrome. Genetic deletion in mice has shown that Gata2 is pivotal to the embryonic generation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). It functions in the embryo during endothelial cell to hematopoietic cell transition to affect hematopoietic cluster, HPC, and HSC formation. Gata2 conditional deletion and overexpression studies show the importance of Gata2 levels in hematopoiesis, during all developmental stages. Although previous studies of cell populations phenotypically enriched in HPCs and HSCs show expression of Gata2, there has been no direct study of Gata2 expressing cells during normal hematopoiesis. In this study, we generate a Gata2Venus reporter mouse model with unperturbed Gata2 expression to examine the hematopoietic function and transcriptome of Gata2 expressing and nonexpressing cells. We show that all the HSCs are Gata2 expressing. However, not all HPCs in the aorta, vitelline and umbilical arteries, and fetal liver require or express Gata2. These Gata2-independent HPCs exhibit a different functional output and genetic program, including Ras and cyclic AMP response element-binding protein pathways and other Gata factors, compared with Gata2-dependent HPCs. Our results, indicating that Gata2 is of major importance in programming toward HSC fate but not in all cells with HPC fate, have implications for current reprogramming strategies.

The DNA binding factor Hmg20b is a repressor of erythroid differentiation

Background In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established. Design and Methods To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene. Results We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic β-like globins and the phospholipase HRAS-like suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts. Conclusions We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the down-regulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic β-like globins.