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Gene Expression Profiling of Ex-vivo expanded Hematopoietic Stem and Progenitor Cells using SCF+TPO+FLT3L and homing factor SDF1

ABSTRACT: Efficient bone marrow homing is a prerequisite for successful engraftment of transplanted HSPC. This study aims at determining factors important in homing of these cells from the blood to the marrow and their re-engraftment. We have isolated nucleated cells from mobilized peripheral blood stem cell harvests (PBSC). CD34+ hematopoietic stem and progenitor cells (HSPC) were enriched from PBSC harvests by immunomagnetic separation using negative selection method. Enriched cells were cultured in vitro for expansion in presence or absence of cytokine (GF: SCF+TPO+FLT3L) and/or chemokine (homing factor SDF1) mixture for 8 days at 370C in humidified atmosphere of 5% CO2 in air. GF+SDF1 –stimulated HSPC showed significantly increased total nucleated ells as well as CD34+ cells as compared to GF-stimulated HSPC as well s unstimulated cultured HSPC. On transplantation of these cells in vivo in mice model who were previously irradiated at sublethal dose of 375cGy, it was observed that GF+SDF1-stimulated HSPC exhibited significantly better engraftment in terms of presence of human CD45+ cells in mouse blood at 6 week post –transplantation. We have compared gene expression profiles of mobilized PBSC harvest cells, enriched CD34+ HSPC population and HSPC cultured in plain media, in presence with GF and in presence of GF+SDF1 derived from mobilized PBSC harvests with respect to genes involved in homing and engraftment capacities.

ORGANISM(S): Homo sapiens

PROVIDER: GSE37334 | GEO | 2016/12/11

SECONDARY ACCESSION(S): PRJNA159589

REPOSITORIES: GEO

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Project description:CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). From each patient only one sample was analyzed. Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays according to manufacturer's instructions. Following quality control and normalization 9 samples of G-CSF-mobilized CD34+ cells were compared with 7 samples of PEG-G-CSF-mobilized cells were compared by significance analysis of microarrays (SAM). The aim was to figure out transcriptional differences resulting from various pharmacokinetics of the same drug (G-CSF: fluctuating serum levels; PEG-G-CSF: continously high serum levels). Peripheral blood stem and progenitor cells (PBSC) are widely used for autologous and allogeneic hematopoietic stem cell transplantation. PBSC can be mobilized into the peripheral blood using cytokines, cytotoxic chemotherapy or a combination of both. Granulocyte colony stimulating factor (G-CSF) has become the most commonly administered cytokine for PBSC mobilization because of its high potency and lack of serious toxicity. Recently, a modified form of recombinant human G-CSF has been introduced. This new compound is pegylated G-CSF (Peg-G-CSF) which possesses a substantially longer half-life than the unconjugated drug because of its reduced renal excretion and therefore provides the basis for continuous G-CSF serum-levels after a single injection. The use of Peg-G-CSF in patients who had received a cytotoxic chemotherapy was accompanied by mobilization kinetics different from those observed in patients who had received unconjugated G-CSF. In particular, more rapid leukocyte recovery and occurrence of CD34-positive cells in the peripheral blood was seen. These findings are presumably related to the more even and continuously high serum level of G-CSF maintained by Peg-G-CSF. Using microarray technology and functional assays, we examined whether pegylation of G-CSF and its different pharmacokinetics results in addition to an earlier leucocyte recovery in transcriptional and functional changes in CD34+ cells obtained from patients who had received either G-CSF or Peg-G-CSF following a standard cytotoxic chemotherapy for PBSC mobilisation. CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays. Following quality control and normalization differentially expressed genes were identified by significance analysis of microarrays (SAM). Comparing both groups 339 genes were significantly differentially expressed (q value <5%; fold change > 1.2). Peg-G-CSF-mobilized CD34+ cells showed an expression pattern of more early progenitor cells as early stem cell markers such as HOX genes were higher expressed and differentiation-associated genes were lower expressed in comparison with G-CSF-mobilized cells. Moreover, Peg-G-CSF-mobilized CD34+ cells had a greater expression level of cell-cycle-promoting genes suggesting a greater cycle activity of these cells. In conclusion, despite the similar active drug component different pharmacokinetics of Peg-G-CSF and G-CSF result in distinct molecular phenotypes reflecting different functional characteristics.

Project description:Peripheral blood stem and progenitor cells (PBSC) are widely used for autologous and allogeneic hematopoietic stem cell transplantation. PBSC can be mobilized into the peripheral blood using cytokines, cytotoxic chemotherapy or a combination of both. Granulocyte colony stimulating factor (G-CSF) has become the most commonly administered cytokine for PBSC mobilization because of its high potency and lack of serious toxicity. Recently, a modified form of recombinant human G-CSF has been introduced. This new compound is pegylated G-CSF (Peg-G-CSF) which possesses a substantially longer half-life than the unconjugated drug because of its reduced renal excretion and therefore provides the basis for continuous G-CSF serum-levels after a single injection. The use of Peg-G-CSF in patients who had received a cytotoxic chemotherapy was accompanied by mobilization kinetics different from those observed in patients who had received unconjugated G-CSF. In particular, more rapid leukocyte recovery and occurrence of CD34-positive cells in the peripheral blood was seen. These findings are presumably related to the more even and continuously high serum level of G-CSF maintained by Peg-G-CSF. Using microarray technology and functional assays, we examined whether pegylation of G-CSF and its different pharmacokinetics results in addition to an earlier leucocyte recovery in transcriptional and functional changes in CD34+ cells obtained from patients who had received either G-CSF or Peg-G-CSF following a standard cytotoxic chemotherapy for PBSC mobilisation. CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays. Following quality control and normalization differentially expressed genes were identified by significance analysis of microarrays (SAM). Comparing both groups 339 genes were significantly differentially expressed (q value <5%; fold change > 1.2). Peg-G-CSF-mobilized CD34+ cells showed an expression pattern of more early progenitor cells as early stem cell markers such as HOX genes were higher expressed and differentiation-associated genes were lower expressed in comparison with G-CSF-mobilized cells. Moreover, Peg-G-CSF-mobilized CD34+ cells had a greater expression level of cell-cycle-promoting genes suggesting a greater cycle activity of these cells. In conclusion, despite the similar active drug component different pharmacokinetics of Peg-G-CSF and G-CSF result in distinct molecular phenotypes reflecting different functional characteristics. Keywords: ordered

Project description:Autologous hematopoietic stem cell transplantation (ASCT) improves survival in multiple myeloma (MM). However, up to 30% of MM patients are unable to collect optimal numbers of peripheral blood (PB) CD34+ hematopoietic stem and progenitor cells (HSPCs) with standard G-CSF mobilization. Motixafortide is a novel cyclic-peptide CXCR4 inhibitor with extended receptor occupancy and enhanced in-vivo activity. The GENESIS Trial is a prospective, phase III, double-blind, placebo-controlled, international, multicenter, superiority trial with 122 adult patients randomized (2:1) to motixafortide+G-CSF or placebo+G-CSF for HSPC mobilization for ASCT in MM (NCT03246529). The primary endpoint of the study was met, with motixafortide+G-CSF enabling 92.5% of patients to collect ≥6x106 CD34+ cells/kg within 2 apheresis procedures versus 26.2% with placebo+G-CSF (p<0.0001). A key, prespecified secondary endpoint was also met, with motixafortide+G-CSF enabling 88.8% of patients to collect ≥6x106 CD34+ cells/kg in 1 apheresis versus 9.5% with placebo+G-CSF (p<0.0001). Motixafortide+G-CSF was safe and well-tolerated, with the most common treatment-emergent adverse events observed being transient, Grade 1/2 injection site reactions (pain: 50%, erythema: 27.5%, pruritis: 21.3%). In addition, extended immunophenotyping and single-cell transcriptional profiling were performed on CD34+ HSPCs mobilized on the GENESIS Trial, as well as a contemporaneous MM cohort undergoing plerixafor+G-CSF mobilization for ASCT and 3 cohorts of allogeneic HSPC donors undergoing single-agent mobilization with motixafortide, plerixafor or G-CSF alone. Motixafortide+G-CSF resulted in a 10.5-fold increase in primitive HSPCs collected versus placebo+G-CSF (p<0.0001); and significantly greater numbers of early stem and progenitors versus both placebo+G-CSF (p<0.0001) and plerixafor+G-CSF (p=0.0327). Motixafortide also preferentially mobilized HSPC subsets with upregulated gene expression pathways associated with enhanced self-renewal, regeneration and quiescence (EGF, NFB/TNF and HBO1). In conclusion, motixafortide+G-CSF mobilized significantly greater CD34+ HSPC numbers within 2 apheresis procedures versus placebo+G-CSF. Motixafortide also preferentially mobilized primitive stem and early progenitor cells with unique transcriptional profiles versus plerixafor and G-CSF.

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Gene Expression Profiling of Ex-vivo expanded Hematopoietic Stem and Progenitor Cells using SCF+TPO+FLT3L and homing factor SDF1

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