Localization of Hematopoietic Stem Cells in Coculture with Mesenchymal Stromal Cells Impacts on Phenotype and Cell Cycle Status

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4774-4774
Author(s):  
Duohui Jing ◽  
Nael Alakel ◽  
Fernando Fierro ◽  
Katrin Mueller ◽  
Martin Bornhaeuser ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Stromal Cells ◽  
The Other ◽  
Cell Contact ◽  
Adherent Cells ◽  
Hematopoietic Stem ◽  
Cell Fractions

Abstract Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became clear that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD34 purified HSC expansion and differentiation demonstrating a promoting impact of MSC on adherent HSC in comparison to non adherent HSC in terms of phenotype, migration capacity and clonogenicity. Performing phase contrast microscopy and confocal microscopy we are able to distinguish HSC which are located on the surface of a MSC monolayer (phase-bright cells) and HSC which are covered by MSC monolayer (phase-dim cells). Both HSC fractions and the non-adherent cells were isolated separately by performing serial washing steps. All three fractions were analyzed at fixed time points during the first week of co-culture in term of cell cycle progression, proliferation, maturation and cell division accompanied differentiation. First we performed propidium iodide (PI) staining for cell cycle analysis revealing that the phase-bright cells contained the highest percentage of G2 cells in comparison to the non adherent cells and the phase-dim cells; 13.9 ±1.0% vs 1.3 ±1.2% vs 2.7 ±2.0%, p<0.001. The data indicate the facilitating impact of MSC on HSC in performing mitosis which is however depending on the location of interaction. When HSC are released into supernatant (non adherent cells) or covered by MSC, G2 phase was significantly down-regulated. Next we studied the proliferation capacity of the separate cell fractions. Consistent with the data of cell cycle, cell number of phase-bright faction increased much faster than the other two fractions during the first 4 days suggesting that the MSC surface in vitro is the predominant location of HSC proliferation. Next we investigated the phenotype of HSC. According to FACS analysis results (CD34+CD38-) phase-dim cells revealed a more immature phenotype in comparison to the non adherent cells and the phase-bright cells. During the first four days 80% of phase-dim cells remained CD34+CD38-, while cells of the phase-bright- and the non adherent fraction exhibited a significant more mature phenotype. Performing cell division tracking using CFSE we were able to show that over time number of divisions of phase-dim cells were significantly diminished in comparison to the other two cell fractions in co-cultures. In addition, phase-dim cells started to lose CD34 at the 7th generation, while non-adherent and phase-bright cells already lost CD34 at the 4th generation. These data suggest that “stemness” of HSC was rather preserved in the cell fraction which was covered by MSC monolayer than in the cell fraction on the surface of MSC. In conclusion we demonstrate HSC in distinct locations in vitro showing different behaviors in terms of phenotype and proliferation. It becomes evident that not only the cell to cell contact matters but also the localization of contact. Further experiments are needed to investigate NOD/SCID repopulation potential of the different cell fractions.

Secreted Mediators of Self-Renewal of Hematopoietic Stem Cells Identified Using Bio-Informatic Analysis of Co-Cultures of HSC and Stromal Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2353-2353
Author(s):  
Baiba Vilne ◽  
Rouzanna Istvanffy ◽  
Christina Eckl ◽  
Franziska Bock ◽  
Olivia Prazeres da Costa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Tissue Growth ◽  
Functional Enrichment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
A Cell

Abstract Abstract 2353 Hematopoiesis is maintained throughout life by the constant production of mature blood cells from hematopoietic stem cells (HSC). One mechanism by which the number of HSC is maintained is self-renewal, a cell division in which at least one of the daughter cells is a cell with the same functional potential as the mother cell. The mechanisms of this process are largely unknown. We have described cell lines that maintain self-renewal in culture. To study possible mechanisms and mediators involved in self-renewal, we performed co-cultures of HSC model cells: Lineage-negative Sca-1+ c-Kit+ (LSK) cells and HSC maintaining UG26–1B6 stromal cells. Microarray analyses were performed on cells prior to co-culture and cells sorted from the cultures. STEM clustering analysis of the data revealed that most changes in gene expression were due to early cell activation. Functional enrichment analysis revealed dynamic changes in focal adhesion and mTOR signaling, as well as changes in epigenetic regulators, such as HDAC in stromal cells. In LSK cells, genes whose products are involved in inflammation, Oxygen homeostasis and metabolism were differentially expressed after the co-culture. In addition, genes involved in the regulaton of H3K27 methylation were also affected. Interestingly, connective tissue growth factor (CTGF), which is involved in TGF-b, BMP and Wnt signaling, was upregulated in both stromal and LSK cells in the first day of co-culture. To study a possible extrinsic role of CTGF as a stromal mediator, we co-cultured siCTGF knockdown stromal cells with wild-type LSK cells. Since self-renewal requires cell division, we focused on cell cycle regulation of LSK cells. We found that knockdown of CTGF in stromal cells downregulates CTGF in LSK cells. In addition, knockdown of stromal CTGF downregulated Ccnd1, Cdk2, Cdkn1a (p21), Ep300 and Fos. On the other hand, decreased CTGF in stromal cells upregulates Cdkn1b (p27) and phosphorylation of Smad2/3. These results show that stromal CTGF regulates the cell cycle of LSK cells. On a functional level, we found that decreased stromal CTGF results in an increased production of MPP and myeloid colony-forming cells in 1-week co-cultures. We will present data showing whether and how a decrease in CTGF in stromal cells affects the maintenance of transplantable HSC. In summary, our current results indicate that reduced expression of CTGF in stromal cells regulates mediators of cell cycle and Smad2/3-mediated signaling in LSK cells, resulting in an increased production of myeloid progenitors. Disclosures: No relevant conflicts of interest to declare.

CD133+ Purified Hematopoietic Stem Cells in Co-Culture with Mesenchymal Stromal Cells - The Cell to Cell Contact Matters.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1421-1421
Author(s):  
Nael Alakel ◽  
Douhui Jing ◽  
Martin Bornhaeuser ◽  
Gerhard Ehninger ◽  
Rainer Ordemann
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Cell Fraction ◽  
Adherent Cell ◽  
Cell Contact ◽  
Adherent Cells ◽  
Facs Analysis ◽  
Hematopoietic Stem ◽  
Migration Capacity

Abstract Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became aware that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD133 purified HSC expansion and differentiation in terms of phenotype, (CXCR4) chemokine receptor expression, migration capacity and clonogenicity. After one week of co-culture with MSC adherent and non-adherent HSC were isolated and analyzed separately. As expected in co-culture experiments with MSC a significant higher expansion of TNC was observed in comparison to the expansion culture without MSC (cytokine driven), 37±6 fold vs. 19±3 fold, p<0.001. However the co-culture expansion was accompanied by a significant increase of differentiation and maturation of the progenitor cells. Therefore we were focusing on the adherent cell fraction of the co-culture system. According to FACS analysis results, adherent HSC in the co-culture display a more immature phenotype than non adherent cells (CD34+CD38−). Next colony assays and LTC-IC were performed showing that the adherent cell fraction contained a significant higher proportion of primary and secondary CFU-C, indicating that this cell fraction contains the majority of clonogenic HSC. The CXCR4 - SDF-1 axis plays an important rule in homing, mobilization and engraftment of the hematopoietic stem cells. Therefore we looked for CXCR4 expression in the two cell fractions using FACS analysis. The adherent cells expressed consistently and significantly more CXCR4 than the non adherent cells; 43±4% vs 15±2%, p<0.001. Data could be confirmed by performing western blot. Due to this data we evaluated the capacity of HSC migration toward SDF-1 gradient using transwell migration assay. The migration capacity of the adherent cells was significantly higher than that of the non adherent cell fraction correlating with the CXCR4 expression; 13±1% vs. 6±1%, p<0.001. We checked the expression of different cell adhesion molecules by FACS analysis. CD44 and CD49e (integrin α5) were significantly higher expressed in adherent cells comparing with non-adherent cells. Our data indicate that the cell-to-cell interaction mediated impact on CD133+ selected HSC leads to an enhanced clonogenic capacity and migratory potential. Further experiments are needed to investigate engraftment and NOD/SCID repopulation potential of the adherent and non-adherent cells after ex-vivo expansion.

scholarly journals Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 512-523 ◽  
Author(s):  
JE Wagner ◽  
D Collins ◽  
S Fuller ◽  
LR Schain ◽  
AE Berson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Surface ◽  
Cell Population ◽  
Size Fraction ◽  
Cell Surface Antigens ◽  
Inhibitory Protein ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte- macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.

scholarly journals Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 365-372 ◽  
Author(s):  
JP Wineman ◽  
S Nishikawa ◽  
CE Muller-Sieburg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Stromal Cell Line ◽  
Cell Maintenance

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

Major Histocompatibility Complex Restriction Between Hematopoietic Stem Cells and Stromal Cells In Vitro

Stem Cells ◽  
2001 ◽  
Vol 19 (1) ◽  
pp. 46-58 ◽  
Author(s):  
Kikuya Sugiura ◽  
Hiroko Hisha ◽  
Junji Ishikawa ◽  
Yasushi Adachi ◽  
Shigeru Taketani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Major Histocompatibility Complex ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Major Histocompatibility ◽  
Hematopoietic Stem ◽  
Histocompatibility Complex

Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1505-1505
Author(s):  
Wendy W. Pang ◽  
Elizabeth A. Price ◽  
Irving L. Weissman ◽  
Stanley L. Schrier
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Expression Profiles ◽  
Frequency Function ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cell Cycle Status

Abstract Abstract 1505 Poster Board I-528 Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations. We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Geminin Regulates Hematopoietic Stem Cell Proliferation and Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1478-1478
Author(s):  
Kathryn M. Shinnick ◽  
Kelly A. Barry ◽  
Elizabeth A. Eklund ◽  
Thomas J. McGarry
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Transcription Factors ◽  
Cell Division ◽  
Dna Replication ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Regulatory Protein ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation

Abstract Abstract 1478 Poster Board I-501 Hematopoietic stem cells supply the circulation with mature blood cells throughout life. Progenitor cell division and differentiation must be carefully balanced in order to supply the proper numbers and proportions of mature cells. The mechanisms that control the choice between continued cell division and terminal differentiation are incompletely understood. The unstable regulatory protein Geminin is thought to maintain cells in an undifferentiated state while they proliferate. Geminin is a bi-functional protein. It limits the extent of DNA replication to one round per cell cycle by binding and inhibiting the essential replication factor Cdt1. Loss of Geminin leads to replication abnormalities that activate the DNA replication checkpoint and the Fanconi Anemia (FA) pathway. Geminin also influences patterns of cell differentiation by interacting with Homeobox (Hox) transcription factors and chromatin remodeling proteins. To examine how Geminin affects the proliferation and differentiation of hematopoietic stem cells, we created a mouse strain in which Geminin is deleted from the proliferating cells of the bone marrow. Geminin deletion has profound effects on all three hematopoietic lineages. The production of mature erythrocytes and leukocytes is drastically reduced and the animals become anemic and neutropenic. In contrast, the population of megakaryocytes is dramatically expanded and the animals develop thrombocytosis. Interestingly, the number of c-Kit+ Sca1+ Lin- (KSL) stem cells is maintained, at least in the short term. Myeloid colony forming cells are also preserved, but the colonies that grow are smaller. We conclude that Geminin deletion causes a maturation arrest in some lineages and directs cells down some differentiation pathways at the expense of others. We are now testing how Geminin loss affects cell cycle checkpoint pathways, whether Geminin regulates hematopoietic transcription factors, and whether Geminin deficient cells give rise to leukemias or lymphomas. Disclosures: No relevant conflicts of interest to declare.

Partial Characterization and In Vitro Expansion of Putative CLL Precursor/Stem Cells Which Are Dependent on Bone Marrow Microenvironment for Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2433-2433
Author(s):  
Medhat Shehata ◽  
Rainer Hubmann ◽  
Martin Hilgarth ◽  
Susanne Schnabl ◽  
Dita Demirtas ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Healthy Persons

Abstract Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p<0,001). These triple positive “putative CLL stem cells” (PCLLSC) co-express CD133 (67%), CD38 (87%), CD127 (52%), CD10 (49%), CD20 (61%), CD23 (96%), CD44 (98%) and CD49d (74%). FISH analysis on 4 patients with documented chromosomal abnormalities detected the corresponding chromosomal aberrations of the mature clone in the sorted CD34+/CD5+/CD19+ and/or CD34+/CD19-/CD5- cells but not in the CD3+ T cells. Multiplex RT-PCR analysis using IgVH family specific primer sets confirmed the clonality of these cells. Morphologically, PCLLSC appeared larger than lymphocytes with narrow cytoplasm and showed polarity and motility in co-culture with human bone marrow stromal cells. Using our co-culture microenvironment model (Shehata et al, Blood 2010), sorted cell fractions (A: CD34+/19+/5+, B: CD34+/19-/5- or C: CD34-/CD19+/5+) from 4 patients were co-cultured with primary autologous human stromal cells. PCLLSC could be expanded in the co-culture to more than 90% purity from fraction A and B but not from fraction C. These cells remained in close contact or migrated through the stromal cells. PCLLSC required the contact with stromal cells for survival and died within 1–3 days in suspension culture suggesting their dependence on bone marrow microenvironment or stem cell niches. RT-PCR demonstrated that these cells belong to the established CLL clone. They also eexpress Pax5, IL-7R, Notch1, Notch2 and PTEN mRNA which are known to play a key role in the early stages of B cells development and might be relevant to the early development of the malignant clone in CLL. Using NOD/SCID/IL2R-gamma-null (NOG) xenogeneic mouse system we co-transplanted CLL cells from 3 patients (5 million PBMC/mouse) together with autologous bone marrow stromal cells (Ratio: 10:1). The percentage of PCLLSC in the transplanted PBMC was 0.18% (range 0.06–0.34%). Using human-specific antibodies, human CD45+ cells were detected in peripharal blood of the mice (mean 0.9 % range 0.47–1.63%) after 2 months of transplantation. More than 90% of the human cells were positive for CD45 and CD5. Among this population, 26% (range 15–35%) of the cells co-expressed CD45, CD19, CD5 and CD34 and thus correspond to the PCLLSC. In conclusion, our data suggest the existence of putative CLL precursors/stem cells which reside within the CD34+ hematopoietic stem cell compartment and carry the chromosomal aberrations of the established CLL clone. These cells could be expanded in vitro in a bone marrow stroma-dependent manner and could be engrafted and significantly enriched in vivo in NOG xenotransplant system. Further characterization and selective targeting and eradication of these cells may pave the way for designing curative therapeutic strategies for CLL. Disclosures: No relevant conflicts of interest to declare.

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 Hematopoietic stem cells differentiate into restricted myeloid progenitors before cell division

2018 ◽  
Author(s):  
Tatyana Grinenko ◽  
Anne Eugster ◽  
Lars Thielecke ◽  
Beata Ramazs ◽  
Anja Krueger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Hematopoietic Stem Cells ◽  
Embryonic Stem ◽  
Erythroid Progenitors ◽  
Multipotent Stem Cells ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

SummaryHematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps that involve the generation of lineage-committed progenitors as well as necessary expansion due to repeated cell divisions. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell tracing approach and Ki67RFP knock-in mice to assess simultaneously divisional history, cell cycle progression, and differentiation of adult HSCs in vivo. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid progenitors, restricted megakaryocyte-erythroid progenitors (PreMEs) and pre-megakaryocyte progenitors (PreMegs), without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with expression of lineage-specific genes that manifested as functional differences between HSCs and restricted progenitors. Thus, HSC fate decisions appear to be uncoupled from physical cell division. Our results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells. Our data, together with separate findings from embryonic stem cells, suggest that cell division and fate choice are independent processes in pluripotent and multipotent stem cells.

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