Protease inhibitors stimulate hematopoiesis and decrease apoptosis and ICE expression in CD34+ cells

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2735-2739 ◽  
Author(s):  
Elaine M. Sloand ◽  
Jaroslaw Maciejewski ◽  
Princy Kumar ◽  
Sonnie Kim ◽  
Aniruddho Chaudhuri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Hiv Infection ◽  
Progenitor Cells ◽  
Protease Inhibitors ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Apoptotic Cell ◽  
Hematologic Toxicity ◽  
Caspase 1 ◽  
Cd34 Cells

Highly active retroviral therapy has been associated with a decline in the frequency of cytopenia in patients with human immunodeficiency virus (HIV) infection. This may result from lower hematologic toxicity of newer antiviral drugs and their increased efficacy against HIV-1. Protease inhibitors, in addition to their effects on HIV replication, appear to affect various cellular functions. Recently, it was reported that ritonavir inhibited caspase-1 expression in normal CD4+ cells. It was hypothesized that protease inhibitors may improve hematopoietic function owing to their direct effects on the bone marrow progenitor cells. When ritonavir was added to methylcellulose cultures of bone marrow cells from HIV-infected patients and normal controls, colony formation increased 2.4-fold (n = 5) in control cultures and 4-fold (n = 5) in cultures of cells from HIV-infected patients. In the presence of ritonavir, cultures of CD34+ cells showed markedly decreased apoptosis in comparison with untreated cultures (45% decrease in apoptotic cell number; n = 6). A synthetic inhibitor of caspase 1 (Ac-Tyr-Val-Ala-Asp-aldehyde [single-letter amino acid codes]), which inhibits activation of several caspases including CPP32 and interleukin 1β–converting enzyme (ICE or caspase 1), also decreased the rate of apoptosis and enhanced colony formation by progenitor cells derived from HIV-infected patients (3-fold; n = 5). In ritonavir-treated samples derived from HIV-infected individuals, the number of cells expressing ICE also decreased. In conclusion, HIV protease inhibitors may, by blocking the caspase-dependent apoptotic pathway, overcome inhibition of hematopoiesis seen in patients with HIV infection, an effect unrelated to their antiviral activity.

Protease inhibitors stimulate hematopoiesis and decrease apoptosis and ICE expression in CD34+ cells

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2735-2739 ◽  
Author(s):  
Elaine M. Sloand ◽  
Jaroslaw Maciejewski ◽  
Princy Kumar ◽  
Sonnie Kim ◽  
Aniruddho Chaudhuri ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Hiv Infection ◽  
Progenitor Cells ◽  
Protease Inhibitors ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Apoptotic Cell ◽  
Hematologic Toxicity ◽  
Caspase 1 ◽  
Cd34 Cells

Abstract Highly active retroviral therapy has been associated with a decline in the frequency of cytopenia in patients with human immunodeficiency virus (HIV) infection. This may result from lower hematologic toxicity of newer antiviral drugs and their increased efficacy against HIV-1. Protease inhibitors, in addition to their effects on HIV replication, appear to affect various cellular functions. Recently, it was reported that ritonavir inhibited caspase-1 expression in normal CD4+ cells. It was hypothesized that protease inhibitors may improve hematopoietic function owing to their direct effects on the bone marrow progenitor cells. When ritonavir was added to methylcellulose cultures of bone marrow cells from HIV-infected patients and normal controls, colony formation increased 2.4-fold (n = 5) in control cultures and 4-fold (n = 5) in cultures of cells from HIV-infected patients. In the presence of ritonavir, cultures of CD34+ cells showed markedly decreased apoptosis in comparison with untreated cultures (45% decrease in apoptotic cell number; n = 6). A synthetic inhibitor of caspase 1 (Ac-Tyr-Val-Ala-Asp-aldehyde [single-letter amino acid codes]), which inhibits activation of several caspases including CPP32 and interleukin 1β–converting enzyme (ICE or caspase 1), also decreased the rate of apoptosis and enhanced colony formation by progenitor cells derived from HIV-infected patients (3-fold; n = 5). In ritonavir-treated samples derived from HIV-infected individuals, the number of cells expressing ICE also decreased. In conclusion, HIV protease inhibitors may, by blocking the caspase-dependent apoptotic pathway, overcome inhibition of hematopoiesis seen in patients with HIV infection, an effect unrelated to their antiviral activity.

Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34+ bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 581-585 ◽  
Author(s):  
Vishwanath Bhattacharya ◽  
Peter A. McSweeney ◽  
Qun Shi ◽  
Benedetto Bruno ◽  
Atsushi Ishida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Vascular Graft ◽  
Bone Marrow Cells ◽  
Venous Blood ◽  
Immunomagnetic Bead ◽  
Composite Grafts ◽  
Cd34 Cells ◽  
Marrow Cells

The authors have shown accelerated endothelialization on polyethylene terephthalate (PET) grafts preclotted with autologous bone marrow. Bone marrow cells have a subset of early progenitor cells that express the CD34 antigen on their surfaces. A recent in vitro study has shown that CD34+ cells can differentiate into endothelial cells. The current study was designed to determine whether CD34+ progenitor cells would enhance vascular graft healing in a canine model. The authors used composite grafts implanted in the dog's descending thoracic aorta (DTA) for 4 weeks. The 8-mm × 12-cm composite grafts had a 4-cm PET graft in the center and 4-cm standard ePTFE grafts at each end. The entire composite was coated with silicone rubber to make it impervious; thus, the PET segment was shielded from perigraft and pannus ingrowth. There were 5 study grafts and 5 control grafts. On the day before surgery, 120 mL bone marrow was aspirated, and CD34+ cells were enriched using an immunomagnetic bead technique, yielding an average of 11.4 ± 5.3 × 106. During surgery, these cells were mixed with venous blood and seeded onto the PET segment of composite study grafts; the control grafts were treated with venous blood only. Hematoxylin and eosin, immunocytochemical, and AgNO3staining demonstrated significant increases of surface endothelialization on the seeded grafts (92% ± 3.4% vs 26.6% ± 7.6%; P = .0001) with markedly increased microvessels in the neointima, graft wall, and external area compared with controls. In dogs, CD34+ cell seeding enhances vascular graft endothelialization; this suggests practical therapeutic applications.

Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 110-114 ◽  
Author(s):  
I McNiece ◽  
R Andrews ◽  
M Stewart ◽  
S Clark ◽  
T Boone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Synergistic Interaction ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.

scholarly journals Inhibition of Immature Erythroid Progenitor Cell Proliferation by Macrophage Inflammatory Protein-1α by Interacting Mainly With a C-C Chemokine Receptor, CCR1

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 605-611 ◽  
Author(s):  
Shao-bo Su ◽  
Naofumi Mukaida ◽  
Jian-bin Wang ◽  
Yi Zhang ◽  
Akiyoshi Takami ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Inflammatory Protein ◽  
Cd34 Cells ◽  
Macrophage Inflammatory Protein ◽  
Marrow Cells

Abstract Several lines of evidence indicate that macrophage inflammatory protein-1α (MIP-1α) modulates the proliferation of hematopoietic progenitor cells, depending on their maturational stages. To clarify the mechanisms for the modulation of hematopoiesis by this chemokine, we examined the expression of a receptor for MIP-1α, CCR1, on bone marrow cells of normal individuals using a specific antibody and explored the effects of MIP-1α on in vitro erythropoiesis driven by stem cell factor (SCF) and erythropoietin (Epo). CCR1 was expressed on glycophorin A-positive erythroblasts in addition to lymphocytes and granulocytes. CCR1+ cells, isolated from bone marrow mononuclear cells (BMMNCs) using a cell sorter, comprised virtually all erythroid progenitor cells in the BMMNCs. Moreover, MIP-1α inhibited, in a dose-dependent manner, colony formation by burst-forming unit-erythroid (BFU-E), but not by colony forming unit-erythroid (CFU-E), in a methylcellulose culture of purified human CD34+ bone marrow cells. Although reverse-transcription polymerase chain reaction (RT-PCR) showed the presence of CCR1, CCR4, and CCR5 transcripts in CD34+ cells in BM, anti-CCR1 antibodies significantly abrogated the inhibitory effects of MIP-1α on BFU-E formation both in a methylcellulose culture and in a single cell proliferation assay of purified CD34+ cells. Although the contribution of CCR4 or CCR5 cannot be completely excluded, these results suggest that MIP-1α–mediated suppression of the proliferation of immature, but not mature erythroid progenitor cells, is largely mediated by CCR1 expressed on these progenitor cells.

scholarly journals Human interleukin-4 enhances stromal cell-dependent hematopoiesis: costimulation with stem cell factor

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2189-2196 ◽  
Author(s):  
U Keller ◽  
MJ Aman ◽  
G Derigs ◽  
C Huber ◽  
C Peschel
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Stromal Cell ◽  
Bone Marrow Cells ◽  
Interleukin 4 ◽  
Adherent Cell ◽  
Hematopoietic Progenitors ◽  
Cd34 Cells

Abstract Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.

scholarly journals Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells.

1992 ◽  
Vol 175 (4) ◽  
pp. 1151-1154 ◽  
Author(s):  
J H Jansen ◽  
J C Kluin-Nelemans ◽  
J Van Damme ◽  
G J Wientjens ◽  
R Willemze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Interleukin 6 ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Marrow Cells

Since monocytes and macrophages that arise during the culture of bone marrow progenitor cells are potential sources of interleukin 6 (IL-6), we investigated whether auto- or paracrine production of this factor is involved in colony formation by normal hematopoietic progenitor cells. We added a polyclonal anti-IL-6 antiserum and a monoclonal anti-IL-6 antibody to cultures of monocyte- and T cell-depleted bone marrow cells. Colony formation was stimulated with granulocyte/monocyte-colony-stimulating factor (GM-CSF), monocyte-CSF, or IL-3. Addition of anti-IL-6 antibody resulted in decreased numbers of monocytic colonies to 40-50% of control values, whereas the numbers of granulocytic colonies were not altered. The inhibitory effect was preserved in cultures of CD34(+)-enriched bone marrow cells. As a second approach, we added a monoclonal antibody directed against the IL-6 receptor to cultures of monocyte- and T cell-depleted bone marrow cells. This antibody almost completely inhibited the growth of monocytic colonies, again without decreasing the number of granulocytic colonies. Finally, the importance of IL-6 in monocytopoiesis was demonstrated in serum-deprived bone marrow cultures: addition of exogenous IL-6 to cultures stimulated with GM-CSF resulted in increased numbers of monocytic colonies. Our results indicate that the permissive presence of IL-6 is required for optimal monocytic colony formation by bone marrow progenitor cells.

Adhesion to E-selectin promotes growth inhibition and apoptosis of human and murine hematopoietic progenitor cells independent of PSGL-1

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1685-1692 ◽  
Author(s):  
Ingrid G. Winkler ◽  
Karen R. Snapp ◽  
Paul J. Simmons ◽  
Jean-Pierre Lévesque
Keyword(s):  
Bone Marrow ◽  
Growth Inhibition ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Mouse Bone Marrow ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

AbstractAlthough both P- and E-selectin are constitutively expressed on bone marrow endothelial cells, their role in the regulation of hematopoiesis has only recently been investigated. We have previously shown that P-selectin glycoprotein ligand-l (PSGL-1/CD162) is expressed by primitive human bone marrow CD34+ cells, mediates their adhesion to P-selectin, and, more importantly, inhibits their proliferation. We now demonstrate that adhesion to E-selectin inhibits the proliferation of human CD34+ cells isolated either from human umbilical cord blood, adult mobilized blood, or steady-state bone marrow. Furthermore, a subpopulation, which does not contain the most primitive hematopoietic progenitor cells, undergoes apoptosis following E-selectin–mediated adhesion. The same phenomenon was observed in cells isolated from mouse bone marrow. Using lineage-negative Sca-1+ c-KIT+ bone marrow cells from PSGL-1–/– and wild-type mice, we establish that PSGL-1 is not the ligand involved in E-selectin–mediated growth inhibition and apoptosis. Moreover, stable transfection of the human myeloid cell line K562 (which does not express PSGL-1) with α(1,3) fucosyltransferase VII alone was sufficient to recapitulate the E-selectin–mediated growth inhibition and apoptosis observed in hematopoietic progenitor cells. These data demonstrate that an E-selectin ligand(s) other than PSGL-1 transduces growth inhibitory and proapoptotic signals and requires posttranslational fucosylation to be functional.

The Hemoglobin-Haptoglobin Receptor (CD163) Is Expressed by a Subpopulation of Erythroid and Granulo-Macrophagic Progenitors and Can Be Stimulated in Vitro and in Vivo by Physiological and Pharmacological Ligands to Stimulate Hematopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1221-1221
Author(s):  
Kathryn Matthews ◽  
Nicole Worsham ◽  
Neeta Rugg ◽  
Jose A. Cancelas ◽  
David Bell
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Abstract 1221 The receptor for the hemoglobin (Hb)-haptoglobin (Hp) complex, CD163, is expressed on the surface of a subpopulation of hematopoietic stem/progenitor cells (HPCs) (Matthews et al, 2006). The purpose of the studies presented here were two-fold – to demonstrate that the CD34+CD163+ double positive population could be isolated from normal adult bone marrow cells and these cells were functional as HPCs and, in addition, that these cells could be stimulated in vivo by ligands to CD163 to affect hematopoiesis. To investigate the clonogenic potential of CD34+/CD163+ HPCs, bone marrow CD34+ cells were examined for CD163 co-expression, sorted by fluorescence activated cell sorting (FACS) and plated into colony-forming assays (CFAs). 4.2% ± 1.4% (n=4) of CD34+ cells were found to co-express CD163 and this population consisted of two distinct sub-populations, CD34++ (hi)CD163+ and CD34+(lo)CD163+, each of which represented approximately half of the total CD34+CD163+ population. All three sorted populations (CD34+(all)CD163−, CD34++(hi) CD163+, CD34+(lo)CD163+) were plated into CFAs (n=4) and were assessed for erythroid and myeloid colony formation. The clonogenic efficiency of CD34++(hi)CD163+ had a 2.5-fold increase in the number CFU-E and CFU-GM when compared to both CD34+ (total) CD163− and CD34+(lo) CD163+ cells. In contrast, CD34+(hi an low)CD163+cells produced fewer BFU-E. To determine how the expression of CD163 expression on progenitor cells may play a role in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb/Hp) as well as an agonistic antibody to CD163 (TBI 304) on HPCs in vivo. NOD-scid IL2R gammanull (NSG) mice (HuMurine Technologies) were engrafted with human CD34+cells and animals with < 30% human CD45+ cells in the peripheral blood were administered either 2 mg Hb/mouse, or 100 or 500 μg/mouse TBI 304 every 4 days. At study termination (day 14), bone marrow cells (BMC) were examined by flow cytometry and enriched for CD34+ cells for enumeration in CFAs. Hb administration resulted in an increase of human CD34+cells ranging from 4% to 7% of BMC and a corresponding 57% increase in colony-forming cells (CFC) when compared to control (PBS-administered) animals. In contrast, TBI 304 produced a dose dependent decrease in CD34+ and CFC, possibly reflecting a depletion of CD34+/CD163+ cells from overstimulation due to the longer circulating antibody. To investigate this, human CD34+ cell engrafted animals were given a single dose of 10 or 100 μg/mouse of TBI 304 and bone marrow cells were examined on day 7. TBI 304 provided a 3.5-fold increase in human CD34+ cells as well as a 1.8 to 6.7-fold increase in bone marrow erythroid lineage engraftment (huGlyA+, huCD36+ and huCD71+) and a 2-fold increase in erythroid and myeloid colony-forming cells. No overall toxicities were observed with the administration of TBI 304 or Hb. We have demonstrated that CD163 is expressed on a population of CD34+ hematopoietic progenitor cells, these cells have increased hematopoietic progenitor activity in vitro and that administration of physiological or pharmacological agonists of the CD163 receptor can measurably stimulate hematopoiesis in vivo. Disclosures: Matthews: Therapure Biopharma: Employment. Bell:Therapure Biopharma: Employment.

A Comparison of Myelopoiesis from Induced Pluripotent Stem Cells with a Mutation in ELANE between Cyclic Neutropenia and Severe Congenital Neutropenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2193-2193
Author(s):  
Satoshi Saito ◽  
Shiho Nishimura ◽  
Miyuki Tsumura ◽  
Yoko Mizoguchi ◽  
Sonoko Sakata ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Cd34 Cells ◽  
Induced Pluripotent ◽  
And Control

Abstract The ELANE is known as the responsible gene for both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). However, relations between mutations in the ELANE gene and abnormal myelopoiesis in the different phenotype of these diseases still remain unclear. It has been reported that induced pluripotent stem cell (iPSC) from an individual patient with SCN (SCN-iPSC) demonstrated maturation arrest of myeloid progenitor cells and poor response to granulocyte-colony stimulating factor as similarly observed in patient's bone marrow cells. Thus, the study on myelopoiesis using disease specific iPSC seems to provide disease pathogenesis as a novel in vitro experimental model. In this study, we established iPSC line from an individual patient with CyN (CyN-iPSC) with heterozygous mutation in ELANE gene (Exon5, R191Q point mutation). Then we compared myelopoiesis among healthy Control-iPS (253G1), SCN-iPS (Exon5, C194X point mutation) , and CyN-iPSC. Undifferentiated colonies derived from CyN-iPSC were staind with pluripotency markers (OCT3/4 and NANOG). CyN-iPSC retained a normal karyotype and ELANE locus mutation of the original samples. In vitro myelopoiesis was examined by using a serum- and feeder-free monolayer hematopoietic culture system. iPSC colonies were cultured on growth factor-reduced Matrigel-coated cell culture dishes in modified Tenneille Serum Replacer 1 (mTeSR™1) medium (StemCell Technologies, Inc.), containing BSA, rh bFGF, rh TGFβ, Lithium Chloride, Pipecolic acid, GABA. Medium was replaced every four days. Then medium was changed to StemPro®-34 SMF Complete Medium plus nutrient supplement (Life technologies Corp.). The iPSC were cultured with BMP4 (80 ng/mL) for four days, and then replaced with VEGF165 (80 ng/mL), bFGF (25.7 ng/mL), and SCF (100 ng/mL) on Day 4. On Day 6, cytokines were replaced with a combination of SCF (50 ng/mL), IL-3 (50 ng/mL), and G-CSF (50 ng/mL). Medium was replaced every 3 - 4 days. No significant difference in the ratio of proliferating CD33+ cells were noted between CyN-iPSCs and Control-iPSCs. CyN-iPSCs showed less capability in the proliferation and maturation for CD15+ cells on days 20 to 40 than Control-iPSCs. The decreased number of CD15+ cells derived from CyN-iPSc implies the defect in mature neutrophil survival. In contrast, CD15+ / CD33+ cells derived from SCN-iPSCs were hardly observed in this culture condition, suggesting the defects of proliferation and maturation in SCN-iPSCs. We next examined the colony formation of CD34+ cells derived from CyN-iPSCs, Control-iPSCs, and SCN-iPSCs. CD34+ cells were obtainded at the day 12 of primary culture of iPSCs and purified by cell sorting using FACS-Aria®. No significant differences in the number of G-colony and GM-colony between CD34+ cells from CyN-iPSCs and Control-iPSCs. In contrast, CD34+ cells from SCN-iPSCs gave rise to the significantly decreased number of G-colony and GM-colony. The observations of myeloid proliferation/maturation and colony formation of CD34+ cells were almost compatible with those obtained from bone marrow cells in patients with SCN and CyN. Furthermore, neutrophils differentiated from CyN-iPSCs showed the excessive cell death, whereas SCN-iPSCs presented the defective myelopoiesis. These results suggest that the analyses using CyN-iPSCs and SCN-iPSCs may be useful tool for investigating the relation of gene mutation and pathophysiology in both diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Separation of functionally distinct human granulocyte-macrophage colony- stimulating factors

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 614-627
Author(s):  
NA Nicola ◽  
D Metcalf ◽  
GR Johnson ◽  
AW Burgess
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Molecular Size ◽  
The Other ◽  
Adherent Cell ◽  
Colony Stimulating Factors ◽  
Gm Csf

Human placental conditioned medium (HPCM) contans colony-stimulating factors (CSFs) required for the growth in vitro of neutrophilic granulocyte-macrophage (GM) and eosinophilic (EO) progenitor cells from human bone marrow. Fractionation of CSFs in HPCM was achieved by manipulation of the elution conditions on a column of phenyl-Sepharose. After equilibration of the phenyl-Sepharose column at high ionic strength (1 M ammonium sulfate), all of the CSF bound; one species of GM-CSF (alpha) and all of the elutable EO-CSF were eluted from the column simply by reducing the salt concentration, whereas the second species of GM-CSF (beta) was free of EO-CSF and was eluted only by increasing the concentration of tehylene glycol in the elution buffer. The two GM-CSFs were functionally distinct. GM-CSF alpha preferentially stimulated colony formation by day 14 of culture, and there was a decreased proportion of neutrophil colonies and increased proportion of macrophage colonies as the strength of the stimulus was decreased; GM- CSF beta, on the other hand, preferentially stimulated colony formation by day 7 of culture, and the proportion of neutrophil colonies was high (average 80%) and independent of the concentration of GM-CSF beta. GM- CSF alpha and GM-CSF beta were indistinguishable on the basis of apparent molecular size on tel filtration columns (molecular weight 30,000), charge properties on isoelectric focusing beds (isoelectric point, 4.9), and were not related to each other as a sialoglycoprotein is related to its asialo form. Adherent cell removal of the target bone marrow cells (to remove colony-stimulating cells) suggested that both GM-CSFs acted directly rather than by stimulating the production of GM- CSF. Mixing and titration experiments indicated that the differences in functional specificities of the two GM-CSFs (and the lack of EO-CSF associated with GM-CSF beta) were not due to the presence of specific inhibitory molecules or lower absolute levels of CSF in one fraction relative to the other. These two species of GM-CSF should be useful in separately enumerating subpopulations of different GM-progenitor cells inhuman hemopoietic disorders.

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