Blood making: learning what to put into the dish

The generation of hematopoietic stem cells (HSCs) from pluripotent stem cell (PSC) sources is a long-standing goal that will require a comprehensive understanding of the molecular and cellular factors that determine HSC fate during embryogenesis. A precise interplay between niche components, such as the vascular, mesenchymal, primitive myeloid cells, and the nervous system provides the unique signaling milieu for the emergence of functional HSCs in the aorta-gonad-mesonephros (AGM) region. Over the last several years, the interrogation of these aspects in the embryo model and in the PSC differentiation system has provided valuable knowledge that will continue educating the design of more efficient protocols to enable the differentiation of PSCs into bona fide, functionally transplantable HSCs. Herein, we provide a synopsis of early hematopoietic development, with particular focus on the recent discoveries and remaining questions concerning AGM hematopoiesis. Moreover, we acknowledge the recent advances towards the generation of HSCs in vitro and discuss possible approaches to achieve this goal in light of the current knowledge.

Download Full-text

Understanding the Journey of Human Hematopoietic Stem Cell Development

Stem Cells International ◽

10.1155/2019/2141475 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 6

Author(s):

Akhilesh Kumar ◽

Saritha S. D’Souza ◽

Abir S. Thakur

Keyword(s):

Stem Cells ◽

Blood Cell ◽

Hematopoietic Stem Cells ◽

Human Blood ◽

Molecular Mechanisms ◽

Inflammatory Responses ◽

Current Knowledge ◽

Hematopoietic Stem ◽

Hematopoietic Development

Hematopoietic stem cells (HSCs) surface during embryogenesis leading to the genesis of the hematopoietic system, which is vital for immune function, homeostasis balance, and inflammatory responses in the human body. Hematopoiesis is the process of blood cell formation, which initiates from hematopoietic stem/progenitor cells (HSPCs) and is responsible for the generation of all adult blood cells. With their self-renewing and pluripotent properties, human pluripotent stem cells (hPSCs) provide an unprecedented opportunity to createin vitromodels of differentiation that will revolutionize our understanding of human development, especially of the human blood system. The utilization of hPSCs provides newfound approaches for studying the origins of human blood cell diseases and generating progenitor populations for cell-based treatments. Current shortages in our knowledge of adult HSCs and the molecular mechanisms that control hematopoietic development in physiological and pathological conditions can be resolved with better understanding of the regulatory networks involved in hematopoiesis, their impact on gene expression, and further enhance our ability to develop novel strategies of clinical importance. In this review, we delve into the recent advances in the understanding of the various cellular and molecular pathways that lead to blood development from hPSCs and examine the current knowledge of human hematopoietic development. We also review howin vitrodifferentiation of hPSCs can undergo hematopoietic transition and specification, including major subtypes, and consider techniques and protocols that facilitate the generation of hematopoietic stem cells.

Download Full-text

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.

Download Full-text

Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition

Cells ◽

10.3390/cells8090951 ◽

2019 ◽

Vol 8 (9) ◽

pp. 951 ◽

Cited By ~ 2

Author(s):

Yong Dong ◽

Chengxiang Xia ◽

Qitong Weng ◽

Tongjie Wang ◽

Fangxiao Hu ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Pluripotent Stem Cells ◽

Culture Condition ◽

Fusion Gene ◽

Nras Mutation ◽

Hematopoietic Stem ◽

Bona Fide

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

Download Full-text

Abstract 22: Identification of the Nature of Cardiac Resident c-kit + Cells

Circulation Research ◽

10.1161/res.119.suppl_1.22 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Chen-Leng Cai ◽

Nishat Sultana ◽

Lu Zhang ◽

Jianyun Yan ◽

Jiqiu Chen ◽

...

Keyword(s):

Stem Cells ◽

Receptor Tyrosine Kinase ◽

Cardiac Injury ◽

Cardiac Stem Cells ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Heart Repair ◽

Bona Fide ◽

Myocardial Marker

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. For more than a decade, c-kit, a receptor tyrosine kinase expressed in certain types of hematopoietic stem cells, has been recognized as a marker of resident CSCs in mammals. It was shown that c-kit + cells are multipotent, with differentiation potential to become cardiomyocytes, endothelial, and smooth muscle cells in vitro and after cardiac injury. Here, we provide new insights into the nature of cardiac resident c-kit + cells. By targeting the c-kit locus with several reporter genes in mice, we unexpectedly found that c-kit + cells rarely co-localizes with cardiac progenitor marker Nkx2.5 or myocardial marker cTnT. Instead, c-kit labels an endocardial population from embryonic stage to adulthood. After acute cardiac injury, the c-kit + cells still retain their endothelial identity and do not become cardiomyocytes. Our study supports the notion that cardiac c-kit + cells are in fact endothelial cells and not CSCs. This finding suggests an urgent need to re-evaluate the mechanisms by which c-kit + cells contribute to heart repair or regeneration given their endothelial identity.

Download Full-text

The hemogenic endothelium: a critical source for the generation of PSC-derived hematopoietic stem and progenitor cells

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03777-y ◽

2021 ◽

Author(s):

Lucas Lange ◽

Michael Morgan ◽

Axel Schambach

Keyword(s):

Stem Cells ◽

De Novo ◽

Cell Types ◽

Hemogenic Endothelium ◽

Hematopoietic Stem ◽

Autologous Cell ◽

Stem And Progenitor Cells ◽

Bona Fide

AbstractIn vitro generation of hematopoietic cells and especially hematopoietic stem cells (HSCs) from human pluripotent stem cells (PSCs) are subject to intensive research in recent decades, as these cells hold great potential for regenerative medicine and autologous cell replacement therapies. Despite many attempts, in vitro, de novo generation of bona fide HSCs remains challenging, and we are still far away from their clinical use, due to insufficient functionality and quantity of the produced HSCs. The challenges of generating PSC-derived HSCs are already apparent in early stages of hemato-endothelial specification with the limitation of recapitulating complex, dynamic processes of embryonic hematopoietic ontogeny in vitro. Further, these current shortcomings imply the incompleteness of our understanding of human ontogenetic processes from embryonic mesoderm over an intermediate, specialized hemogenic endothelium (HE) to their immediate progeny, the HSCs. In this review, we examine the recent investigations of hemato-endothelial ontogeny and recently reported progress for the conversion of PSCs and other promising somatic cell types towards HSCs with the focus on the crucial and inevitable role of the HE to achieve the long-standing goal—to generate therapeutically applicable PSC-derived HSCs in vitro.

Download Full-text

Constitutive Expression of the ‘Lymphoid Enhancer Factor 1‘ (Lef-1) Perturbs Early Hematopoietic Development and Induces a Lethal Myeloproliferative Syndrome in a Subset of Transplanted Mice.

Blood ◽

10.1182/blood.v106.11.644.644 ◽

2005 ◽

Vol 106 (11) ◽

pp. 644-644

Author(s):

Konstantin Petropoulos ◽

Farid Ahmed ◽

Christina Schessl ◽

Natalia Arseni ◽

Aniruddha Deshpande ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

T Cell ◽

Progenitor Cells ◽

Constitutive Expression ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Myeloproliferative Syndrome

Abstract Lef-1 is a nuclear protein of the Lef/Tcf family of transcription factors, known to be a key component of the Wnt/β-catenin signalling pathway. Lef-1 is crucially linked to normal B- and T-cell development. Furthermore, its aberrant expression has been associated with T-cell lymphoma and CLL. However, there are few data about the potential role of this transcription factor in normal hematopoietic stem cell and progenitor development. Aim of this project was to clarify the expression pattern of Lef-1 in early hematopoietic progenitor cells and to test whether constitutive expression of this transcription factor affects early hematopoietic development. Analysis of Lef-1 expression by semi-quantitative RT-PCR and Real Time PCR demonstrated Lef-1 expression in both lymphoid (B220+, CD4+, CD8+) and myeloid (Gr1+, Mac1+) subpopulations, but also in highly purified hematopoietic stem cells (Sca1+/Kit+/Lin−). In order to prove functional relevance of Lef-1 expression, constitutive expression of Lef-1 and of a constitutive active Lef-1 mutant (CA-Lef-1; with a Lef-1 activating β-catenin domain) was induced in primary murine bone marrow (BM) cells by retroviral gene transfer, using a MSCV based retroviral construct with an IRES-GFP cassette. At the level of clonogenic progenitor cells, both Lef-1 and CA-Lef-1 increased the colony forming potential of progenitors in vitro by more than 2-fold compared to the empty vector control (n=4, p<0.03). At the level of the short-term repopulating stem cell, Lef-1 remarkably increased the size and the number of spleen colonies resulting in a 8fold increase in the CFU-S frequency compared to the control (median 120 CFU-S/1x105 versus 15 CFU-S/1x105 cells, respectively; p< 0.001; Lef-1 n=5, control n=13). To assess the impact of Lef-1 on long-term repopulating stem cells mice were transplanted with BM cells transduced either with Lef-1 or CA-Lef-1. Both Lef-1 constructs severely perturbed normal hematopoietic development inducing a reduction of lymphoid cells with an inversion of the lymphoid/myeloid ratio (ratio 0.03 vs. 5.8 in the non-transduced compartment) and accumulation of neutrophils in the peripheral blood (97 % Gr1 positive cells versus 15 % in the non-transduced compartment) as well as in the spleen (lymphoid/myeloid ratio 0.2 vs. 6.9 and 87 % versus 12 % Gr1+ cells). 3 mice (1 Lef-1 and 2 CA-Lef-1 mice) succumbed to a lethal myeloproliferative syndrome, one mouse (Lef-1) developed acute leukemia, which was readily transplantable into secondary and tertiary recipients and showed indefinite IL-3 dependent cell growth in vitro. Taken together, these data show that ordered expression of Lef-1 plays a key role in early hematopoietic development and that deregulation of this transcription factor favors the development of myeloid malignancies.

Download Full-text

Ectopic HOXB4 Expression Expands the Pool of Hemogenic Endothelium-Initiating Progenitor Cells during Pluripotent Stem Cell Differentiation

Blood ◽

10.1182/blood.v124.21.3496.3496 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3496-3496

Author(s):

Nadine Teichweyde ◽

Lara Kasperidus ◽

Peter A Horn ◽

Hannes Klump

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Pluripotent Stem Cell ◽

Stem Cell Differentiation ◽

Great Promise ◽

Hemogenic Endothelium ◽

Hematopoietic Development

Abstract Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for future somatic gene and cell therapy. So far, HSCs capable of long-term multilineage reconstitution in mice have only been obtained when the homeodomain transcription factor HOXB4 was ectopically expressed during pluripotent stem cell differentiation (Kyba et al. Cell 109(1): 29-37, 2002; Pilat et al. Proc Natl Acad Sci USA 102(34): 12101-12106, 2005; Lesinski et al. Stem Cells Transl Med 1(8): 581-591, 2012). However, the primary "target" cell of HOXB4 during hematopoietic development, in vitro, is not yet known. Its identification is a prerequisite for unambiguously identifying the molecular circuits driving HSC development, at least in vitro. To pin down this cell, we retrovirally expressed HOXB4 or a Tamoxifen-inducible HOXB4-ERT2 fusion protein in different reporter and knock-out mouse embryonic stem cell (ESC) lines. For these experiments, ESCs were differentiated for 6 days as embryoid bodies (EBs), dissociated and subsequently cocultured on OP9 stroma cells in medium supplemented with 100 ng/ml mSCF, 40 ng/ml mTPO, 100 ng/ml hFlt3L and 40 ng/ml hVEGF (STFV) for further 3 days. Use of a Runx1(-/-) ESC-line containing a doxycycline-inducible Runx1 coding sequence (“iRunx1”; kindly provided by G. Lacaud, Manchester) uncovered that HOXB4 acts during formation of the hemogenic endothelium (HE) from which HSCs arise. Without Runx1 induction, which arrests hematopoietic development at the HE-stage, ectopic HOXB4 expression mediated an approximately 30-fold increase in the number of circular endothelial, bona fide HE-sheets being Flk1+VE-Cadherin+Tie2+ (mean values: control: 11+/-4.8 n=7; HOXB4: 301+/-47 n=7; P<0.0001, unpaired, 2-sided Student´s t-test) and expressing Sox17 and Lmo2. This observation suggested an expansion of HE progenitors, detectable from day 5 of EB differentiation on. Determination of their frequencies within the VE-Cadherin+ population revealed a HOXB4-mediated increase from 1:360 cells (control) up to 1:15 cells (HOXB4; 95% C.I. = 1:12-1:21). After additional Runx1 induction, the endothelial cells morphologically underwent an Endothelial-to-Hematopoietic Transition (EHT) as verified at the single cell level by time-lapse microscopy. Concomitantly, they upregulated transcription of Gfi1, Gfi1b and Pu.1, initiated surface expression of the pan-hematopoietic marker CD45 and generated hematopoietic colony forming cells (CFC), thus proving their identity as real hemogenic endothelial cells. Taken together, our results strongly suggest that HOXB4 first and foremost promotes hematopoiesis by substantially increasing the number of hemogenic endothelium progenitors during mouse pluripotent stem cell differentiation. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

10.1101/2021.10.03.462906 ◽

2021 ◽

Author(s):

Yasmin Natalia Serina Secanechia ◽

Isabelle Bergiers ◽

Matt Rogon ◽

Christian Arnold ◽

Nicolas Descostes ◽

...

Keyword(s):

Developmental Process ◽

Ex Vivo ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Hematopoietic Stem ◽

Hematopoietic Development ◽

Stem And Progenitor Cells ◽

Bona Fide ◽

Endothelial Precursors

ABSTRACTRecent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.

Download Full-text

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.

Download Full-text

Hematopoietic Development of Multipotent Germline Stem Cells Derived from Neonatal Mouse Testis In Vitro and In Vivo.

Blood ◽

10.1182/blood.v106.11.519.519 ◽

2005 ◽

Vol 106 (11) ◽

pp. 519-519 ◽

Cited By ~ 1

Author(s):

Momoko Yoshimoto ◽

Toshio Heike ◽

Hsi Chang ◽

Shiro Baba ◽

Hisanori Fujino ◽

...

Keyword(s):

Stem Cells ◽

Es Cells ◽

Embryonic Stem ◽

Neonatal Mouse ◽

Mouse Testis ◽

Germline Stem Cell ◽

Facs Analysis ◽

Hematopoietic Stem ◽

Hematopoietic Development

Abstract Recently multipotent stem cells have been established from neonatal mouse testis (Shinohara et al, Cell 2004). This multipotent germline stem cell (mGS) is very similar to embryonic stem cells. It differentiates into various types of somatic cells in vitro and produces teratomas after inoculation into mice. Here we show the characteristics of hematopoietic development from mGS cells. mGS cells were maintained on mitomycin C treated mouse embryonic fibroblasts. In undifferentiated state, surface markers were almost the same among mGS, ES and EG cell; E-cadherin+, β1 integrin+, CD31+ and c-kit+. For induction, undifferentiated mGS cells were cultured on OP9 stromal cell line. Four days after induction, we detected and sorted FLK1+ cells as much as ES cells. Flk1+ cells were further cultured on OP9 with various cytokines. Erythroid, myeloid, lymphoid, megakaryocyte and mast cells as well as endothelial cells and beating cells were obtained in the same manner as the induction of ES cell and EG cells. mGS cells had colony forming abilities including mix and magakaryocyte colonies. The number of mixed colony was dependent on the combination of cytokines. In erythroid differentiation, mGS showed the two waves of embryonic and definitive erythroid production, which was proved by RT-PCR and immunochemistry. When hematopoietic cells derived from GFP+ mGS cells that were cultured on OP9 for 6 days were transplanted into BM of NODγ mice directly, GFP+ cells were detected in the BM and Spleen by FACS analysis and PCR 4 months after transplantation. FACS analysis showed that GFP+ cells were detected in the side population when BM cells of transplanted mice were stained with Hoechst33324. Immunostaining of the slice of BM showed some GFP+ cells were attached to the endosteal region that is thought to be the niche for hematopoietic stem cells. Thus, mGS cells have as much capabilities of blood cell production as ES cells and were showed having hematopoietic reconstitution ability. Considering mGS cells can be obtained from postnatal mice, they have strong advantages in research for clinical application.

Download Full-text