scholarly journals Stroma-Contact Prevents Loss of Hematopoietic Stem Cell Quality During Ex Vivo Expansion of CD34+ Mobilized Peripheral Blood Stem Cells

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Dimitri A. Breems ◽  
Ellen A.W. Blokland ◽  
Karen E. Siebel ◽  
Angelique E.M. Mayen ◽  
Lilian J.A. Engels ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Ex Vivo ◽  
Peripheral Blood Stem Cells ◽  
Soluble Factors ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
Ex Vivo Culture ◽  
Mobilized Peripheral Blood ◽  
Graft Quality

Abstract Stroma-supported long-term cultures (LTC) allow estimation of stem cell quality by simultaneous enumeration of hematopoietic stem cell (HSC) frequencies in a graft using the cobblestone area forming cell (CAFC) assay, and the ability of the graft to generate progenitors in flask LTC (LTC-CFC). We have recently observed that the number and quality of mobilized peripheral blood stem cells (PBSC) was low in patients having received multiple rounds of chemotherapy. Moreover, grafts with low numbers of HSC and poor HSC quality had a high probability to cause graft failure upon their autologous infusion. Because ex vivo culture of stem cells has been suggested to present an attractive tool to improve hematological recovery or reduce graft size, we have studied the possibility that such propagation may affect stem cell quality. In order to do so, we have assessed the recovery of different stem cell subsets in CD34+ PBSC after a 7-day serum-free liquid culture using CAFC and LTC-CFC assays. A numerical expansion of stem cell subsets was observed in the presence of interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact, stromal soluble factors, or combined addition of FLT3-ligand and thrombopoietin improved this parameter. In contrast, ex vivo culture severely reduced the ability of the graft to produce progenitors in LTC while stromal soluble factors partly abrogated this quality loss. The best conservation of graft quality was observed when the PBSC were cultured in stroma-contact. These data suggest that ex vivo propagation of PBSC may allow numerical expansion of various stem cell subsets, however, at the expense of their quality. In addition, cytokine-driven PBSC cultures require stroma for optimal maintenance of graft quality.

scholarly journals Stroma-Contact Prevents Loss of Hematopoietic Stem Cell Quality During Ex Vivo Expansion of CD34+ Mobilized Peripheral Blood Stem Cells

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Dimitri A. Breems ◽  
Ellen A.W. Blokland ◽  
Karen E. Siebel ◽  
Angelique E.M. Mayen ◽  
Lilian J.A. Engels ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Ex Vivo ◽  
Peripheral Blood Stem Cells ◽  
Soluble Factors ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
Ex Vivo Culture ◽  
Mobilized Peripheral Blood ◽  
Graft Quality

Stroma-supported long-term cultures (LTC) allow estimation of stem cell quality by simultaneous enumeration of hematopoietic stem cell (HSC) frequencies in a graft using the cobblestone area forming cell (CAFC) assay, and the ability of the graft to generate progenitors in flask LTC (LTC-CFC). We have recently observed that the number and quality of mobilized peripheral blood stem cells (PBSC) was low in patients having received multiple rounds of chemotherapy. Moreover, grafts with low numbers of HSC and poor HSC quality had a high probability to cause graft failure upon their autologous infusion. Because ex vivo culture of stem cells has been suggested to present an attractive tool to improve hematological recovery or reduce graft size, we have studied the possibility that such propagation may affect stem cell quality. In order to do so, we have assessed the recovery of different stem cell subsets in CD34+ PBSC after a 7-day serum-free liquid culture using CAFC and LTC-CFC assays. A numerical expansion of stem cell subsets was observed in the presence of interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact, stromal soluble factors, or combined addition of FLT3-ligand and thrombopoietin improved this parameter. In contrast, ex vivo culture severely reduced the ability of the graft to produce progenitors in LTC while stromal soluble factors partly abrogated this quality loss. The best conservation of graft quality was observed when the PBSC were cultured in stroma-contact. These data suggest that ex vivo propagation of PBSC may allow numerical expansion of various stem cell subsets, however, at the expense of their quality. In addition, cytokine-driven PBSC cultures require stroma for optimal maintenance of graft quality.

Granulocyte-Derived Cationic Peptides (GDCPs) Present in Leucophoresis Products Enhance Homing of Hematopoietic Stem Cells (HSCs) to SDF-1 Gradient; Potential Implications for Accelerated Recovery of Hematopoiesis After Transplantation of Mobilized Peripheral Blood Stem Cells (PBSC).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 371-371
Author(s):  
HakMo Lee ◽  
Wan Wu ◽  
Marcin Wysoczynski ◽  
Magdalena Kucia ◽  
Mary J. Laughlin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Cationic Peptides ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
Anaphylatoxin C3a ◽  
Mobilized Peripheral Blood

Abstract Abstract 371 Current strategies to accelerate hematopoietic reconstitution after transplantation, include transplantation of greater numbers of HSC or ex vivo expansion of harvested HSC before transplant. However, the number of HSC availabel for allogeneic or autologous transplantation can be low (e.g., umbilical cord blood, poor mobilizers) and strategies to expand HSC and maintain equivalent engraftment capability ex vivo are limited. We reported that some compounds present in leucopheresis products [(e.g., platelet-derived microparticles (Blood 2001, 98: 3143)] and some complement cascade cleavage fragments, e.g., anaphylatoxin C3a (Blood 2005, 101, 3784), enhance the homing responses of HSC to SDF-1 gradient. We recently noted that small cationic peptides released from activated granulocytes (beta2-defensin and cathelicidin) positively prime responsiveness of murine and human HSC to SDF-1 gradient (Leukemia 2009; in press). Accordingly, both compounds enhanced transwell migration of HSC to low threshold doses of SDF-1. This phenomenon was not receptor-dependent, as agonists of membrane receptors that may bind beta2-defensin (FPRL-1), cathelicidin (CCR6) - FPRL-1 agonist, and MIP-3alpha, respectively, did not show similar priming effects. This could be explained by affected distribution of membrane lipids by cationic peptides. In support of this notion, an inhibitor of cell membrane raft formation (methyl-b-cyclodextran) inhibited the priming effect of both compounds, indicating this effect is dependent on CXCR4 incorporation into lipid rafts. Direct confocal analysis of CXCR4 and lipid raft colocalization in the presence or absence of cationic peptides confirmed these findings. Because leucopheresis products are enriched in activated granulocytes that release beta2-defensin and cathelicidin, we tested whether this may explain why mobilized peripheral blood stem cells (PBSC) engraft faster compared to HSC isolated directly from bone marrow (BM) in a murine BM transplant model. Accordingly, syngeneic BMMNCs were exposed ex vivo to beta2-defensin or cathelicidin for 30 minutes and subsequently transplanted into lethally irradiated recipients. We noted that animals transplanted with BM cells primed by those cationic peptides showed accelerated recovery of platelets and neutrophils by ∼3-5 days compared to unprimed control cells. We envision that small cationic peptides, which primarily possess antimicrobial functions and are harmless to mammalian cells, could be clinically applied to prime human HSC before transplantation. This novel approach would be particularly important in cord blood transplantation, where the number of HSC availabel for transplantation is usually limited. We postulate that this promising strategy warrants further investigations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hematopoietic stem cells in the blood after stem cell factor and interleukin-11 administration: evidence for different mechanisms of mobilization

Blood ◽  
1995 ◽  
Vol 86 (12) ◽  
pp. 4674-4680 ◽  
Author(s):  
P Mauch ◽  
C Lamont ◽  
TY Neben ◽  
C Quinto ◽  
SJ Goldman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Cytotoxic Agents ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
Interleukin 11

Peripheral blood stem cells and progenitor cells, collected during recovery from exposure to cytotoxic agents or after cytokine administration, are being increasingly used in clinical bone marrow transplantation. To determine factors important for mobilization of both primitive stem cells and progenitor cells to the blood, we studied the blood and splenic and marrow compartments of intact and splenectomized mice after administration of recombinant human interleukin-11 (rhlL-11), recombinant rat stem cell factor (rrSCF), and IL-11 + SCF. IL-11 administration increased the number of spleen colony- forming units (CFU-S) in both the spleen and blood, but did not increase blood long-term marrow-repopulating ability (LTRA) in intact or splenectomized mice. SCF administration increased the number of CFU- S in both the spleen and blood and did not increase the blood or splenic LTRA of intact mice, but did increase blood LTRA to normal marrow levels in splenectomized mice. The combination of lL-11 + SCF syngeristically enhanced mobilization of long-term marrow-repopulating cells from the marrow to the spleen of intact mice and from the marrow to the blood of splenectomized mice. These data, combined with those of prior studies showing granulocyte colony-stimulating factor mobilization of long-term marrow repopulating cells from the marrow to the blood of mice with intact spleens, suggest different cytokine- induced pathways for mobilization of primitive stem cells.

Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2271-2286 ◽  
Author(s):  
M. Rosenzweig ◽  
T.J. MacVittie ◽  
D. Harper ◽  
D. Hempel ◽  
R.L. Glickman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
High Level

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

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