The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short- term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.

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Expression and Function of Murine Receptor Tyrosine Kinases, TIE and TEK, in Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v89.12.4317 ◽

1997 ◽

Vol 89 (12) ◽

pp. 4317-4326 ◽

Cited By ~ 71

Author(s):

Michihiro Yano ◽

Atsushi Iwama ◽

Hitoshi Nishio ◽

Junko Suda ◽

Goro Takada ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Stem Cell Marker ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

And Function

Abstract Two highly related receptor tyrosine kinases, TIE and TEK, comprise a family of endothelial cell-specific kinase. We established monoclonal antibodies against them and performed detailed analyses on their expression and function in murine hematopoietic stem cells (HSCs). TIE and TEK were expressed on 23.7% and 33.3% of lineage marker-negative, c-Kit+ and Sca-1+ (Lin− c-Kit+ Sca-1+) HSCs that contain the majority of day-12 colony-forming units-spleen (CFU-S) and long-term reconstituting cells, but not committed progenitor cells. Lin− c-Kit+ Sca-1+ cells were further divided by the expression of TIE and TEK. TIE+ and TEK+ HSCs as well as each negative counterpart contained high proliferative potential colony-forming cells and differentiated into lymphoid and myeloid progenies both in vitro and in vivo. However, day-12 CFU-S were enriched in TIE+ and TEK+ HSCs. Our findings define TIE and TEK as novel stem cell marker antigens that segregate day-12 CFU-S, and provide evidence of novel signaling pathways that are involved in the functional regulation of HSCs at a specific stage of differentiation, particularly of day-12 CFU-S.

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In Vitro Expansion of Fetal Liver Hematopoietic Stem Cells

10.21203/rs.3.rs-156554/v1 ◽

2021 ◽

Author(s):

Rashmi Bhardwaj ◽

Lalit Kumar ◽

Deepika Chhabra ◽

N K Mehra ◽

Atul Sharma ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Mononuclear Cells ◽

Culture Media ◽

Fetal Liver ◽

Antigen Expression ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Fetal liver hematopoietic stem cells because of their proliferative potential have been considered appropriate for management of aplastic anemia. Bone marrow recovery was possible in some cases; engraftment potential of these cells however, was unsatisfactory, possibly due to the availability of smaller number of these cells from a single fetus. Present study was undertaken to see if fetal liver hematopoietic stem cells could be expanded in vitro. Mononuclear cells were isolated and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ cells were separated by magnetic cell sorting positive selection method using flow cytometry. Hematopoietic stem cells (CD34+) were cultured by using 5 cytokines, stem cell factor, granulocyte macrophages-colony stimulating factor, interlukin-6, Fms-related tyrosine kinase 3 and erythropoietin, in 4 different combinations along with supplements, in serum free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in combination of media, supplements and 4 cytokines, namely SCF, Flt-3, IL6 and Epo to yield large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

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In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells

Blood ◽

10.1182/blood.v80.12.3044.bloodjournal80123044 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3044-3050 ◽

Cited By ~ 2

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

Y Miura ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Function ◽

Bone Marrow Cells ◽

Synergistic Effects ◽

Single Cells ◽

Hematopoietic Stem

c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker- negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c- kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c- kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.

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Extracellular HMGB1 Enhances Proliferation, Differentiation and Migration of Human CD34+ Hematopoietic Stem Cells in Vitro

Blood ◽

10.1182/blood.v112.11.4744.4744 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4744-4744

Author(s):

Xingbing Wang ◽

Xin Chen ◽

Weihua Ren ◽

Xiucai Xu ◽

Kaidi Song ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Proliferation And Differentiation ◽

Cord Blood Cd34 ◽

And Migration

Abstract High-mobility group box 1 protein (HMGB1) is a chromatin protein and acts as a cytokine involved in inflammation, cell proliferation, differentiation, migration and stem cell recruitment. So far, little is known about its effect on hematopoietic stem cells (HSCs). In this study, we investigated whether receptors for HMGB1 are expressed on human CD34+ HSCs, and whether HMGB1 could affect HSCs proliferation, differentiation and migration in vitro. As examined by FACS analysis and RT-PCR, cord blood CD34+ HSCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products), TLR2 (Toll-like receptor 2) and TLR4. To study the effects of HMGB1 on CD34+ HSCs proliferation and differentiation, freshly isolated cord blood CD34+ HSCs were cultured for 7 days in medium alone or in the presence of HMGB1. Flow cytometric analysis showed that HMGB1 (50ng/ml) can induce the differentiation of CD34+ HSCs along the granulo-monocytic (CD14+, CD13+) lineage and erythropoiesis (CD71+). In contrast, HMGB1 did not induce the expression of CD41, a marker for megakaryocyte lineage. The numbers of cells cultured in the presence of HMGB1 were always increased in comparison with controls. Furthermore, higher numbers of granulomonocytic progenitors (CFU-GM), erythroid progenitors (BFU-E), and CFU-MIX were confirmed by CFC assays in a HMGB1 dose dependent manner after 14-day culture. The results suggest that HMGB1 enhances CD34+ HSCs proliferation and differentiation. We next assessed the effects of HMGB1 on HSCs migration by chemotaxis assay using Boyden chambers. HMGB1 dose-dependently increased the chemotactic migration of CD34+ HSCs. A neutralizing anti-RAGE antibody significantly blocked the HMGB1-induced migration of HSCs, whereas neutralizing TLR2 and TLR4 antibodies did not significantly influence HMGB1-stimulated HSCs migration, suggesting that the migratory effect of HMGB1 on human HSCs is predominantly mediated by RAGE. In summary, our results provide the first report of HMGB1 receptors expression profile of human cord blood CD34+ HSCs and demonstrate that HMGB1 can increase the proliferation and migration of HSCs and directly induce of HSCs along myeloid differentiation and erythropoiesis in vitro. Further studies will be needed to clarify the mechanism of HMGB1 activation and the physiological function of HMGB1 in HSCs in vivo.

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In vitro expansion of fetal liver hematopoietic stem cells

Scientific Reports ◽

10.1038/s41598-021-91272-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rashmi Bhardwaj ◽

Lalit Kumar ◽

Deepika Chhabra ◽

N. K. Mehra ◽

Atul sharma ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Mononuclear Cells ◽

Culture Media ◽

Fetal Liver ◽

Antigen Expression ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Gm Csf

AbstractFetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

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In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells

Blood ◽

10.1182/blood.v80.12.3044.3044 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3044-3050 ◽

Cited By ~ 354

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

Y Miura ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Function ◽

Bone Marrow Cells ◽

Synergistic Effects ◽

Single Cells ◽

Hematopoietic Stem

Abstract c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker- negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c- kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c- kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.

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Identification of Phospholipase C Gamma 1 As a Key Regulator of Erythropoiesis

Blood ◽

10.1182/blood.v120.21.4744.4744 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4744-4744

Author(s):

Tina M Schnoeder ◽

Patricia Arreba-Tutusaus ◽

Inga Griehl ◽

Daniel B Lipka ◽

Florian H Heidel ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

I 11 ◽

Erythroid Development

Abstract Abstract 4744 Erythropoiesis is a complex multistage process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors. Regulation of survival, expansion and differentiation of erythroid progenitors is dependent on a well-coordinated cohort of transcription factors and an intricate network of finely tuned regulatory signalling pathways. In vivo and in vitro studies have highlighted erythropoietin receptor (EpoR) signaling through JAK2 tyrosine kinase as a crucial regulator of erythropoiesis. This leads to the subsequent activation of downstream effectors such as STAT5, MAPK, and PI-3K/Akt pathways. However, detailed knowledge about signalling pathways involved in EPO/EpoR induced differentiation of erythroid progenitors remain elusive. Phosphatidylinositol-specific phospholipase C gamma1 (PLCg1) is known to act as key mediator of calcium-signalling that can substitute for PI-3K/AKT signalling in oncogenic models. Moreover, its loss is associated with lack of erythropoiesis in a straight knockout mouse model. As it is tempting to speculate on the role of Plcg1/Ca-signalling downstream of EpoR/JAK in regulation of erythroid development we aimed to investigate its influence on differentiation and proliferation of hematopoietic cells in vitro and in vivo. Using different cellular models (Ba/F3, 32D) stably transfected with EpoR and wildtype JAK2 we could provide evidence that PLCg1 is a downstream target of EpoR/JAK2 signalling. Knockdown of PLCg1 led to a decreased proliferation of PLCg1-deficient cells compared to control cells whereas survival of these cells was not affected. In contrast, other downstream targets of EpoR signalling were not affected by PLCg1 knockdown. In order to assess specifically its role in erythroid development, we used the murine pro-erythroblast cell line I-11 as well as primary fetal liver cells (FLC). The I-11 cell line was isolated from p53-deficient fetal livers and is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation; primary FLC were harvested at E13.5. PLCg1 knockdown by using RNA-interference technology led to a significant delay in erythroid differentiation and accumulation of immature erythroid progenitors (e.g. pro-erythroblasts) as assessed by cytology and flow cytometry technology. In addition, we tested the colony-forming potential of PLCg1-deficient I-11 and fetal liver cells compared to controls. Colony formation was significantly impaired in both - I-11 and primary FLC - when compared to control cells (shRNA-scr). We performed gene-expression analysis by Q-RT-PCR on sorted hematopoietic stem and progenitor cells and found a higher expression in MEP compared to GMP or CMP. To clarify, whether the effects of Plcg1 knockdown are restricted to erythroid development at the stage of MEP or erythroid progenitors, we aimed to investigate adult hematopoietic stem cells in erythroid development. We infected lineage-depleted/erythroid-enriched (Gr1-, B220-, CD3/4/8, CD19-/ IL7Ra- negative) bone marrow cells with either PLCg1 or control shRNA. Using flow cytometry analysis to examine differentiation we could observe a reduction of megakaryocyte/erythroid progenitor cells (MEP) in PLCg1 knockdown cells compared to control cells while development of other lineages (e.g. GMP) remained unaffected. Currently, competitive repopulation assays investigating the repopulation and differentiation capacity of hematopoietic stem cells after Plcg1 knockdown (or scr controls) are under way to explore the role of Plcg1 signalling in hematopoietic and erythroid development in vivo. Taken together, our findings presume PLCg1 to be a key regulator in erythroid development and understanding of its relevance in development and maintenance of normal hematopoiesis will be a crucial prerequisite for targeting this important pathway in myeloproliferative disease. Disclosures: No relevant conflicts of interest to declare.

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Endomucin, a CD34-like sialomucin, marks hematopoietic stem cells throughout development

Journal of Experimental Medicine ◽

10.1084/jem.20051325 ◽

2005 ◽

Vol 202 (11) ◽

pp. 1483-1492 ◽

Cited By ~ 48

Author(s):

Azusa Matsubara ◽

Atsushi Iwama ◽

Satoshi Yamazaki ◽

Chie Furuta ◽

Ryutaro Hirasawa ◽

...

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Signal Sequence ◽

Time Frame ◽

Mouse Bone Marrow ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

High Level

To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34−c-Kit+Sca-1+Lin− (CD34−KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)–HSCs were exclusively present in the Endomucin+CD34−KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45−CD41+Endomucin+ fraction at day 10.5 of gestation (E10.5) and in the CD45+CD41+Endomucin+ fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.

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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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