Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)–transfected human stromal cells

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 532-540 ◽  
Author(s):  
Yutaka Kawano ◽  
Masayoshi Kobune ◽  
Miki Yamaguchi ◽  
Kiminori Nakamura ◽  
Yoshinori Ito ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Ex Vivo ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Stromal Cell Line ◽  
Human Telomerase

We developed a new human stromal cell line that could expand human hematopoietic progenitor/stem cells. Primary human bone marrow stromal cells were infected with retrovirus containing the human telomerase catalytic subunit (hTERT) gene, resulting in increased population doubling and the acquisition of cell immortalization. Characteristics of the hTERT-transduced stromal (hTERT-stromal) cells were identical with those of the primary stromal cells in terms of morphologic appearance and expression of surface antigens. Human cord blood (CB) CD34+ cells were expanded by coculture with primary stromal or hTERT-stromal cells in the presence of stem cell factor, thrombopoietin, and Flk-2/Flt-3 ligand under serum-free condition. The degree of expansion of CD34+ cells and total number of colony-forming units in culture (CFU-Cs) after 2 weeks' coculture with the hTERT-stromal cells were nearly the same as those after 2 weeks' coculture with primary stromal cells (CD34+ cells, 118-fold ± 8-fold versus 117-fold ± 13-fold; CFU-Cs, 71-fold ± 5-fold versus 67-fold ± 5-fold of initial cell number). CB expansion on hTERT-stromal cells occurred at a similar rate through 7 weeks. In contrast, the rate of CB expansion on primary stromal cells had drastically declined at 7 weeks. In nonobese diabetic/severe combined immunodeficiency (SCID) mice, the degree of engraftment of SCID-repopulating cells that had been cocultured with hTERT-stromal cells for 4 weeks was significantly higher than that of precocultured CB cells. These results indicate that this hTERT-stromal cell line could be useful for ex vivo expansion of hematopoietic progenitor/stem cells and for analyzing the microenvironment of human bone marrow.

scholarly journals Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 365-372 ◽  
Author(s):  
JP Wineman ◽  
S Nishikawa ◽  
CE Muller-Sieburg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Stromal Cell Line ◽  
Cell Maintenance

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stromal Cell Line ◽  
Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

scholarly journals Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4116-4124 ◽  
Author(s):  
L Croisille ◽  
I Auffray ◽  
A Katz ◽  
B Izac ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Adherent Cells ◽  
Long Term Culture ◽  
Bone Marrow Cultures ◽  
Stromal Cell Line ◽  
Clonogenic Cells

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units- granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.

N-rasm-Transduced Fibroblasts Facilitate the Expansion and Engraftment of Umbilical Cord Blood Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3196-3196
Author(s):  
Alla Dolnikov ◽  
Tiffany Holmes ◽  
Sylvie Shen ◽  
Robert Knight ◽  
Tracey A. O’Brien
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitor ◽  
Soluble Factors ◽  
Cd34 Cells

Abstract Clinical trials using ex-vivo expanded umbilical cord blood (UCB) cells have thus far failed to demonstrate significant improvement in speed of hematopoietic recovery suggesting better understanding of self-renewal, proliferation, homing and engraftment capabilities of cord blood is needed. Recent progress has been made in the identification and understanding of the cellular niche for hematopoietic stem cells (HSCs) within bone marrow however attempts to identify soluble factors that regulate HSC engraftment to bone marrow have been less successful. We have demonstrated that N-rasm transduced NIH 3T3 fibroblasts release soluble factors that support the ex-vivo expansion of UCB CD34+ hematopoietic progenitor cells (HPCs). UCB CD34+ cells were cultured in combination with cytokines : TPO, SCF, IL-6 and flt3-ligand(all at 100ng/ml). Significant (7-fold) expansion of total nucleated cells was seen on day 19 in cultures complemented with medium produced by N-rasm -transduced fibroblasts (N-rasm medium), compared to control cells (GFP- transduced fibroblasts (GFP medium)). Maximum expansion of UCB CD34+ cells (2-fold) and the most primitive sub-population of CD34+ CD117+ cells (2-fold) were seen on day 4 in N-rasm medium cells compared to GFP medium cells. Furthermore, increased numbers of cycling cells were observed in UCB CD34+ cell cultures supplemented with N-rasm medium. Significant (3-fold) expansion of CD34+CXCR4+ cells enriched with the NOD/SCID mice engrafting cells, was also observed. This increased expansion of CD34+CXCR4+ cells correlated with a 3-fold increased engraftment of human UCB cells in a NOD/SCID mouse model confirming retention of engraftment capability of expanded cells. This data demonstrates that N-rasm -transduced fibroblasts secrete soluble factors that promote ex-vivo expansion and in-vivo engraftment of primitive human hematopoietic progenitor/stem cells derived from UCB. Proteins that are secreted by N-rasm -transduced fibroblasts that activate hematopoiesis are currently being identified. Our data demonstrates the potential of N-rasm-transduced fibroblasts as a reservoir for the discovery of novel secreted proteins that regulate ex-vivo expansion and engraftment of human UCB HSCs and may represent a potential novel expansion strategy allowing greater use UCB as a stem cell source for allogeneic transplantation.

scholarly journals Connexin-43-type gap junctions mediate communication between bone marrow stromal cells

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 38-45 ◽  
Author(s):  
K Dorshkind ◽  
L Green ◽  
A Godwin ◽  
WH Fletcher
Keyword(s):  
Bone Marrow ◽  
Gap Junctions ◽  
Cell Line ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Cell Communication ◽  
Marrow Stromal Cells ◽  
Stromal Cell Line ◽  
Cell Cell

Several morphologic studies have suggested that gap junctions exist between bone marrow stromal cells. This possibility was examined by analysis of stromal cells present in the adherent layer of primary long- term lymphoid bone marrow cultures and in additional studies using a stromal cell line. Results showing that the fluorescent dye lucifer yellow, when microinjected into a single stromal cell, transferred between most other contacting stroma and that stromal cells were electronically coupled provided support that cell-cell communication occurs between these microenvironmental elements. Additional studies showed that transcripts for connexin (Cx) 43, but not for Cx26 or Cx32, were present in a stromal cell line. To examine the potential for regulated cell-cell communication between the stroma, cells were treated with interleukin-1 (IL-1), a cytokine known to affect stromal cell function, and the effects on dye transfer were examined. IL-1 treatment resulted in a reversible decrease in the ability of dye to transfer between stromal cells in contact. Taken together, these studies show that gap junctions exist between stromal cells and that their permeability can be regulated. However, gap junction-mediated cell-cell communication could not be shown between the stroma and developing lymphoid cells.

scholarly journals Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

Blood ◽  
1993 ◽  
Vol 81 (2) ◽  
pp. 365-372 ◽  
Author(s):  
JP Wineman ◽  
S Nishikawa ◽  
CE Muller-Sieburg
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Stromal Cell Line ◽  
Cell Maintenance

Abstract We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).

scholarly journals Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1185-1192 ◽  
Author(s):  
LG Billips ◽  
D Petitte ◽  
K Dorshkind ◽  
R Narayanan ◽  
CP Chiu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Kit Ligand ◽  
Interleukin 7 ◽  
Stromal Cell Line ◽  
Differentiation And Proliferation

Abstract Newly formed B lymphocytes are a population of rapidly renewed cells in the bone marrow of mammals and their steady state production presumably depends on a cascade of regulatory cells and cytokines. Although considerable information has been forthcoming about the role of interleukin-7 (IL-7) in potentiating pre-B-cell proliferation, few studies have addressed the possibility that multiple cytokines are involved in the progression of early events in cellular differentiation and proliferation in this hematopoietic lineage. Our laboratory previously described pre-B-cell differentiation mediated by the bone marrow stromal cell line S17. In this study, we further delineate the role of stromal cells in differentiation and proliferation of pre-B cells. These experiments show that the stromal cell line S17 potentiates the proliferative effect of IL-7 on B-lineage cells and that this S17-derived potentiator can be replaced with recombinant kit- ligand (KL). Our results further show that pre-B-cell formation from B220-, Ig- progenitor cells and expression of mu heavy chain of immunoglobulin is uniquely dependent on the presence of S17 stromal cells and cannot be reproduced with IL-7, KL, or costimulation with both IL-7 and KL. These data contribute to a rapidly evolving model of stromal cell regulation of B-cell production in the marrow and suggest unique roles for IL-7, KL, and as yet uncharacterized stromal cell- derived lymphokines in this process.

scholarly journals A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assays

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 2916-2924 ◽  
Author(s):  
C Issaad ◽  
L Croisille ◽  
A Katz ◽  
W Vainchenker ◽  
L Coulombel
Keyword(s):  
Stem Cells ◽  
Cell Line ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Proliferative Potential ◽  
Short Term ◽  
Stromal Cell Line ◽  
Colony Assays

Abstract Analysis of molecular mechanisms associated with stem cell commitment and differentiation requires an in vitro assay that identifies the most primitive hematopoietic stem cells in human bone marrow. Such primitive stem cells usually do not form colonies in short-term semisolid assays and are best identified by their ability to initiate sustained hematopoiesis when they are cocultured with competent stromal cells. In this study, we investigated whether a murine marrow stromal cell line (MS-5) that supports colony-forming unit-spleen (CFU-S) maintenance would permit, both in short-term colony assays and long-term cultures, the development of primitive human stem cells sorted on the basis of their high expression of CD34 and lack of expression of CD38 antigen. In short-term colony assays, this population included almost exclusively primitive progenitor cells. MS-5 cells synergized with any combination of interleukin-3, Steel factor, granulocyte colony- stimulating factor, agar-leukocyte conditioned medium, and erythropoietin and increased at least twofold both the cloning efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore, MS-5 cells triggered the development of multipotent blast cell progenitors with a high proliferative potential, which in these conditions represented 1% to 2% of CD34++/CD38- cells. When MS-5 cells were substituted by human stromal cells or when growth factor combinations were used in the absence of stromal cells, much lower numbers of CFU-blast were detected. This selective action of MS-5 on early progenitors was also observed when MS-5 cells were used as feeders in long-term cultures of CD34++/CD38- cells. Murine cells promoted the expansion of high proliferative potential primitive progenitor cells up to 3 months, although they did not support their differentiation in mature clonogenic progenitors or terminally differentiated cells. Sustained hematopoiesis in these longterm cultures was accounted for by 2% to 5% of initial CD34++/CD38- cells as estimated by limiting dilution experiments. Mechanisms by which murine stromal cells act specifically on human primitive stem cells are unclear, but from our data this effect is unlikely to be explained solely by known species cross-reactive growth factors. Further manipulation of this long-term coculture system should prove useful in identifying stromal molecules regulating commitment and differentiation of early human progenitor cells.

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