Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region

Hematopoietic stem cells (HSCs) emerge during embryogenesis and maintain hematopoiesis in the adult organism. Little is known about the embryonic development of human HSCs. We demonstrate that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region, specifically in the dorsal aorta, and only later appear in the yolk sac, liver, and placenta. AGM region cells transplanted into immunodeficient mice provide long-term high level multilineage hematopoietic repopulation. Human AGM region HSCs, although present in low numbers, exhibit a very high self-renewal potential. A single HSC derived from the AGM region generates at least 300 daughter HSCs in primary recipients, which disseminate throughout the entire recipient bone marrow and are retransplantable. These findings highlight the vast regenerative potential of the earliest human HSCs and set a new standard for in vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine.

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A Long-Term Culture System for the Expansion of Hematopoietic Stem Cells from Embryonic Yolk Sac with the Capacity to Seed Erythroid and Lymphoid Development In Vitro and to Reconstitute the Lymphoid Compartment in Severe Combined Immunodeficient Mice

Artificial Organs ◽

10.1111/j.1525-1594.1996.tb00644.x ◽

2008 ◽

Vol 20 (10) ◽

pp. 1093-1109 ◽

Cited By ~ 3

Author(s):

Hong Ji ◽

Xian-Zhong Yu ◽

Thomas E. Wagner

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Culture System ◽

Yolk Sac ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Lymphoid Development ◽

Development In Vitro

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Ontogeny stage-independent and high-level clonal expansion in vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor

Blood ◽

10.1182/blood-2011-04-350066 ◽

2011 ◽

Vol 118 (16) ◽

pp. 4366-4376 ◽

Cited By ~ 16

Author(s):

Sanja Sekulovic ◽

Maura Gasparetto ◽

Véronique Lecault ◽

Corinne A. Hoesli ◽

David G. Kent ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Single Cells ◽

Hematopoietic Stem ◽

Image Tracking ◽

Large Numbers ◽

High Level

Abstract Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin−Sca-1+c-kit+ cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd–transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.

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CD66e Enrichment Enhances Repopulation of Human Long-Term Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2021-148235 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2156-2156

Author(s):

Kuiying Ma ◽

Riguo Fang ◽

Lingling Yu ◽

Yongjian Zhang ◽

Chao Li ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Surface Markers ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Differentiation Pattern ◽

Human Hscs

Abstract Gene-modified hematopoietic stem cells (HSCs) therapy has demonstrated remarkable success for the treatment of inherited blood disorders. As the origin of hematologic hierarchy, HSCs play an essential role in sustaining life-long hematopoiesis. HSCs identification via reliable and robust bio-markers could facilitate the development of HSC gene therapy. Previous studies showed that long-term hematopoietic stem cells (LT-HSCs) were enriched in the Lin -CD34 +CD38 -CD45RA -CD90 +CD49f + population which could support long-term hematopoietic reconstitution. However, several of these surface markers proved to be unreliable when ex vivo culturing, such as CD38 and CD49f. Thus, HSCs characterization is still hindered by lacking bona-fide bio-markers, and consequently identification of long-term HSCs still needs time-consuming in vivo transplantation. To this end, we performed in vitro screening and comprehensive functional evaluation to identify a novel surface marker of human HSCs. During initial screening, a cell surface antigen screen panel (including 242 human cell surface markers) and human CD34 and CD90 antibodies were used to perform flow cytometry analysis on CD34 + HSPCs enriched from umbilical cord blood. Compared with CD34 + cell population, we found that CD66 (a,c,d,e), CD200 and CD48 positive cells were more enriched in CD34 +CD90 + subset. Previous studies indicated that HSCs cannot be maintained during in vitro culturing. By tracking these candidate surface markers based on this principle, CD66e was selected as the potential HSCs bio-marker. Next, we examined the in vivo hematologic repopulating potential of HSCs by limiting dilution assay (LDA) on immune-deficient mouse model. We sorted CD66e + and CD66e - subsets from CD34 +CD90 +CD45RA - subpopulation, and transplanted into irradiated NOD-scid Il2rg −/− (NPG) mice respectively. At week16 post-transplantation, in contrast to the CD66e - group, CD66e + cells exhibited significantly higher reconstitution in peripheral blood (PB), bone marrow (BM) and spleen. Engraftment dynamics revealed that the CD66e - group were only capable of reconstitution 4 weeks post transplantation, even at the highest initial cell dose. Moreover, the CD66e - group displayed impaired multi-lineage differentiation pattern, especially in PB and BM samples, while the CD66e + group presented a robust multi-lineage reconstitution. Notably, LDA results showed that the CD66e + cells within CD34 +CD90 +CD45RA - population contained 1 out of 529 SCID repopulating cells (SRC), almost 60-fold greater than the CD66e - fraction. To further investigate the long-term repopulating potential of the CD66e + cells, we performed the secondary transplantation collected from the BM cells of primary recipients. CD66e + cells presented significant higher repopulating activity than CD66e- subset in the secondary recipients. These findings reveal that the major cells with homing and long-term reconstitution capacity among CD34 +CD90 +CD45RA - cells were CD66e positive. In order to determine the transcriptional profile of CD66e + cells, we performed RNA-sequencing analysis using the population of CD34 + cells, CD34 +CD90 +CD45RA - cells, CD66e + and CD66e - cells within CD34 +CD90 +CD45RA - subset. Remarkably, compared with other groups, the CD66e + cells displayed a bias toward the signature of HSC and early progenitors such as LMPP and CLP. Moreover, gene set enrichment analysis showed that hematopoietic lineage and long-term potentiation-related genes were highly enriched in the CD66e + cells. Further qRT-PCR experiment confirmed that several HSC-related genes were significantly higher expressed in CD34 +CD90 +CD45RA -CD66e + cells, compared to CD66e - population or CD34 + HSPCs, suggesting that the gene expression profile of CD66e + cells is reminiscent of HSC signature. Altogether, we demonstrate that CD66e is a robust functional HSC bio-marker that CD66e-positive population among CD34 +CD90 +CD45RA - cells exhibit typical HSC signature, enhanced in vivo engraftment potential and robust multilineage differentiation pattern, which will provide an invaluable tool to investigate the origin of human HSCs, paving the way for the therapeutic application. Figure 1 Figure 1. Disclosures Fang: EdiGene, Inc.: Current Employment.

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Using mitochondrial activity to select for potent human hematopoietic stem cells

Blood Advances ◽

10.1182/bloodadvances.2020003658 ◽

2021 ◽

Vol 5 (6) ◽

pp. 1605-1616

Author(s):

Jiajing Qiu ◽

Jana Gjini ◽

Tasleem Arif ◽

Kateri Moore ◽

Miao Lin ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hematopoietic Cell Transplantation ◽

Mitochondrial Activity ◽

Mitochondrial Morphology ◽

Blood Disorders ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Human Hscs

Abstract Hematopoietic cell transplantation is a critical curative approach for many blood disorders. However, obtaining grafts with sufficient numbers of hematopoietic stem cells (HSCs) that maintain long-term engraftment remains challenging; this is due partly to metabolic modulations that restrict the potency of HSCs outside of their native environment. To address this, we focused on mitochondria. We found that human HSCs are heterogeneous in their mitochondrial activity as measured by mitochondrial membrane potential (MMP) even within the highly purified CD34+CD38−CD45RA−CD90+CD49f+ HSC population. We further found that the most potent HSCs exhibit the lowest mitochondrial activity in the population. We showed that the frequency of long-term culture initiating cells in MMP-low is significantly greater than in MMP-high CD34+CD38−CD45RA−CD90+ (CD90+) HSCs. Notably, these 2 populations were distinct in their long-term repopulating capacity when transplanted into immunodeficient mice. The level of chimerism 7 months posttransplantation was >50-fold higher in the blood of MMP-low relative to MMP-high CD90+ HSC recipients. Although more than 90% of both HSC subsets were in G0, MMP-low CD90+ HSCs exhibited delayed cell-cycle priming profile relative to MMP-high HSCs. These functional differences were associated with distinct mitochondrial morphology; MMP-low in contrast to MMP-high HSCs contained fragmented mitochondria. Our findings suggest that the lowest MMP level selects for the most potent, likely dormant, stem cells within the highly purified HSC population. These results identify a new approach for isolating highly potent human HSCs for further clinical applications. They also implicate mitochondria in the intrinsic regulation of human HSC quiescence and potency.

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High-level correction of the sickle mutation amplified in vivo during erythroid differentiation

10.1101/432716 ◽

2018 ◽

Cited By ~ 6

Author(s):

Wendy Magis ◽

Mark A. DeWitt ◽

Stacia K. Wyman ◽

Jonathan T. Vu ◽

Seok-Jin Heo ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Genetic Disorders ◽

Erythroid Differentiation ◽

Hematopoietic Stem ◽

Curative Therapy ◽

Human Hscs ◽

High Level

ABSTRACTSickle Cell Disease (SCD), one of the world’s most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction of the sickle mutation in more than 30% of long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol that is directly applicable to a clinical setting. We further find that in vivo erythroid differentiation markedly enriches for corrected ß-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at curative levels with a streamlined protocol that is ready to be translated into a therapy.ONE SENTENCE SUMMARYCas9-mediated correction of the sickle mutation in human hematopoietic stem cells can be accomplished at curative levels.

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Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.728057 ◽

2021 ◽

Vol 9 ◽

Author(s):

Wanbo Tang ◽

Jian He ◽

Tao Huang ◽

Zhijie Bai ◽

Chaojie Wang ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Lineage Tracing ◽

Genetic Lineage ◽

Hematopoietic Stem ◽

Genetic Lineage Tracing

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

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Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽

10.1182/blood-2011-01-330514 ◽

2011 ◽

Vol 117 (18) ◽

pp. 4773-4777 ◽

Cited By ~ 115

Author(s):

Hal E. Broxmeyer ◽

Man-Ryul Lee ◽

Giao Hangoc ◽

Scott Cooper ◽

Nutan Prasain ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

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Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v94.5.1623 ◽

1999 ◽

Vol 94 (5) ◽

pp. 1623-1636 ◽

Cited By ~ 72

Author(s):

Chu-Chih Shih ◽

Mickey C.-T. Hu ◽

Jun Hu ◽

Jeffrey Medeiros ◽

Stephen J. Forman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

In Vitro Culture ◽

Culture System ◽

Ex Vivo ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

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