Gene Transfer in Hematopoietic Progenitor Cells Mediated by an SV40 Based Episomal Vector Carrying A S/MAR Element.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5264-5264
Author(s):  
Eirini Papapetrou ◽  
Panos Ziros ◽  
Ilina Mitcheva ◽  
Aglaia Athanassiadou ◽  
Nicholas Zoumbos
Keyword(s):  
Stem Cells ◽  
Gene Transfer ◽  
Progenitor Cell ◽  
K562 Cells ◽  
Hematopoietic Progenitor Cell ◽  
Egfp Expression ◽  
Hematopoietic Progenitor ◽  
Episomal Vector ◽  
Cd34 Cells

Abstract Background/Aims: Replicating episomal vectors present a potential alternative to currently used oncoretroviral vectors for gene transfer in hematopoietic progenitor/stem cells. Their main advantage is that they can persist in the recipient nucleus as independent units, without integrating into the host’s genome, eliminating thus the risk of insertional mutagenesis. In the present study we explored the capacity of a recently developed SV40-based episomal vector, pEPI-eGFP, to stably transfect hematopoietic progenitor cell lines and primary cells, in order to evaluate its potential for therapeutic applications. pEPI-eGFP contains the enhanced green fluorescent protein (eGFP) cDNA and a Scaffold/Matrix Attachment Region (S/MAR) and it does not code for any proteins of viral origin. These unique properties qualify pEPI-eGFP as an attractive vehicle for gene therapy applications. Results: The vector was maintained as a stable episome in K562 cells for at least 100 generations and supported long-term EGFP expression, even in cells cultured in non-selective medium. Methylation-dependent cleavage assays demonstrated the vector’s ability to self-replicate in K562 cells and MEL cells, whereas its episomal status was confirmed by Southern blotting and plasmid rescue in E. coli. The vector was also maintained in primary human fibroblasts for at least 30 passages with and without selection pressure. Transfection of CD34+ cells from umbilical cord blood with pEPI-eGFP was feasible by electroporation with an efficiency of up to 30%, as estimated by flow cytometric evaluation of eGFP expression 24–48 h post-transfection. Cytokine prestimulation of CD34+ cells did not enhance transfection efficiency, whereas transfection of post-mitotic cells, such as dendritic cells was also feasible. This suggests that efficient transfection with the vector does not require cell cycling. PCR with eGFP-specific primers in single CFU colonies derived from CD34+/eGFP+/PI− FACS-sorted cells demonstrated the presence of the vector in ~20% of the colonies. Conclusion: Our results demonstrate that pEPI-2 exhibits a considerable potential as a gene transfer vector for hematopoietic cells, as it confers long-term transgene expression in hematopoietic progenitor cell lines as a stable episome and can efficiently transfect unstimulated CD34+ cells. Further studies, both in vitro and in vivo, are required to assess the long-term maintenance in hematopoietic progenitor/stem cells and their progeny as well as other features of the vector, in order to evaluate its overall efficacy and possibly improve its performance.

Hematopoietic Progenitor Cell (HPC) Collection Is Feasible in Previously Transplanted Multiple Myeloma Patients and Plerixafor Improves Collection

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4127-4127
Author(s):  
Xenofon Papanikolaou ◽  
Lisa Tyler ◽  
Bart Barlogie ◽  
Michele Cottler-Fox
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Median Time ◽  
Progenitor Cell ◽  
Median Number ◽  
Hematopoietic Progenitor Cell ◽  
Cell Transplant ◽  
Hematopoietic Progenitor ◽  
Platelet Recovery ◽  
Cd34 Cells

Abstract Abstract 4127 Autologous hematopoietic progenitor cell transplant (aHPCT) as salvage treatment in relapse is a therapeutic modality proven to induce response and improve overall survival (OS) in Multiple Myeloma (MM). However, many previously transplanted MM patients who might benefit from this intervention no longer have cryopreserved stem cells in storage. For these patients, it is important to know if HPC can be collected again adequate to support further aHPCT. Records of MM patients treated at the Myeloma Institute for Research and Therapy (MIRT), University of Arkansas for Medical Sciences, between 06/22/2005 – 11/16/2011 were reviewed to identify patients undergoing attempted remobilization following at least one previous aHPCT. HPC collection was performed using a Spectra (TerumoBCT) apheresis device to perform large volume leukapheresis (30L processed). Collection was guided by our predictive formula (Rosenbaum ER et al., Cytotherapy 2012;14(4) :461–6) and was continued until the collection goal was met or the daily collection contained less than 0.5×106 CD34+ cells/kg. We identified 48 patients (median age 58, range 38–82) who underwent at least one HPC collection, for a total of 98 collections (19 patients underwent one collection, 17 two collections, 6 three collections, 3 four collections, 3 five collections). The median time from previous APSCT was 3.25 years (range 0–8.8). Mobilization regimen information was available in all collections but two (Table 1). Plerixafor (Mozobil®) was used in 34 patients, for a total of 44 collections. For the total cohort of patients the median number of CD34+ cells per collection was 3.15×106 CD34+ cells/kg (0.1– 21.56), with a median of 4 days for each collection (1–10). No statistically significant correlation was seen for age, sex, number of previous aHPCT, time elapsed from previous aHPCT, or chemotherapy regimen used for previous aHPCT relative to the number of CD34+ cells collected. While the median number of CD34+ collected cells with or without plerixafor did not differ significantly (3.15 vs. 3.35×106 CD34+ cells/kg, p=0.701), the addition of plerixafor yielded a statistically significant increase if there was a poor previous mobilization (1.73 vs. 2.94×106 CD34+ cells/kg, p=0.004). Of 48 patients collected, 30 then underwent a subsequent aHPCT. Of these, 16 received cells procured after an aHPCT and 14 with cells procured prior to an aHPCT. No treatment related mortality was recorded in these aHPCT. Median number of CD34+ infused cells was 5.12×106 CD34+ cells/kg and 4.70×106 CD34+ cells/kg (p=0.583) for the before and after aHPCT procured stem cells. Median time for neutrophil engraftment (>500/mm3) was 10 days in both (p=0.897). Median time for platelet engraftment >20.000/mm3 was 12 and 11 days respectively (p=0.234), while for platelet engraftment >50.000/mm3 it was 24 and 15 days, with the difference being statistical significant (p=0.035). Nevertheless, all patients but one eventually achieved adequate platelet recovery (>100.000/mm3). In conclusion, collection of CD34+ cells following aHPCT is feasible and the addition of plerixafor can yield enough cells for a subsequent aHPCT even in cases where the initial attempt failed. HPC collected after a previous aHPCT provide a safe graft for another transplant. Table 1 Mobilization Type Number of Collections CD34+ cells×106/kg Median (range) Days for each collection median (range) Chemotherapy+G-CSF 30 3.19 (0.25–16.7) 4 (2–10) Chemotherapy+G–CSF +plerixafor 7 4.51 (1.68–11.98) 5 (2–7) G–CSF+/–GM–CSF+/–Epo 23 2.69 (0.52–16.93) 3 (3–5) G–CSF+plerixafor 36 2.84 (0.1–16.45) 4 (1–5) Disclosures: No relevant conflicts of interest to declare.

Optimal Proliferation of a Hematopoietic Progenitor Cell Line Requires Either Costimulation With Stem Cell Factor or Increase of Receptor Expression That Can Be Replaced by Overexpression of Bcl-2

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2569-2577 ◽  
Author(s):  
Huei-Mei Huang ◽  
Jian-Chiuan Li ◽  
Yueh-Chun Hsieh ◽  
Hsin-Fang Yang-Yen ◽  
Jeffrey Jong-Young Yen
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Progenitor Cell ◽  
Stem Cell Differentiation ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Progenitor Cell Line

Abstract In vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor  subunit (IL5R) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5–dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5R is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.

Long-term “in vitro” proliferating mouse hematopoietic progenitor cell lines

2010 ◽  
Vol 130 (1-2) ◽  
pp. 32-35 ◽  
Author(s):  
Patricia Vegh ◽  
Jana Winckler ◽  
Fritz Melchers
Keyword(s):  
Cell Lines ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor

Evidence for Efficacy and Safety of Lentiviral Mediated Gene Transfer in T Cells and CD34+ Cells from Wiskott-Aldrich Syndrome Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3279-3279
Author(s):  
Samantha Scaramuzza ◽  
Sara Trifari ◽  
Francesco Marangoni ◽  
Silvana Martino ◽  
Ayse Metin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Gene Transfer ◽  
Lentiviral Vector ◽  
Efficacy And Safety ◽  
Wiskott Aldrich Syndrome ◽  
Cd34 Cells ◽  
Was Gene

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.

Hematopoietic Progenitor Cell Transplantation from Parental Donors for Children with Sickle Cell Anemia and Stroke.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3393-3393
Author(s):  
Paul Woodard ◽  
Gregory Hale ◽  
Wing Leung ◽  
Raymond Barfield ◽  
Kimberly Kasow ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Transplantation ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Progenitor Cell ◽  
Chronic Gvhd ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Pilot Studies ◽  
Cd34 Cells

Although allogeneic hematopoietic progenitor cell transplantation (HPCT) can be curative for sickle cell anemia (SCA), most patients lack an HLA matched sibling donor or matched unrelated donor. A 2002 multidisciplinary conference held at St. Jude Children’s Research Hospital reached consensus that pilot studies using parental donors were reasonable and ethical. Subsequently, eight children with a history of clinically overt stroke were transplanted on two sequential pilot studies. Peripheral blood progenitor cells were obtained from parents with sickle cell trait. Conditioning was i.v. busulfan (targeted to Css 900 ng/ml) q 6 hours x 4 days, fludarabine 150–200 mg/m2, and OKT3/methylprednisolone and infusion of CD34+ HPCT for 3 patients. Five patients received i.v. busulfan (targeted to Css 900 ng/ml) x 4 days, cyclophosphamide 200 mg/kg, thiotepa 10 mg/kg, OKT3/methylprednisolone, and infusion of both CD34+ cells and CD3+ cells with a fixed T-cell addback of 1.0–1.5 x 105 CD3+ cells/kg. Six children received pre-transplant immunosuppression with hydroxyurea and azathioprine. The median follow-up of eight patients is 1.4 years (range 2 months–4 years). Five of eight had durable donor engraftment,4 of whom are alive and free of SCA post-HPCT. Rejection occurred in 4 patients and was successfully reversed with additional CD34+ cells in one of three patients. GVHD occurred in patients receiving a fixed T-cell addback or DLI: two patients had grade II acute graft-versus-host disease (aGVHD), one grade III aGVHD, and three patients developed chronic GVHD, including a fatal case of bronchiolitis obliterans organizing pneumonia (BOOP) and fungal sepsis. One engrafted patient developed medulloblastoma 2 years post-HPCT and is in remission after treatment. Further investigation revealed that this child inherited a novel germline p53 mutation. In this preliminary experience, haploidentical HPCT for children with SCA and stroke was associated with significant graft rejection and chronic GVHD. The addition of pre-transplant hydroxyurea and azathioprine, increased intensity of conditioning, and the use of T-cell addback to the graft did not improve engraftment in the second cohort. While offering the possibility of cure, haploidentical HPCT for SCA as performed in this experience is associated with significant toxicity and should only be pursued in the context of a rigorously designed and controlled prospective clinical trial.

MT1-MMP and RECK Inversely Regulate Hematopoietic Progenitor Cell Egress.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1259-1259
Author(s):  
Abraham Avigdor ◽  
Yaron Vagima ◽  
Polina Goichberg ◽  
Shoham Shivtiel ◽  
Melania Tesio ◽  
...  
Keyword(s):  
Steady State ◽  
Progenitor Cell ◽  
Scid Mice ◽  
Hematopoietic Progenitor Cell ◽  
Chimeric Mice ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.

RUNX1c Expression Identifies an Enriched Subpopulation of Hematopoietic Progenitor Cells Derived From Human Pluripotent Stem Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2294-2294
Author(s):  
Patrick I Ferrell ◽  
Mitali Adlakha ◽  
Dan S. Kaufman
Keyword(s):  
Stem Cells ◽  
Progenitor Cell ◽  
Hematopoietic Cells ◽  
Sleeping Beauty ◽  
Hematopoietic Progenitor Cell ◽  
Reporter System ◽  
Hematopoietic Progenitor ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Qrt Pcr

Abstract Abstract 2294 The ability of human embryonic stem cells (hESCs) to differentiate into any cell lineage makes them an important resource for studies of developmental biology. hESC-derived hematopoietic stem cells (HSCs) developed through in vitro culture systems could provide an unlimited supply of material for replacement of defective blood lineages in vivo. However, to date, there has been only limited ability to isolate functional HSCs from hESCs. Runx1 is a key hematopoietic transcription factor vital to the development of the early embryo. Previous studies have demonstrated the onset of Runx1c to correlate directly with the emergence of definitive HSCs in the murine embryo. Similarly, expression of RUNX1c increases during differentiation of hESCs to hematopoietic cells. This particular feature makes RUNX1c an especially attractive tool for tracking the development of potential HSCs from hESCs. Therefore, we have created a novel reporter system in hESCs wherein both 1 kb of the basal promoter as well as the +24 intronic enhancer of RUNX1c drive expression of the tdTomato fluorochrome. Transgenic hESC lines stably expressing tdTomato from RUNX1c regulatory elements were generated using the Sleeping Beauty transposon system. Successfully engineered clones were selected based on constitutive expression of a GFPzeocin fusion protein which was also included in the transgene. To induce differentiation, the spin-embryoid body (EB) method was used with fully defined media. Differentiating cells were analyzed for tdTomato expression. Sorted tdTomato+ cells were evaluated for RUNX1c expression and hematopoietic stem/progenitor cell potential (CFC assay). Undifferentiated RUNX1c reporter hESC clones were uniformly tdTomato-negative. These clones are shown to express the tdTomato transgene concurrent with the expression of endogenous RUNX1c, as analyzed by flow cytometry and qRT-PCR. Expression was first seen at day 12 with a peak between days 14–16 of differentiation. Cells sorted for expression of tdTomato showed a significant increase of endogenous RUNX1c expression compared to tdTomato-negative cells. Using fluorescence microscopy, we were able to detect tdTomato+ cells with hematopoietic morphology budding from the EBs after 12–15 days. Together, these results validate the accuracy of this reporter system. tdTomato+ cells were found to be enriched for hematopoietic progenitor potential, as demonstrated by increased ability to form hematopoietic colonies in methylcelluose (430 +/− 143 per 105 cells) as compared to both tdTomato negative (131 +/− 15 per 105 cells) and unsorted cells (163 +/− 28 per 105 cells). Interestingly and unexpectedly, flow cytometric analysis demonstrated that only 5–10% of CD34+CD45+ cells expressed tdTomato. The first CD45+ population was found to lack tdTomato expression. Likewise, the first emerging tdTomato+ cells did not express CD45. Of the extracellular markers examined in this study, CD31 and CD43 were found to be expressed on all tdTomato+ cells, while CD31+ and CD43+ populations, though initially tdTomato−, were found to gradually increase in tdTomato expression to 80% and 90% by day 20, respectively. Emerging CD34+ cells lack tdTomato expression with an increase to around 10% of cells, plateauing at day 15. While CD45+ cells initially lack tdTomato expression, they gain expression over time, until days 19–22, when 50–90% of CD45+ cells co-express tdTomato (RUNX1c). Therefore, this hESC RUNX1c reporter system appears to accurately define timing of RUNX1c expression. The finding that emerging RUNX1c+ cells have increased hematopoietic progenitor cell potential mimics what has been found in mice. This, combined with our finding that only a fraction of CD34+CD45+ cells express Runx1c, suggests that one reason hESC-derived hematopoietic cells fail to engraft in immunodeficient mice is that only a small percent of differentiated cells that express hematopoietic surface antigens actually have hematopoietic stem/progenitor cell activity. Current studies focus on more in-depth analysis of tdTomato expressing cells using both single-cell qRT-PCR as well as LTC-IC and engraftment assays to test for functionality. Furthermore, we are currently testing an iPSC line stably expressing the same RUNX1c:tdTomato transgene to test how its RUNX1c expression in iPSCs compares to hESCs during hematopoietic differentiation. Disclosures: No relevant conflicts of interest to declare.

Heparan Sulfate Proteoglycan Expression Is Induced During Early Erythroid Differentiation of Multipotent Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 2884-2897 ◽  
Author(s):  
Zofia Drzeniek ◽  
Georg Stöcker ◽  
Barbara Siebertz ◽  
Ursula Just ◽  
Timm Schroeder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Progenitor Cell ◽  
Erythroid Differentiation ◽  
Proteoglycan Synthesis ◽  
Hematopoietic Progenitor Cell ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Heparan sulfate (HS) proteoglycans of bone marrow (BM) stromal cells and their extracellular matrix are important components of the microenvironment of hematopoietic tissues and are involved in the interaction of hematopoietic stem and stromal cells. Although previous studies have emphasized the role of HS proteoglycan synthesis by BM stromal cells, we have recently shown that the human hematopoietic progenitor cell line TF-1 also expressed an HS proteoglycan. Immunochemical, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis of this HS proteoglycan showed that it was not related to the syndecan family of HS proteoglycans or to glypican. To answer the question of whether the expression of HS proteoglycans is associated with the differentiation state of hematopoietic progenitor cells, we have analyzed the proteoglycan synthesis of several murine and human hematopoietic progenitor cell lines. Proteoglycans were isolated from metabolically labeled cells and purified by several chromatographic steps. Isolation and characterization of proteoglycans from the cell lines HEL and ELM-D, which like TF-1 cells have an immature erythroid phenotype, showed that these cells synthesize the same HS proteoglycan, previously detected in TF-1 cells, as a major proteoglycan. In contrast, cell lines of the myeloid lineage, like the myeloblastic/promyelocytic cell lines B1 and B2, do not express HS proteoglycans. Taken together, our data strongly suggest that expression of this HS proteoglycan in hematopoietic progenitor cell lines is associated with the erythroid lineage. To prove this association we have analyzed the proteoglycan expression in the nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction of erythroid or granulocytic differentiation. Our data show that HS proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells. In contrast, during granulocytic differentiation, no expression of HS proteoglycans was observed.

Optimal Proliferation of a Hematopoietic Progenitor Cell Line Requires Either Costimulation With Stem Cell Factor or Increase of Receptor Expression That Can Be Replaced by Overexpression of Bcl-2

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2569-2577 ◽  
Author(s):  
Huei-Mei Huang ◽  
Jian-Chiuan Li ◽  
Yueh-Chun Hsieh ◽  
Hsin-Fang Yang-Yen ◽  
Jeffrey Jong-Young Yen
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Progenitor Cell ◽  
Stem Cell Differentiation ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Progenitor Cell Line

In vitro proliferation of hematopoietic stem cells requires costimulation by multiple regulatory factors whereas expansion of lineage-committed progenitor cells generated by stem cells usually requires only a single factor. The distinct requirement of factors for proliferation coincides with the differential temporal expression of the subunits of cytokine receptors during early stem cell differentiation. In this study, we explored the underlying mechanism of the requirement of costimulation in a hematopoietic progenitor cell line TF-1. We found that granulocyte-macrophage colony-stimulating factor (GM-CSF) optimally activated proliferation of TF-1 cells regardless of the presence or absence of stem cell factor (SCF). However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells and required costimulation of SCF for optimal proliferation. The synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor  subunit (IL5R) in TF-1 cells by genetic selection or retroviral infection also resumed optimal proliferation due to correction of the defect in apoptosis suppression. Exogenous expression of an oncogenic anti-apoptosis protein, Bcl-2, conferred on TF-1 cells an IL-5–dependent phenotype. In summary, our data suggested SCF costimulation is only necessary when the expression level of IL5R is low and apoptosis suppression is defective in the signal transduction of IL-5. Expression of Bcl-2 proteins released the growth restriction of the progenitor cells and may be implicated in leukemia formation.

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