Expansion of Human Cord Blood Primitive Progenitors in Serum-Free Media Using Human Bone Marrow Mesenchymal Stem Cells (original) (raw)
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The comparison of different protocols for expansion of umbilical-cord blood hematopoietic stem cells
Journal of Cellular and Molecular Medicine, 2004
Hematopoiesis is maintained by the activity of multipotent stem cells, which have the dual capacity to self-renew and to differentiate into all of the blood cell lineages. The major challenge of stem cells based regenerative therapy is to expand ex vivo the primitive compartment to increase transplantable stem cells number. The present study was designed to evaluate several culture systems for in vitro maintenance of umbilical cord blood stem cells. The influences of different growth conditions such as stromal feeder layer, cytokines supplement and placental conditioned medium (PCM) have been evaluated over a relatively short period of time on CD34+ cell expansion and maintenance of clonogenic progenitors. When cells were expanded on feeder layer in the presence of added cytokines and PCM on average a 2.96-fold increase of CD34+CD71- and a 3.13-fold increase of CD34+HLA-DR- was observed. The total number of colony forming cells (35±2.65) indicated also that the yield of clonogenic progenitors obtained with a combination of all factors was two folds higher than each of these factors alone and ten time above control (3.67± 2.52). In conclusion, the results of our study clearly show that the ex vivo expansion of hematopoietic progenitor cells obtained from human umbilical cord blood is dependent on controlled experimental conditions, which might be helpful when designing culture systems for clinical applications.
Expansion of Non-Enriched Cord Blood Stem/Progenitor Cells CD34+ CD38-Using Liver Cells
2005
culture were examined with the goal of generating a suitable protocol for expanding hematopoietic stem cells for patient transplantation. Using primary fetal liver cells, we established a serum-free culture system to expand human primitive stem/progenitors cells. Non-enriched cord blood CD34 + cells were cultured on a monolayer of mouse primary fetal liver cells in the presence of trombopoietin, flt3/flk2 ligand, and/or stem cell factor, IL-6 and IL-3 under serum-free conditions. After 1 or 2 weeks of culture, cells were examined for clonogenic progenitors and percentage of CD34 + CD38cells. In the presence of trombopoietin, flt3/flk2 ligand, and stem cell factor, fetal liver cells supported expansion of CD34 + cells more than 10 to 20 fold. In addition, colony forming unit-cell assay was expanded more than 5-and 10-fold after 1 and 2 weeks of culture, respectively. These results strongly suggest that fetal liver cells may be a suitable feeder layer for expansion of hematopoietic progenitors from umbilical cord blood in vitro. Iran. Biomed. J. 9 (3): 111-116, 2005
Acta Haematologica, 2007
Umbilical cord blood is a promising source of hematopoietic stem cells (HSC) for allogeneic transplantation. However, graft rejection and delayed engraftment remain major limitations, both of which are related to a limited number of stem cells in the cord blood graft. Ex vivo expansion of HSC has been suggested as one of the ways of overcoming the challenges caused by a limited hematopoietic cell number from cord blood stem cell transplantation. In this study, we quantified and characterized an ex vivo expansion capacity of cord blood-derived HSC in a liquid culture system under different conditions. These conditions included: the combinations and concentrations of hematopoietic growth factors [stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and erythropoietin (EPO)], placental conditioning medium (PCM), and stromal cell support. During culture, the mean nucleated cell count, the mean CD34+ cell count, fold expansion, viability, clonogenic assays and immunophenotypic characterization were performed on day 0, day 7, day 12 and day 14 on the expanded cellular product. The maximum expansion was achieved using GF2 (SCF + IL-3 + GM-CSF) with stromal cell support. The mean CD34+ cell expansion on days 7 and 12 was 16.25- and 21.4-fold (5.2-32), respectively, and the mean nucleated cell expansion was 15.1- and 21-fold (18.1-23.2). The mean nucleated cell viability on day 12 was 87.9% (85.6-92.5). After 12 days, granulocyte-macrophage colony-forming units CFU-GEMM showed a 20.4-fold increase. A 21.4-fold increase in the CD34+ cells and a 20-fold increase in the CFU-GEMM should provide enough cells from a single cord blood unit to reduce the period of cytopenia after single unit cord blood transplantation. Even if there was some doubt about the long-term repopulating capacity of the expanded cells part of the collected umbilical cord cells (25%) could be expanded till day 12 after transplanting the major part (75%) of the collection.
International Journal of Molecular Sciences
Umbilical cord blood (UCB) serves as a source of hematopoietic stem and progenitor cells (HSPCs) utilized in the regeneration of hematopoietic and immune systems, forming a crucial part of the treatment for various benign and malignant hematological diseases. UCB has been utilized as an alternative HSPC source to bone marrow (BM). Although the use of UCB has extended transplantation access to many individuals, it still encounters significant challenges in selecting a histocompatible UCB unit with an adequate cell dose for a substantial proportion of adults with malignant hematological diseases. Consequently, recent research has focused on developing ex vivo expansion strategies for UCB HSPCs. Our results demonstrate that co-cultures with the investigated mesenchymal stromal cells (MSCs) enable a 10- to 15-fold increase in the cellular dose of UCB HSPCs while partially regulating the proliferation capacity when compared to HSPCs expanded with early acting cytokines. Furthermore, the ...
Bone Marrow Transplantation, 2006
One factor limiting the therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is the low cell dose of the graft. This is associated with an increased incidence of delayed or failed engraftment. Cell dose can be increased and the efficacy of CB transplantation potentially improved, by ex vivo CB expansion before transplantation. Two ex vivo CB expansion techniques were compared: (1) CD133 + selection followed by ex vivo liquid culture and (2) co-culture of unmanipulated CB with bone-marrow-derived mesenchymal stem cells (MSCs). Ex vivo culture was performed in medium supplemented with granulocyte colony-stimulating factor, stemcell factor and either thrombopoietin or megakaryocyte growth and differentiation factor. Expansion was followed by measuring total nucleated cell (TNC), CD133 + and CD34 + cell, colony-forming unit and cobblestone area-forming cell output. When compared to liquid culture, CB-MSC co-culture (i) required less cell manipulation resulting in less initial HPC loss and (ii) markedly improved TNC and HPC output. CB-MSC co-culture therefore holds promise for improving engraftment kinetics in CB transplant recipients.
Clinical and Laboratory Haematology, 2007
Progenitor cells (CD34+) can be isolated from umbilical cord blood and used to correct or reconstitute various cell lines within the haematopoietic and endothelial cell lineage. The main disadvantage of this procedure relates to the low volume of blood that can be collected after the umbilical cord has been clamped, which limits the number of progenitor cells available for treatment. This limitation, however, can be overcome by expanding CD34+ cells ex vivo. Our aim was to perform a controlled study to determine if the ex-vivo proliferation of umbilical cord CD34+ cells is enhanced when they are placed in a system that mimics the bone marrow microenvironment. For this purpose, CD34+ cells were isolated from umbilical cord blood using a magnetic cell sorting kit and seeded in platforms containing different cocktails of cytokines with and without a three-dimensional (3D) biomatrix. Results from this study suggest that the number of viable cells can double after 1 week in any of the culture platforms and that the 3D biomatrix does not enhance cell proliferation.
Journal of Cellular Biochemistry, 2011
Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal-based serum-free culture system to evaluate the effect of different initial CD34 þ cell enrichments (Low: 24 AE 1.8%, Medium: 46 AE 2.6%, and High: 91 AE 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34 þ and CD34 þ CD90 þ expression, we have identified early activation of CD34 expression on CD34 À cells in Low and Medium conditions, prior to cell division (35 AE 4.7% and 55 AE 4.1% CD34 þ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34 þ /CD34 þ CD90 þ cell yield (High: 14 AE 1.0/3.5 AE 1.4-fold; Medium:22 AE 2.0/3.4 AE 1,0-fold; Low:31 AE 3.0/4.4 AE 1.5-fold) after a 7-day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34 þ cell recovery for each strategy, on overall CD34 þ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34 þ cell numbers after 7 days of expansion (30 AE 2.0 vs. 13 AE 1.0 Â 10 6 cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex-vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost-effective expansion of HSC for cellular therapy.
UMBILICAL CORD BLOOD AS A SOURCE OF HEMATOPOIETIC STEM CELLS: FROM RESEARCH TO CLINICAL APPLICATION
The clonogenic capacity of human umbilical cord blood (UCB) has been evaluated in several studies which found high numbers of primitive hematopoietic progenitor cells. Recently, UCB progenitor cells were shown to possess significant advantages over bone marrow (BM) in terms of proliferative capacity and immunologic reactivity. Therefore UCB has come to be considered an attractive source of hematopoietic stem cells for both research and clinical applications. UCB has been used in the treatment of diseases potentially curable by bone marrow transplantation (BMT). Seventy-one transplants have been performed world-wide using UCB cells, and the results have been reported to the International Cord Blood Transplant Registry (ICBTR). Since UCB cells appear to be less alloreactive than BM cells, studies are under way to determine the feasibility of UCB banking for use in unrelated transplants. Because of the limited volume of UCB that can be obtained in a single collection, studies have been carried out to determine the most successful procedures for collection and fractionation of UCB and to quantify precisely the progenitor/stem cell content.