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

Blood ◽  
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

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

Blood ◽  
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.

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

Haematologica ◽  
2018 ◽  
Vol 104 (5) ◽  
pp. 894-906 ◽  
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

scholarly journals Modeling the human bone marrow niche in mice: From host bone marrow engraftment to bioengineering approaches

2018 ◽  
Vol 215 (3) ◽  
pp. 729-743 ◽  
Author(s):  
Ander Abarrategi ◽  
Syed A. Mian ◽  
Diana Passaro ◽  
Kevin Rouault-Pierre ◽  
William Grey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Models ◽  
Hematopoietic Cells ◽  
Human Growth ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Stem And Progenitor Cells

Xenotransplantation of patient-derived samples in mouse models has been instrumental in depicting the role of hematopoietic stem and progenitor cells in the establishment as well as progression of hematological malignancies. The foundations for this field of research have been based on the development of immunodeficient mouse models, which provide normal and malignant human hematopoietic cells with a supportive microenvironment. Immunosuppressed and genetically modified mice expressing human growth factors were key milestones in patient-derived xenograft (PDX) models, highlighting the importance of developing humanized microenvironments. The latest major improvement has been the use of human bone marrow (BM) niche–forming cells to generate human–mouse chimeric BM tissues in PDXs, which can shed light on the interactions between human stroma and hematopoietic cells. Here, we summarize the methods used for human hematopoietic cell xenotransplantation and their milestones and review the latest approaches in generating humanized BM tissues in mice to study human normal and malignant hematopoiesis.

A Stro-1+ human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system

2006 ◽  
Vol 34 (10) ◽  
pp. 1353-1359 ◽  
Author(s):  
Raquel Gonçalves ◽  
Cláudia Lobato da Silva ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
Graça Almeida-Porada
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Culture System ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Feeder Layer ◽  
Hematopoietic Stem ◽  
Serum Free

scholarly journals Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells

2010 ◽  
Vol 38 (1) ◽  
pp. 46-54 ◽  
Author(s):  
C. Chang I. Lee ◽  
Jared E. Christensen ◽  
Mervin C. Yoder ◽  
Alice F. Tarantal
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Clonal Analysis ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Stem And Progenitor Cells

Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Stem And Progenitor Cells ◽  
Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Deletion of proapoptotic Puma selectively protects hematopoietic stem and progenitor cells against high-dose radiation

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4707-4714 ◽  
Author(s):  
Lijian Shao ◽  
Yan Sun ◽  
Zhonghui Zhang ◽  
Wei Feng ◽  
Yongxing Gao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Cells ◽  
High Dose ◽  
Adverse Side Effect ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Novel Strategy ◽  
Induced Apoptosis ◽  
Dose Radiation

Abstract Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted, but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here, we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell–autonomous manner. Unexpectedly, loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly, null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation, thereby accelerating hematopoietic regeneration. Consistent with these findings, Puma is required for radiation-induced apoptosis in HSCs and HPCs, and Puma is selectively induced by irradiation in primitive hematopoietic cells, and this induction is impaired in Puma-heterozygous cells. Together, our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy.

scholarly journals Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Bo O Zhou ◽  
Lei Ding ◽  
Sean J Morrison
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Leptin Receptor ◽  
Hematopoietic Cells ◽  
Perivascular Niche ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Function ◽  
Angiopoietin 1

Hematopoietic stem cells (HSCs) are maintained by a perivascular niche in bone marrow but it is unclear whether the niche is reciprocally regulated by HSCs. Here, we systematically assessed the expression and function of Angiopoietin-1 (Angpt1) in bone marrow. Angpt1 was not expressed by osteoblasts. Angpt1 was most highly expressed by HSCs, and at lower levels by c-kit+ hematopoietic progenitors, megakaryocytes, and Leptin Receptor+ (LepR+) stromal cells. Global conditional deletion of Angpt1, or deletion from osteoblasts, LepR+ cells, Nes-cre-expressing cells, megakaryocytes, endothelial cells or hematopoietic cells in normal mice did not affect hematopoiesis, HSC maintenance, or HSC quiescence. Deletion of Angpt1 from hematopoietic cells and LepR+ cells had little effect on vasculature or HSC frequency under steady-state conditions but accelerated vascular and hematopoietic recovery after irradiation while increasing vascular leakiness. Hematopoietic stem/progenitor cells and LepR+ stromal cells regulate niche regeneration by secreting Angpt1, reducing vascular leakiness but slowing niche recovery.

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