Normal hematopoietic stem/progenitor cells isolated from human bone marrow are susceptible to glucocorticoids-induced apoptosis via the regulation of signal transduction pathways involved in cell cycle

Human bone marrow endothelial cells immortalized with the T antigen of SV40 (TrHBMEC) have previously been characterized by us with regard to their properties that are similar to primary marrow endothelial cells and their utility as a model system. We now report that TrHBMEC express a recently discovered signal transduction molecule termed RAFTK (related adhesion focal tyrosine kinase), also called Pyk2 or CAK-β. RAFTK, the second member of the focal adhesion kinase (FAK) family, is known to be activated in response to calcium flux in neuronal cells and integrin stimulation in megakaryocytes and B cells. We have studied the effects of cytokines on RAFTK activation in TrHBMEC. Treatment of TrHBMEC with the vascular endothelial growth factor (VEGF ), as well as the VEGF-related protein (VRP), the recently identified ligand for the FLT-4 receptor, resulted in enhanced tyrosine phosphorylation of RAFTK. Similar changes in RAFTK phosphorylation were observed upon stimulation of TrHBMEC with basic fibroblast growth factor (bFGF ) or oncostatin M (OSM). Stimulation of these cells with growth factors also resulted in an increase in RAFTK activity and the c-Jun NH2 -terminal kinase (JNK). RAFTK coimmunoprecipitated with the cytoskeletal protein paxillin through its C-terminal proline-rich domain in TrHBMEC. These results suggest that, in marrow endothelium, activation of RAFTK by VEGF, VRP, OSM, and bFGF represents a new element in the signal transduction pathways used by these growth factors and likely acts to coordinate signaling from their surface receptors to the cytoskeleton, thereby modulating cell growth and function.

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Flt3high and Flt3low CD34+Progenitor Cells Isolated From Human Bone Marrow Are Functionally Distinct

Blood ◽

10.1182/blood.v91.6.1947.1947_1947_1958 ◽

1998 ◽

Vol 91 (6) ◽

pp. 1947-1958 ◽

Cited By ~ 1

Author(s):

Katharina S. Götze ◽

Manuel Ramı́rez ◽

Kelly Tabor ◽

Donald Small ◽

William Matthews ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Suspension Culture ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Suspension Cultures ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Numbers ◽

Marrow Cells

We generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin− marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38− marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34−CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit–granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3highcells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin−Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin−Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.

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Flt3high and Flt3low CD34+Progenitor Cells Isolated From Human Bone Marrow Are Functionally Distinct

Blood ◽

10.1182/blood.v91.6.1947 ◽

1998 ◽

Vol 91 (6) ◽

pp. 1947-1958 ◽

Cited By ~ 54

Author(s):

Katharina S. Götze ◽

Manuel Ramı́rez ◽

Kelly Tabor ◽

Donald Small ◽

William Matthews ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Suspension Culture ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Suspension Cultures ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Numbers ◽

Marrow Cells

Abstract We generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin− marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38− marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34−CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit–granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3highcells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin−Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin−Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.

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Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic Lineage Commitment of Human Bone Marrow Progenitor Cells

Blood ◽

10.1182/blood.v94.6.1971.418k34_1971_1978 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1971-1978 ◽

Cited By ~ 3

Author(s):

Gösta Bergh ◽

Mats Ehinger ◽

Inge Olsson ◽

Sten Eirik W. Jacobsen ◽

Urban Gullberg

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Differentiation ◽

Progenitor Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Lineage Commitment ◽

Flt3 Ligand ◽

Monocytic Differentiation ◽

Cd34 Cells

The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

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In Vitro Hematopoietic Lineage Interconversion from Human Bone Marrow Stem and Progenitor Cells.

Blood ◽

10.1182/blood.v104.11.4160.4160 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4160-4160

Author(s):

Ling Chen ◽

Stephanie Jean-Noel ◽

Kevin Hall ◽

Ying Shi ◽

Griffin P. Rodgers

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Surface ◽

Progenitor Cells ◽

Hematopoietic Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Two Populations

Abstract The cell surface antigen, CD133, marks a fraction of hematopoietic stem and progenitor cells and has been successfully used to study their differential biology. To evaluate the differentiating capacity of stem/progenitor cells, we cultivated purified normal human bone marrow CD133 selected cells for 2 weeks with erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF) to induce erythroid or myeloid differentiation, respectively. After the second week of cultivation, we reversed the seeding environment of the two populations by placing EPO treated cells into a G-CSF environment and G-CSF treated cells into an EPO environment for an additional 2-week culture. The cells produced in the culture were phenotypically defined by morphology and flow cytometry, and genotypically by RNA and proteomic analyses. Three-color flow cytometry was used for identifying CD133+ progenitors, CD36+ erythroid and CD13+ myeloid cells, as shown in Table 1. The morphology of the cultured cells, assessed by Wright-Giemsa staining, is consistent with the conversion of cellular specific markers. Rapid analysis of gene expression demonstrated co-expression of 76% of 266 genes analyzed among the erythroid and myeloid lineages. Furthermore, proteomic analysis exhibited the sharing of 33% of 9518 expressed protein spots assayed in the two populations after the first 2-week culture, and 32% after 2 weeks of the switch culture. Our data clearly demonstrate that the committed erythroid and myeloid precursors are able to change their fate and can switch into the opposite cell type by a conversion pathway under a specifically defined condition. We termed this switch as interconversion, considering conversion of hematopoietic cells to non-hematopoietic cells. Furthermore, the observations presented in this study show that cytokines used can improve the conversion. We are developing a mathematical model describing the kinetics of hematopoietic stem/progenitor cell transitions into specific lineages, along with the conversion of committed cells based on multiple potential energy wells corresponding to different cell states and cytokines. Table 1. Expression of cell surface markers after 4-week culture D0 1 week 2 weeks 4 weeks CD expression (%) E G E G E2w →G2w → G2w E2w Data are presented as a mean of at least 2 experiments. E: EPO; G: G-CSF; E2w or G2w: EPO or G-CSF treatment for two weeks. CD133+ 96.19 15.74 13.6 0.24 0.36 0.01 0.63 CD36+ 0 60.37 27.39 96.37 25.87 45.41 68.54 CD13+ 0.43 35.41 57.29 24.41 92.1 85.87 37.76 CD133+ / CD36+ 0.44 22.24 15.97 0.12 0.18 1.55 7.65 CD133+ / CD13+ 1.24 19.43 13.36 0.36 1.09 13.31 14.92 CD36+ / CD13+ 0.09 41.25 17.80 23.69 54.1 46.60 79.41

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Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic Lineage Commitment of Human Bone Marrow Progenitor Cells

Blood ◽

10.1182/blood.v94.6.1971 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1971-1978 ◽

Cited By ~ 24

Author(s):

Gösta Bergh ◽

Mats Ehinger ◽

Inge Olsson ◽

Sten Eirik W. Jacobsen ◽

Urban Gullberg

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Differentiation ◽

Progenitor Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Lineage Commitment ◽

Flt3 Ligand ◽

Monocytic Differentiation ◽

Cd34 Cells

Abstract The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

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Long noncoding RNAs of single hematopoietic stem and progenitor cells in healthy and dysplastic human bone marrow

Haematologica ◽

10.3324/haematol.2018.208926 ◽

2018 ◽

Vol 104 (5) ◽

pp. 894-906 ◽

Cited By ~ 10

Author(s):

Zhijie Wu ◽

Shouguo Gao ◽

Xin Zhao ◽

Jinguo Chen ◽

Keyvan Keyvanfar ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Noncoding Rnas ◽

Long Noncoding Rnas ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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Modulation of Interaction of Human Osteoprogenitor Cells with Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v124.21.2933.2933 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2933-2933

Author(s):

Frank Akwaa ◽

Rakhil Rubinova ◽

Benjamin J Frisch ◽

Mark W LaMere ◽

Kristen Marie O'Dwyer ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Progenitor Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Stem ◽

Osteoprogenitor Cells ◽

Stem And Progenitor Cells ◽

Osteolineage Cells ◽

Supportive Cells

Abstract Osteoprogenitor cells (OPCs) are marrow microenvironmental cells known to modulate hematopoietic stem and progenitor cells (HSPCs). Specifically, OPCs regulate HSPCs in response to Parathyroid hormone (PTH) treatment in murine models. However, the role of OPCs in human HSPC regulation and whether human OPCs can be manipulated is poorly understood. Niche stimulation is an appealing strategy to aid in the treatment of hematopoietic dysfunction. Myelodysplastic syndromes (MDS) are clonal disorders with ineffective hematopoiesis resulting in cytopenias and risk of transformation to acute leukemia (AML). In mouse models, disruption of the osteolineage cells can contribute to initiation of ineffective hematopoiesis with phenotypic features of MDS. Our long term goal is to utilize microenvironmental stimulation as a therapeutic tool to improve hematopoietic disorders. We hypothesized that human cells isolated from the marrow fraction containing spicules harbor HSPC supportive cells, which can be manipulated to improve HSPC support. Moreover we hypothesized that OPC number and function is impaired by dysplasia-initiated microenvironmental disruption as a potential mechanism for reduced support of HSPCs and ineffective hematopoiesis. Our objective was to isolate human bone marrow spicule associated cells (SACs) and define their ability to support HSPCs, determine the impact of PTH treatment of SAC/HSPCs interactions and characterize dysplasia-induced osteolineage changes in human MDS and AML bone marrow. To achieve this objective, we used normal as well as MDS/AML patient-derived OPCs using a mouse-human co-culture system. Human bone marrow SACs isolated by collagenase digestion were either used for co-culture, analyzed with flow cytometry or cultured in mineralization media in limited dilutions. To assess the potential impact of PTH on human OPC interaction with HSPCs, we developed a 7 day co-culture of human bone marrow SACs treated with either vehicle or PTH, with mouse Lineage- Sca1+ c-Kit+ (LSK) hematopoietic progenitor cells. At the end of the co-culture, all cells present were used for competitive transplantation. Transplant experiments demonstrated that PTH treatment of the human bone marrow SACs leads to improved function of the co-cultured LSK cells as demonstrated by significantly improved engraftment of the LSK cells after transplant into irradiated C57/bl6 recipient mice when sampled at pre-specified time points over a 20-week period (N=12, 2-way ANOVA; p < 0.05). Flow cytometry analysis showed that mature (Lin- CD31- CD146+ CD105-) and immature osteolineage (Lin- CD31- CD146+ CD105+) cells were present in SACs and more abundant compared to within BMMCs (1% vs 0.1% and 0.24% vs 0.12% for the same patient). Notably, the putative HSC-supportive MSC pool was increased in SACs vs BMMCs (0.052% vs 0.019%). The presence of OPCs was functionally confirmed using colony forming unit osteoblasts (CFU-OBs). CFU-OB frequency was calculated using L-Calc TM (StemCell technologies). Among normal donors the frequency of CFU-OBs was low in marrow donors >50 years old compared to <50 years old donors (2.240e-005 ± 3.300e-006 N=2 vs. 0.0001146 ± 4.163e-005 N=9). We identified non-statistically significant decrease in the frequency of CFU-OBs in bone marrow SACs from MDS patients compared to normal donors (1.090e-005 ± 1.400e-006 N=2 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.179); and similar decrease in frequency of osteoprogenitor cells in the bone marrow aspirates from AML patients compared to normal donors (2.303e-005 ± 9.371e-006 N=3 vs. 5.024e-005 ± 1.277e-005 N=8; p= 0.251). These data support our hypothesis that OPCs in patients with MDS and AML are negatively impacted compared to normal bone marrow. These data demonstrate that human SACs contain HSPC-supportive cells which can be stimulated to improve HSPC function. Human SACs comprise MSCs and osteolineage cells including osteoprogenitor cells. Aging decreases OPC pools in SACs. Our data in our small sample also suggest that dysplastic bone marrow microenvironment may negatively impact OPCs, which may in turn decrease OPC support of HPSCs. PTH treatment in our in-vitro model shows the potential to improve the interaction between the OPCs and HSPCs, resulting in amelioration of HSC function. Together these data suggest a strategy where targeting the MDS microenvironment may add to the currently available treatment modalities. Disclosures Calvi: Fate Therapeutics: Patents & Royalties.

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