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.

scholarly journals A gene transfer strategy for making bone marrow cells resistant to trimetrexate

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2579-2587 ◽  
Author(s):  
HT Spencer ◽  
SE Sleep ◽  
JE Rehg ◽  
RL Blakley ◽  
BP Sorrentino
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Retroviral Vectors ◽  
Hematopoietic Progenitor ◽  
High Level ◽  
Variant Enzyme ◽  
Marrow Cells

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y- containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.

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.

scholarly journals Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 661-669 ◽  
Author(s):  
EF Srour ◽  
JE Brandt ◽  
RA Briddell ◽  
S Grigsby ◽  
T Leemhuis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Hla Dr

Abstract Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

scholarly journals T lymphocyte differentiation in vitro from adult human prethymic CD34+ bone marrow cells.

1993 ◽  
Vol 177 (6) ◽  
pp. 1531-1539 ◽  
Author(s):  
G E Tjønnfjord ◽  
O P Veiby ◽  
R Steen ◽  
T Egeland
Keyword(s):  
Bone Marrow ◽  
T Lymphocyte ◽  
Bone Marrow Cells ◽  
Cell Receptor ◽  
Thymic Tissue ◽  
Adult Human ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Marrow Cells

Pluripotent lymphohematopoietic stem cells are probably confined to bone marrow cells expressing CD34 surface molecules. To investigate the capacity of adult human CD34+ bone marrow cells to differentiate along the T lymphoid lineage, we plated purified CD34+ cells from healthy adults in liquid culture on adherent thymic stromal cells prepared from HLA- or blood group-mismatched postnatal thymic tissue. We show that purified CD34+CD3-CD4-CD8- bone marrow cells contained progenitors with the ability to differentiate into CD4+ and CD8+ T lymphocytes expressing surface (s)CD3 and T cell receptor alpha/beta in vitro. These progenitors were found in the CD34+CD2+sCD3-CD4-CD8-, CD34+CD7+sCD3-CD4-CD8-, and CD34+CD2+CD7+sCD3-CD4-CD8-, as well as in the CD34+CD2-sCD3-CD4-CD8-, CD34+CD7-sCD3-CD4-CD8-, and CD34+CD2-CD7-sCD3-CD4-CD8- subsets, indicating that T lymphocyte progenitors sensitive to signals mediated by thymic stroma in vitro are not restricted to CD34+ cells already coexpressing early T lymphocyte-associated markers. Finally, we show that T lymphopoiesis was enhanced by c-kit ligand.

scholarly journals The Chemokine SDF-1 Is a Chemoattractant for Human CD34+ Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34+ Progenitors to Peripheral Blood

1997 ◽  
Vol 185 (1) ◽  
pp. 111-120 ◽  
Author(s):  
A. Aiuti ◽  
I.J. Webb ◽  
C. Bleul ◽  
T. Springer ◽  
J.C. Gutierrez-Ramos
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor ◽  
Transient Elevation

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38− or CD34+/DR−) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

Therapeutic Targeting of CD163 on Hematopoietic Progenitor Cells By Novel Monoclonal Antibody TBI 304H Demonstrates an Increase in Erythropoiesis in Humanized Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1163-1163
Author(s):  
Kathryn Matthews ◽  
Irina Eberle-Ayres ◽  
Katherine Lu ◽  
Nishi Singh ◽  
Murray J Cutler ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Single Dose ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Dose Dependent

Abstract Hemoglobin (Hb) is known to stimulate erythropoiesis, a process that may be mediated by CD163. CD163 is a receptor for the hemoglobin-haptoglobin (Hb-Hp) complex expressed on monocyte/macrophages as well as a subpopulation of human CD34+ hematopoietic progenitor cells (HPCs). We have demonstrated that administration of ligands to the CD163 receptor can measurably stimulate erythropoiesis in human CD34+ cell-engrafted severe-combined immunodeficiency (SCID) mice. To better elucidate the role of CD163 in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb-Hp) as well as a stimulatory antibody, TBI 304H, on HPCs in vivo. SCID mice engrafted with human CD34+ cells were used as a model system were used to investigate the effect of Hb and anti-CD163 monoclonal antibodies (TBI 304 and TBI 304H) on human hematopoiesis in vivo. In an initial study, NOD-SCID IL2R gammanull (NSG) mice were engrafted with human CD34+ cells and animals with < 30% human CD45+ cells in the peripheral blood were administered 2 mg Hb/mouse, or 100 or 500 µg/mouse TBI 304 every 4 days for a total of four doses. At study termination on day 14, bone marrow cells (BMCs) were examined by flow cytometry and CD34+ cells were recovered from the BMCs for enumeration in colony-forming assays. Hemoglobin administration resulted in an increase of human CD34+ cells ranging from 4% to 7% of BMCs and a corresponding 57% increase in colony-forming cells (CFCs) over control animals. In contrast, the monoclonal antibody (mAb), TBI 304, produced a dose-dependent decrease in CD34+ and colonies, possibly reflecting a depletion of CD34+/CD163+ cells as a result of overstimulation due to the much longer circulating half-life of the mAb compared to Hb.. To confirm this hypothesis, human CD34+ cell engrafted animals were given only a single dose of 10 or 100 µg/mouse of TBI 304 and BMCs were examined earlier 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 colony-forming cells. The ability of TBI 304 to stimulate erythropoiesis in preclinical models led to the creation of an anti-CD163 mAb suitable for human clinical use. TBI 304H was generated by grafting the complementarity-determining regions derived from TBI 304 onto a humanized IgG4 framework without altering antigen specificity. An IgG4 framework, as an antibody without Fc effector function, was deemed the most suitable for an agonistic mAb. In the single dose, 7 day Hu-SCID model human CD34+ cells were mobilized from the mouse bone marrow by TBI 304H, as reflected by dose dependent decreases in huCD34+, huCD71+, and huGlyA+ cells in the mouse marrow. At the highest dose tested (500 µg/mouse) the decrease in human HPCs was similar to that found in animals administered Hb (2 mg/mouse). In this model, human hematopoiesis derived from the engrafted human CD34+ cells is not sustained and these date may reflect a mobilization of human HPCs through stimulation by an anti-CD163 antibody. Therapure has received U.S. FDA approval to conduct a Phase I trial of the novel therapeutic antibody TBI 304H. The Phase I clinical trial is a single-center, open-label, intra-subject escalating dose study, which will evaluate the safety, tolerability and pharmacokinetics of TBI 304H following administration to subjects experiencing chemotherapy-induced anemia. Disclosures Matthews: Therapure Biopharma: Employment. Eberle-Ayres:Therapure Biopharma: Employment. Lu:Therapure Biopharma: Employment. Singh:Therapure Biopharma: Employment. Cutler:Therapure Biopharma: Employment. Bell:Therapure Biopharma: Employment.

Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2476-2482 ◽  
Author(s):  
JM Molina ◽  
DT Scadden ◽  
M Sakaguchi ◽  
B Fuller ◽  
A Woon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Immunodeficiency Virus ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Marrow Cells

The pathogenesis of the hematologic abnormalities commonly observed in patients with acquired immunodeficiency syndrome (AIDS) is incompletely understood. We report here that in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from six patients with AIDS was not significantly different from that of normal human immunodeficiency virus (HIV) seronegative donors: 25.3 +/- 5 CFU-GM per 5 x 10(4) low density marrow cells and 33.5 +/- 5 BFU-E were observed in AIDS patients versus 32.7 +/- 5 CFU-GM and 42.1 +/- 5 BFU-E in controls. Furthermore, no HIV-DNA in individual colonies (CFU-GM and BFU-E) could be detected using the polymerase chain reaction (PCR) technique, although HIV-1 DNA was detected in peripheral blood mononuclear cells from the same patients. Similarly, normal bone marrow cells exposed in vitro to different isolates of HIV or recombinant purified HIV-1 envelope glycoprotein (gp) 120 did not exhibit any difference in growth of CFU-GM or BFU-E as compared with mock exposed bone marrow cells. HIV- 1 DNA could not be detected by the PCR technique in individual colonies derived from HIV exposed marrow. This study suggests that committed myeloid and erythroid progenitors from AIDS patients are responsive to hematopoietic growth factors in vitro and do not appear to contain HIV- 1 DNA. Also, HIV or its envelope gp did not alter the growth of hematopoietic progenitor cells in vitro. No evidence of HIV infection of progenitor cells could be demonstrated. Impaired hematopoiesis in patients with AIDS may not be related to direct effects of HIV on committed progenitor cells.

Impairment of lymphopoiesis and myelopoiesis in mice reconstituted with bone marrow–hematopoietic progenitor cells expressing SDF-1–intrakine

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2074-2080 ◽  
Author(s):  
Nobuyuki Onai ◽  
Yan-yun Zhang ◽  
Hiroyuki Yoneyama ◽  
Toshio Kitamura ◽  
Sho Ishikawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
B Lymphopoiesis ◽  
Hematopoietic Progenitor ◽  
Fcm Analysis ◽  
Marrow Cells

Both SDF-1 and CXCR4 disruption are lethal to mice at the embryonic stage and cause abnormalities in B lymphopoiesis, myelopoiesis, cardiogenesis, vasculogenesis, and cerebellar development. To investigate the role of SDF-1 and CXCR4 in hematopoiesis during the adult stage, mice reconstituted with bone marrow–derived hematopoietic progenitor cells transduced with either the SDF-1 or a genetically modified SDF-1–intrakine gene using a retroviral expression vector were analyzed. Flow cytometric (FCM) analysis showed a dramatic reduction of CXCR4 expression on the cells of intrakine-transduced mice, whereas CCR7 and CCR1 expression was unchanged or marginally decreased on splenocytes. Migration of splenocytes and bone marrow cells to SDF-1 was markedly suppressed in intrakine-transduced mice. FCM analysis of bone marrow cells of intrakine-transduced mice exhibited decreased numbers of pro-B (B220+ CD43+), pre-B (B220+CD43−), and immature B (B220+IgM+) cells and a decreased number of granulocytes/myeloid (Gr1+ CD11b+) cells. Impaired B lymphopoiesis and myelopoiesis in intrakine-transduced mice were confirmed by an in vitro colony-forming assay of bone marrow cells. In contrast, B lymphopoiesis and myelopoiesis were enhanced in SDF-1–transduced mice. Interestingly, T-cell maturation in the thymus was impaired both in intrakine- and SDF-1–transduced mice, suggesting that SDF-1 and CXCR4 play an important role in T lymphopoiesis as well as in B lymphopoiesis and myelopoiesis in adults. These results demonstrate an essential role of CXCR4 and its ligand SDF-1 in adult hematopoiesis, and they indicate the intrakine method as a powerful tool for functional analysis of chemokines/chemokine receptors in vivo and as a potential therapeutic approach for acquired immunodeficiency syndrome.

scholarly journals Fas-Mediated Apoptosis of the Hematopoietic Progenitor Cells in Mice Infected With Murine Cytomegalovirus

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3565-3573 ◽  
Author(s):  
Takehiko Mori ◽  
Kiyoshi Ando ◽  
Kazuo Tanaka ◽  
Yasuo Ikeda ◽  
Yasuhiro Koga
Keyword(s):  
Progenitor Cells ◽  
Fold Increase ◽  
Murine Cytomegalovirus ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Mcmv Infection ◽  
Cd34 Cells ◽  
Colony Forming Units

Abstract The effects of cytomegalovirus (CMV) infection on hematopoietic progenitor cells in vivo were investigated to elucidate the pathogenesis of CMV-induced myelosuppression. BALB/c mice were inoculated with 0.2LD50 of murine CMV (MCMV). Lineage marker negative, c-kit positive (Lin−c-kit+) and Lin−CD34+ cells, which are both phenotypically defined as hematopoietic progenitor cells, showed a significant reduction in number on day 3 postinfection (pi). Moreover, the reduction in the number of day-14 colony-forming units-spleen (CFU-S), another indicator to identify hematopoietic progenitor cells, was noted on day 3 pi. To clarify the mechanism of such depletion, we examined the cells undergoing apoptosis in the Lin− populations and found a 15-fold increase in the apoptosis-induction of these cells. Furthermore, an increase in the expression level of Fas, which mediates apoptosis, was observed in such Lin−c-kit+ and Lin−Sca-1+ cells on day 3 pi. In vitro treatment with the anti-Fas antibody accelerated the apoptosis in Lin− cells, but not in the uninfected control cells, thus indicating that the upregulated Fas on Lin− cells is directly related to the acceleration of apoptosis found in these cells in vivo. These results suggest that MCMV infection reduces the number of hematopoietic progenitor cells in bone marrow at least in part due to Fas-mediated apoptosis, and this phenomenon is thus considered to contribute to CMV-induced myelosuppression.

scholarly journals Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 661-669 ◽  
Author(s):  
EF Srour ◽  
JE Brandt ◽  
RA Briddell ◽  
S Grigsby ◽  
T Leemhuis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Hla Dr

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

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