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.

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

Abstract 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.

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.

scholarly journals 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

Abstract 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.

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 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.

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 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.

scholarly journals Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1473-1481 ◽  
Author(s):  
LS Rusten ◽  
SE Jacobsen ◽  
O Kaalhus ◽  
OP Veiby ◽  
S Funderud ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.

scholarly journals 939 Autocrine/paracrine factors in cancer associated fibroblasts drive immunosuppression through impaired myeloid cell functions

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A985-A985
Author(s):  
Nikita Sharma ◽  
Priya Govindaraju ◽  
Shermineh Bradford ◽  
Yarong Wang ◽  
Brianna Flynn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Progenitor Cells ◽  
Tumor Antigen ◽  
Bone Marrow Cells ◽  
Pancreatic Stellate Cells ◽  
Hematopoietic Progenitor Cells ◽  
Cancer Associated Fibroblasts ◽  
Hematopoietic Progenitor ◽  
Marrow Cells

BackgroundCancer associated fibroblasts (CAFs) promote tumorigenesis by secreting immunosuppressive cytokines, stimulating angiogenesis, and supporting the growth of tumor cells. Through their interactions with immune cells, CAFs are known to directly impact the functionality of T cells and macrophages. However, CAF interaction with dendritic cells (DCs) and DC progenitor cells and its impact on DC function is relatively understudied and was the main focus of this study.MethodsTwo types of coculture systems were used in this study. For the human system, fibroblasts from lung squamous cell carcinoma (LUSC) were cocultured with MUTZ3 cells (hematopoietic progenitor cells) in the presence of DC differentiation stimuli, sometimes followed by DC maturation stimuli. For the mouse coculture system, activated (YPSC-c) and inactivated (PSC-b) pancreatic stellate cells (PSCs) were isolated from the pancreas of C57BL/6 mice by the density gradient method and co-cultured in the presence of bone marrow cells in the presence of DC differentiation and maturation stimuli. For human tumor antigen processing and cross presentation assay MART1 peptide (10mer and 20mer) was used.ResultsCo-culture of human and murine hematopoietic progenitor cells with fibroblasts (human LUSC CAFs and murine PSC results in decrease in differentiation and maturation of DCs. DCs differentiated and matured in the presence of fibroblasts have impaired ability to process and present tumor antigen to T cells. In the presence of PSC fibroblasts DC differentiation from murine bone marrow cells is skewed more towards MDSC and macrophages. In contrast to inactivated PSC-b, activated PSC-c influence DC differentiation in a contact dependent manner. Furthermore, PSC-b and PSC-c show transcriptionally distinct signatures which translate to unique secretory profiles as measured by Luminex. Analysis of the conditioned media from the coculture demonstrated that PSC-c secrete (among others) CXCL1, IL6, and CCL5 chemo/cytokines. These and other factors may play an important role in mediating fibroblast induced suppression of DC differentiation from monocytes.ConclusionsOur study demonstrates that cancer associated fibroblasts, or their precursors directly impact DC differentiation and antigen presentation via cytokines that could be targeted therapeutically to improve DC expansion and activity in the tumor microenvironment.

Bone Marrow Endothelial Cell Support of Primitive Hematopoietic Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3573-3573
Author(s):  
Xiaoying Zhou ◽  
Dilani Rosa ◽  
Cynthia Cunningham ◽  
Gregor B. Adams
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Functional Role ◽  
Hematopoietic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Vascular Niche

Abstract Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in specialized microenvironments known as the stem cell niche. Many reports have found the HSCs to be resident next to the endosteal surface of bone where cells of the osteoblastic lineage are a key component of the so-called endosteal niche. However, HSCs have also been found to reside adjacent to sinusoidal blood vessels. These observations have led to the proposal that HSCs in the adult BM may also reside in a vascular niche. However, the functional role of the vascular niche in hematopoiesis remains to be determined. We wished to evaluate the role that BM endothelial cells (BMECs) play in HSC physiology. To examine this we cultured BMEC-enriched cells in vitro, identified by expression of CD31, Tie-2, VE-cadherin and LDL uptake. We compared these cells to spleen derived ECs and BM stromal cells (BMSCs) in their ability to support primitive hematopoietic cells for extended periods in in vitro culture. We found that BMECs were superior in their ability to support the cobblestone area forming cell activity of VEGF-R1+ HSCs than spleen ECs or BMSCs. We also found that the number of cobblestone area cells was markedly reduced when VEGF-R1− HSCs were cultured on any of the supportive cell layers, however this may be due to an intrinsic difference between these cells as a much higher proportion of VEGF-R1+ HSCs were found to be in the G0 phase of the cell cycle than VEGF-R1− cells. To evaluate the supportive role of BMECs, spleen ECs or BMSCs on hematopoietic progenitor cells (HPC) we cultured purified primitive cells on these supportive layers and the total number of colony-forming unit-culture (CFU-C) cells were examined after 4-days or 7-days co-culture with the feeder cells. The results showed that BMECs or spleen ECs can promote the generation of CFU-C from VEGFR1+ HSCs or VEGFR1− HSCs, yet the tot al number of CFU-C produced from VEGFR1+ HSCs was greater than that from VEGFR1- HSCs. However, both of these cell types were able to support the generation of CFU-Cs to a greater degree than BMSCs. To examine the mechanism of enhanced support of VEGF-R1+ HSCs by the BMECs, we performed real-time PCR analysis for the expression of the VEGF-R1 ligands. Both BMECs and spleen ECs were found to express VEGF-A and –B to similar levels, however the expression of placental growth factor was higher in the BMECs. Whether the increased expression of this factor plays a functional role in the support of the HSCs in currently being evaluated. Our findings suggest that the ECs from BM or spleen can promote the proliferation of hematopoietic progenitor cells, while BMECs can maintain the long-term culture of VEGFR1+ HSCs in vitro. The functional relevance of this in vivo is currently being investigated.

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