Modeling human yolk sac hematopoiesis with pluripotent stem cells

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac–derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

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Hematopoietic specification from human pluripotent stem cells: current advances and challenges toward de novo generation of hematopoietic stem cells

Blood ◽

10.1182/blood-2013-07-474825 ◽

2013 ◽

Vol 122 (25) ◽

pp. 4035-4046 ◽

Cited By ~ 75

Author(s):

Igor I. Slukvin

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Pluripotent Stem Cells ◽

De Novo ◽

Human Pluripotent Stem Cells ◽

Cellular Reprogramming ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Cellular Pathways

Abstract Significant advances in cellular reprogramming technologies and hematopoietic differentiation from human pluripotent stem cells (hPSCs) have already enabled the routine production of multiple lineages of blood cells in vitro and opened novel opportunities to study hematopoietic development, model genetic blood diseases, and manufacture immunologically matched cells for transfusion and cancer immunotherapy. However, the generation of hematopoietic cells with robust and sustained multilineage engraftment has not been achieved. Here, we highlight the recent advances in understanding the molecular and cellular pathways leading to blood development from hPSCs and discuss potential approaches that can be taken to facilitate the development of technologies for de novo production of hematopoietic stem cells.

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GATA2 deficiency and human hematopoietic development modeled using induced pluripotent stem cells

Blood Advances ◽

10.1182/bloodadvances.2018017137 ◽

2018 ◽

Vol 2 (23) ◽

pp. 3553-3565 ◽

Cited By ~ 13

Author(s):

Moonjung Jung ◽

Stefan Cordes ◽

Jizhong Zou ◽

Shiqin J. Yu ◽

Xavi Guitart ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Nk Cells ◽

Pluripotent Stem Cells ◽

Bone Marrow Failure ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Gata2 Deficiency ◽

Induced Pluripotent

Abstract GATA2 deficiency is an inherited or sporadic genetic disorder characterized by distinct cellular deficiency, bone marrow failure, various infections, lymphedema, pulmonary alveolar proteinosis, and predisposition to myeloid malignancies resulting from heterozygous loss-of-function mutations in the GATA2 gene. How heterozygous GATA2 mutations affect human hematopoietic development or cause characteristic cellular deficiency and eventual hypoplastic myelodysplastic syndrome or leukemia is not fully understood. We used induced pluripotent stem cells (iPSCs) to study hematopoietic development in the setting of GATA2 deficiency. We performed hematopoietic differentiation using iPSC derived from patients with GATA2 deficiency and examined their ability to commit to mesoderm, hemogenic endothelial precursors (HEPs), hematopoietic stem progenitor cells, and natural killer (NK) cells. Patient-derived iPSC, either derived from fibroblasts/marrow stromal cells or peripheral blood mononuclear cells, did not show significant defects in committing to mesoderm, HEP, hematopoietic stem progenitor, or NK cells. However, HEP derived from GATA2-mutant iPSC showed impaired maturation toward hematopoietic lineages. Hematopoietic differentiation was nearly abolished from homozygous GATA2 knockout (KO) iPSC lines and markedly reduced in heterozygous KO lines compared with isogenic controls. On the other hand, correction of the mutated GATA2 allele in patient-specific iPSC did not alter hematopoietic development consistently in our model. GATA2 deficiency usually manifests within the first decade of life. Newborn and infant hematopoiesis appears to be grossly intact; therefore, our iPSC model indeed may resemble the disease phenotype, suggesting that other genetic, epigenetic, or environmental factors may contribute to bone marrow failure in these patients following birth. However, heterogeneity of PSC-based models and limitations of in vitro differentiation protocol may limit the possibility to detect subtle cellular phenotypes.

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Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from human induced pluripotent stem cells

10.1101/2021.04.25.441298 ◽

2021 ◽

Author(s):

Naritaka Tamaoki ◽

Stefan Siebert ◽

Takuya Maeda ◽

Ngoc-Han Ha ◽

Meghan L. Good ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Circulatory System ◽

Yolk Sac ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Induced Pluripotent

The human definitive yolk sac is an important organ supporting the early developing embryo through nutrient supply and by facilitating the establishment of the embryonic circulatory system. However, the molecular and cellular biology of the human yolk sac remains largely obscure due to the lack of suitable in vitro models. Here, we show that human induced pluripotent stem cells (hiPSCs) co-cultured with various types of stromal cells as spheroids self-organize into yolk sac-like organoids without the addition of exogenous factors. Yolk sac-like organoids recapitulated a yolk sac specific cellular complement and structures as well as the functional ability to generate definitive hematopoietic progenitor cells (HPCs). Furthermore, sequential hemato-vascular ontogenesis could be observed during organoid formation. Notably, our organoid system can be performed in a scalable, autologous, and xeno-free condition, thereby providing an important model of human definitive yolk sac development and allows for efficient bulk generation of hiPSC-derived HPCs.

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Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro

Blood ◽

10.1182/blood-2003-03-0729 ◽

2003 ◽

Vol 102 (13) ◽

pp. 4345-4353 ◽

Cited By ~ 46

Author(s):

Weiming Li ◽

Scott A. Johnson ◽

William C. Shelley ◽

Michael Ferkowicz ◽

Paul Morrison ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Endothelial Cell ◽

Ex Vivo ◽

Yolk Sac ◽

Hematopoietic Progenitor Cell ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem

AbstractThe embryonic origin and development of hematopoietic and endothelial cells is highly interdependent. We hypothesized that primary endothelial cells from murine yolk sac and para-aortic splanchnopleura (P-Sp) may possess the capacity to expand hematopoietic stem cells (HSCs) and progenitor cells ex vivo. Using Tie2-GFP transgenic mice in combination with fluorochrome-conjugated monoclonal antibodies to vascular endothelial growth factor receptor-2 (Flk1) and CD41, we have successfully isolated pure populations of primary endothelial cells from 9.5-days after coitus (dpc) yolk sac and P-Sp. Adult murine bone marrow Sca-1+c-Kit+lin- cells were cocultured with yolk sac or P-Sp Tie2-GFP+Flk-1+CD41- endothelial cell monolayers for 7 days and the total number of nonadherent cells increased 47- and 295-fold, respectively, and hematopoietic progenitor counts increased 9.4- and 11.4-fold, respectively. Both the yolk sac and P-Sp endothelial cell cocultures facilitated long-term (> 6 months) HSC competitive repopulating ability (2.8- to 9.8-fold increases, respectively). These data suggest that 9.5-dpc yolk sac- and P-Sp-derived primary Tie2-GFP+Flk-1+CD41- endothelial cells possess the capacity to expand adult bone marrow hematopoietic progenitor cell and HSC repopulating ability ex vivo. (Blood. 2003;102:4345-4353)

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Evidence of Increased Hemangioblastic and Early Hematopoietic Potential in Chronic Myeloid Leukemia (CML)-derived Induced Pluripotent Stem Cells (iPSC)

10.1101/2020.06.17.140202 ◽

2020 ◽

Author(s):

G. Telliam ◽

O. Féraud ◽

S. Baykal-Köse ◽

F. Griscelli ◽

J. Imeri ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Signaling Pathway ◽

Pluripotent Stem Cells ◽

Embryoid Body ◽

Embryonic Stem ◽

Blast Cell ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Induced Pluripotent

ABSTRACTHemangioblasts derived from mesodermal lineage are the earliest precursors of hematopoietic stem cells and endothelial cells. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are the only experimental systems in which these cells can be assayed and quantified. We show here using CML-derived iPSC and blast-cell colony forming (Bl-CFC) assays that hemangioblasts are highly expanded in CML derived iPSC as compared to human H1-ESC-derived hemangioblasts. BCR-ABL signaling pathway is intact in these cells with evidence of CRK-L phosphorylation which is reduced by the use of Imatinib. Hematopoietic progenitor assays generated using blast-CFC demonstrates also a highly increased hematopoietic progenitor potential of these cells as compared to H1-ESC. The same results were also obtained using hematopoietic progenitor assays via embryoid body formation. In CML iPSC, we have also found a significant reduction of Aryl Hydrocarbon Receptor (AHR) expression which is involved in hematopoietic quiescence. Further inhibition of AHR using StemRegenin (SR1), an AHR antagonist, led to an increase of blast-cell colonies in CML iPSC whereas the use of an AHR agonist inhibited blast cell colonies. Thus, our results show for the first time, the possibility of establishment of a myeloproliferative phenotype using patient-derived iPSC and the presence of a major expansion hemangioblast compartment and derived hematopoietic progenitors in this context. They also suggest that the AHR signaling pathway could represent a novel druggable target in CML.

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Hematopoietic Development From Human Induced Pluripotent Stem Cells.

Blood ◽

10.1182/blood.v114.22.2530.2530 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2530-2530

Author(s):

Matthias Grauer ◽

Martina Konantz ◽

Nina I. Niebuhr ◽

Lothar Kanz ◽

In-Hyun Park ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Genetic Modification ◽

Pluripotent Stem Cells ◽

Ips Cells ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Human Ips Cells ◽

Blood Formation ◽

Induced Pluripotent

Abstract Abstract 2530 Poster Board II-507 A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generate pluripotent stem cells. Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) human pluripotent stem cell lines (Park et al, 2008). Current research is focusing on improving reprogramming protocols (e.g. circumventing the use of retroviral technology and oncoproteins) and methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. We have asked whether these signaling pathways patterning blood fate are conserved during hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data showed robust differentiation of iPS cells to mesoderm and to blood lineages, comparable to reports on differentiation of human ESC in this system. We detected robust formation of CD34+ (28.9±12), CD45+ (26.8±13.4) and CD34+CD45+ (16.1±13.7) cells, and a high incidence of CFU-initiating cells in functional colony assays, predominantly displaying myeloid but also some mixed CFU-GEMM activity. Similar to our findings in mouse ESC, mesodermal and hematopoietic genes were expressed in waves, and expression was augmented by supplementation of cultures with BMP4. Mesodermal markers (e.g. BRACHYURY ) were induced at day 2, and declined after day 9, when hematopoietic markers (SCL) appeared, indicating conversion of mesoderm to progenitors of the blood lineage. Expression of all three human CDX genes (CDX1, CDX2 and CDX4) peaked at day 6, suggesting that the function of CDX genes to pattern preformed mesoderm to blood fate may be conserved in human embryogenesis. Ongoing experiments in our laboratory focus on genetic modification of human iPS cells to study effects of specific genes during human emrbyonic hematopoiesis. Furthermore we have succeeded in transducing iPS cells with lentiviruses that allow GFP expression and puromycin selection, thus indicating feasibility for genetic modification. Taken together, our results show robust hematopoietic differentiation of human iPS cells and suggest that genetically modified in vitro differentiating iPS cells can be used to study human developmental hematopoiesis. Characterizing genetic pathways governing human embryonic blood formation will direct differentiation of induced pluripotent stem cells into repopulating hematopoietic stem cells, enabling generation of isogenic cell replacement therapies. Moreover, this experimental approach enables modeling of hematologic diseases, opening up a novel platform for gradual studies of genetic mechanisms during disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

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Evi-1 Regulates Myelopoiesis and Hematopoietic Stem Cell Development in Zebrafish and Human Pluripotent Stem Cells

Blood ◽

10.1182/blood.v118.21.1281.1281 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1281-1281

Author(s):

Martina Konantz ◽

Matthias Grauer ◽

Sarah Grzywna ◽

Martijn Brugman ◽

Lothar Kanz ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Pluripotent Stem Cells ◽

Zebrafish Embryo ◽

Human Pluripotent Stem Cells ◽

Hematopoietic Stem ◽

Hematopoietic Development

Abstract Abstract 1281 The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Over the last years, Evi-1 evolved as one of the most potent oncogenes associated with human and murine myeloid leukemia. More recent studies in knockout mice suggest also involvement of Evi-1 in the regulation of developmental hematopoiesis, but the role of Evi-1 in this context is poorly understood. Here, we analyzed zebrafish embryo and human pluripotent stem cells (PSC) to understand how Evi-1 modulates early hematopoietic development. We examined the hematopoietic development in zebrafish embryo by in situ hybridization (ISH) for hematopoietic markers. The zebrafish homologue evi-1 was shown to be expressed in co-localization with scl in the posterior blood islands, indicating a role during early blood development. We also performed loss-of-function studies were by injecting morpholino oligonucleotides (MO) in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. Inhibition of evi-1 was confirmed in MO-injected versus control embryos. N=100 zebrafish embryos were analyzed per experiment in each group. To control for off-target effects, two separate MO were designed and injected. MO mediated evi-1 knockdown severely reduced numbers of circulating blood cells and induced hemorrhages. ISH performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells (measured by pu.1 expression), while no changes were observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Moreover, analyses at 36 hours and 5 days post fertilization showed strong reduction of runx1+/cmyb+ cells and rag1+ lymphoid cells, indicating a role of evi-1 in developing hematopoietic stem cells (HSC). Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects hematopoietic stem cell proliferation through regulation of Gata2. To test whether Gata2 is a putative downstream regulator of Evi-1 in our system, we performed a rescue experiment and co-injected gata2 mRNA in evi-1 MO treated fish. Indeed, ectopic gata2 rescued the impaired myeloid phenotype, as shown by re-occurrence of mpo, l-plastin as well as pu.1 expressing cells. To assess whether these molecular interactions are conserved during human developmental hematopoiesis, we surveyed in vitro differentiating human pluripotent stem cells (PSC) genetically modified to suppress EVI-1. EVI-1 expression was detected during differentiation of human PSC in embryoid bodies, especially around day 9 when hematopoietic progenitors start to emerge in this system. Treatment with EVI-1 shRNA strongly reduced the generation of myeloid colonies from human PSC in vitro as well as the numbers of emerging CD34+ and CD45+ cells. Molecularly, EVI-1 suppression inhibited the expression of PU.1 and GATA2 during the course of development, while leaving SCL and GATA1 expression unaltered. Taken together, our data suggest that, in both fish and human, Evi-1 regulates embryonic myelopoiesis through interactions with Gata2 and independently of Gata1 and embryonic erythropoiesis. Moreover, Evi-1 appears crucial for HSC development. Currently ongoing experiments in our laboratory focus on the further elucidation of the molecular mechanisms underlying the Evi-1 effects during developmental hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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