scholarly journals Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1591-1591
Author(s):  
Juliana M. Xavier ◽  
Lauremilia Ricon ◽  
Karla Priscila Vieira ◽  
Longhini Ana Leda ◽  
Carolina Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

scholarly journals PTPN11D61Y/+; Vavcre+ Animals Commonly Succumb to Leukemia Relapse Despite Robust Engraftment of Transplanted Wild-Type Hematopoietic Stem and Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 496-496
Author(s):  
Stefan P. Tarnawsky ◽  
Mervin C. Yoder ◽  
Rebecca J. Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Stem And Progenitor Cells ◽  
Leukemia Relapse ◽  
Conditioning Regimens

Juvenile Myelomonocytic Leukemia (JMML) is a rare childhood myelodysplastic / myeloproliferative overlap disorder. JMML exhibits myeloid populations with mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. While allogeneic hematopoietic stem cell transplant (HSCT) is the treatment of choice for children with JMML, 50% of children succumb to leukemia relapse; however, the mechanism leading to this high relapse rate is unknown. We hypothesized that the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, leading to poor engraftment of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and thus promoting leukemia relapse. Using Vav1 promoter-directed Cre, we generated a mouse model of JMML that conditionally expresses gain-of-function PTPN11D61Yin utero during development. While PTPN11D61Y/+; VavCre+embryos did not demonstrate in utero lethality, we observed a modest reduction of PTPN11D61Y/+; VavCre+ mice at the time of weaning compared to predicted Mendelian frequencies. Further, surviving PTPN11D61Y/+; VavCre+ mice developed elevated peripheral blood leukocytosis and monocytosis as early as 4 weeks of age compared to PTPN11+/+; VavCre+ controls. To address the hypothesis that an aberrant bone marrow microenvironment in the PTPN11D61Y/+ mice leads to poor engraftment of wild-type donor cells following transplant, we examined engraftment of wild-type hematopoietic stem and progenitor cells (HSPCs) in the PTPN11D61Y/+; VavCre+ mice and monitored animals for disease relapse. 16-24 week-old diseased PTPN11D61Y/+; VavCre+ and control PTPN11+/+; VavCre+ mice were lethally irradiated (11 Gy split dose) and transplanted with 5 x 105 CD45.1+ wild-type bone marrow low density mononuclear cells (LDMNCs), which simulates a limiting stem cell dose commonly available in a human HSCT setting. 6 weeks post-HSCT, PTPN11D61Y/+; VavCre+recipients demonstrated an unexpected elevated CD45.1+ donor cell contribution in peripheral blood compared to the control PTPN11+/+; VavCre+ recipients. However, despite superior engraftment in the PTPN11D61Y/+; VavCre+ recipients, these mice had a significantly shorter median survival post-HSCT due to a resurgence of recipient CD45.2-derived leukemic cells. We repeated the experiment using a high dose of CD45.1+ LDMNCs (10 x 106 cells) to determine if providing a saturating dose wild-type cells could prevent the relapse of recipient-derived leukemogenesis and normalize the survival of the PTPN11D61Y/+; VavCre+recipients. While this saturating dose of wild-type cells resulted in high peripheral blood chimerism in both the PTPN11D61Y/+; VavCre+ and PTPN11+/+; VavCre+ recipients, the PTPN11D61Y/+; VavCre+ animals nevertheless demonstrated significantly reduced overall survival. When we examined the cause of mortality in the HSCT-treated PTPN11D61Y/+; VavCre+mice, we found enlarged spleens, hypercellular bone marrow, and enlarged thymuses. Flow cytometry revealed that the majority of cells in the peripheral blood, bone marrow, and spleen were recipient-derived CD45.2+ CD4+ CD8+ T cells. To verify that the disease was neoplastic in origin, secondary transplants into CD45.1/.2 recipients were performed from two independent primary PTPN11D61Y/+; VavCre+and two independent primary PTPN11+/+; VavCre+ controls. Secondary recipients of bone marrow from PTPN11D61Y/+; VavCre+ animals rapidly succumbed to a CD45.2-derived T-cell acute lymphoid leukemia (T-ALL). Previous studies demonstrated that wild-type PTPN11 is needed to protect the integrity of the genome by regulating Polo-like kinase 1 (Plk1) during the mitosis of the cell cycle (Liu et al., PNAS, 2016). We now demonstrate that even when PTPN11 mutant animals are provided with saturating doses of wild-type HSCs, dysregulated residual recipient cells are able to produce relapsed disease. Collectively, these studies highlight the propensity of residual mutant PTPN11 cells to transform after being subjected to mutagenic agents that are commonly used for conditioning regimens prior to allogeneic HSCT. These findings suggest that modified pre-HSCT conditioning regimens bearing reduced mutagenicity while maintaining adequate cytoreductive efficacy may yield lower post-HSCT leukemia relapse in children with PTPN11mutations. Disclosures No relevant conflicts of interest to declare.

The RNA Editing Enzyme ADAR1 Is Required for Survival of Differentiating Hematopoietic Progenitor Cells in Adult Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1395-1395
Author(s):  
Feng Xu ◽  
Qingde Wang ◽  
Hongmei Shen ◽  
Hui Yu ◽  
Yanxin Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Rna Editing ◽  
Cell Population ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Low Level ◽  
Adult Mice

Abstract Adenosine Deaminases Acting on RNA (ADAR) are RNA-editing enzymes converting adenosine residues into inosine (A-to-I) in many double-stranded RNA substrates including coding and non-coding sequences as well as microRNAs. Disruption of the ADAR1 gene in mice results in fetal liver, but not yolk sac, defective erythropoiesis and death at E11.5 (Wang Q et al, Science 2000). Subsequently, a conditional knockout mouse model confirmed these findings and showed massively increased cell death in the affected organs (Wang Q et al, JBC 2004). However, the actual impact of ADAR1 absence on definitive or adult hematopoiesis has not been examined. To define the role of ADAR1 in adult hematopoiesis, we first examined the expression of ADAR1 in different hematopoietic stem/progenitor cell subsets isolated from bone marrow by real-time RT-PCR. ARAR1 was present in hematopoietic stem cells (HSCs) at relatively low level and increased in hematopoietic progenitor cells (HPCs). A series of functional hematopoietic assays were then undertaken. A conditional deletion of ADAR1 was achieved by transducing Lin− or Lin−cKit+ bone marrow cells from ADAR1-lox/lox mice with a MSCV retroviral vector co-expressing Cre and GFP. PCR analysis confirmed the complete deletion of ADAR1 in the transduced cells within 72 hours after the transduction. This system allowed us to evaluate the acute effect of ADAR1 deletion in a specific hematopoietic cell population. Following 4 days of in vitro culture after transduction, the absolute number of Lin− Sca1+ cells in the Cre transduced group was similar to the input number; however the differentiating Lin+ cells significantly decreased whereas both the Lin−Sca1+ and Lin+ cells in the vector (MSCV carrying GFP alone) transduced group increased during culture. Moreover, the colony forming cell (CFC) assay showed much fewer and smaller colonies that contained dead cells from the gene deleted group as compared to those from the control group (p<0.001). The TUNEL assay showed a dramatic increase of apoptosis in the Lin+ population but not in the Lin− cells. Given the mixed genetic background of the ADAR1-lox/lox mice, repopulation of the transduced hematopoietic cells in vivo was examined in immunodeficient mice. Sublethally irradiated (3.5 Gy) NOD/SCID-γcnull recipient were transplanted with either 1.5 × 105 Cre or vector transduced Lin− ADAR1-lox/lox cells. Multi-lineage engraftment in peripheral blood was monitored monthly. While the vector transduced cells were able to constitute more than 90% in multiple lineages of the peripheral blood at 1 to 3 months, Cre-transduced cells were virtually undetectable at all the time points (n=9 to 13, p<0.001). A similar result was found in the hematopoietic organs, including the bone marrow, spleen and thymus. Interestingly, however, the Lin−Sca1+cKit+ cell population was preserved in the Cre transduced group despite the very low level of total donor-derived cells in the bone marrow (n=6 to 7, p<0.01). Consistently, the single cell culture experiment demonstrated that there was no significant difference between ADAR−/− and wild-type HSCs in terms of survival and division during the first 3 days of culture. Taken together, our current study demonstrates nearly absolute requirement of ADAR1 for hematopoietic repopulation in adult mice and it is also suggested that ADAR1 has a preferential effect on the survival of differentiating progenitor cells over more primitive cells.

Absolute density of different sources of hematopoietic progenitor cells: Bone marrow, peripheral blood stem cell and umbilical cord blood

Cytotherapy ◽  
2017 ◽  
Vol 19 (1) ◽  
pp. 128-130 ◽  
Author(s):  
Anelise Bergmann Araújo ◽  
Melissa Helena Angeli ◽  
Gabrielle Dias Salton ◽  
Juliana Monteiro Furlan ◽  
Tissiana Schmalfuss ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Absolute Density ◽  
Different Sources

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

Chemotactic and chemokinetic activities of stem cell factor on murine hematopoietic progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4100-4108 ◽  
Author(s):  
N Okumura ◽  
K Tsuji ◽  
Y Ebihara ◽  
I Tanaka ◽  
N Sawai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stem Cell Factor ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Murine Bone Marrow ◽  
Checkerboard Assay

We investigated the effects of stem cell factor (SCF) on the migration of murine bone marrow hematopoietic progenitor cells (HPC) in vitro using a modification of the checkerboard assay. Chemotactic and chemokinetic activities of SCF on HPC were evaluated by the numbers of HPC migrated on positive and negative gradients of SCF, respectively. On both positive and negative gradients of SCF, HPC began to migrate after 4 hours incubation, and their numbers then increased time- dependently. These results indicated that SCF functions as a chemotactic and chemokinetic agent for HPC. Analysis of types of colonies derived from the migrated HPC showed that SCF had chemotactic and chemokinetic effects on all types of HPC. When migrating activities of other cytokines were examined, interleukin (IL)-3 and IL-11 also affected the migration of HPC, but the degrees of each effect were lower than that of SCF. The results of the present study demonstrated that SCF is one of the most potent chemotactic and chemokinetic factors for HPC and suggest that SCF may play an important role in the flow of HPC into bone marrow where stromal cells constitutively produce SCF.

scholarly journals Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells.

1994 ◽  
Vol 180 (3) ◽  
pp. 1177-1182 ◽  
Author(s):  
H W Snoeck ◽  
D R Van Bockstaele ◽  
G Nys ◽  
M Lenjou ◽  
F Lardon ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Interferon Gamma ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Ifn Gamma ◽  
Hematopoietic Stem ◽  
First Time ◽  
Dose Dependent

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony-forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1234-1234
Author(s):  
Robert S Welner ◽  
Giovanni Amabile ◽  
Deepak Bararia ◽  
Philipp B. Staber ◽  
Akos G. Czibere ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cells ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

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