Expansion Of Cord Blood-derived CD34 Research Articles

Potential of Polycaprolactone Nanofiber Scaffold for Ex Vivo Expansion of Cord Blood-Derived CD34+ Hematopoietic Stem Cells

An efficient and practical ex vivo expansion of cord blood hematopoietic stem cells as an alternative source of HSC transplantation is crucial in understanding the potential of HSC transplantation in treating or supportive therapy in a variety of hematologic and non-hematologic disorders.

A hyaluronic acid/graphene oxide hydrogel for enhanced ex vivo expansion of cord blood-derived CD34+ cells

HA/GO hydrogels were prepared by methacrylated GO (MeGO) and methacrylated HA (MeHA) via an in-situ free radical polymerization, and then a series of HA/GO hydrogels with highly porous structure were obtained. All hydrogels exhibited excellent water absorption ability. Additionally, the elastic moduli of hydrogels were clearly enhanced by GO. CD34+ cells cultured on HA/GO-0.05 hydrogel showed higher expansion of CD34+ cells and CD34+CD38− cells compared to the other three hydrogels. Therefore, HA/GO-0.05 hydrogels have a potential for clinical-applied ex vivo expansion of hematopoietic stem cells.

Biomimetic Macroporous PCL Scaffolds for Ex Vivo Expansion of Cord Blood-Derived CD34+ Cells with Feeder Cells Support.

Ex vivo expansion of hematopoietic stem cells (HSCs) with most current methods can hardly satisfy clinical application requirement. While in vivo, HSCs efficiently self-renew in niche where they interact with 3D extracellular matrix and stromal cells. Therefore, co-cultures of CD34+ cells and mesenchyme stem cells derived from human amniotic membrane (hAMSCs) on the basis of biomimetic macroporous three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds are developed, where scaffolds and hAMSCs are applied to mimic structural and cellular microenvironment of HSCs. The influence of scaffolds, feeder cells, and contact manners on expansion and stemness maintenance of CD34+ cells is investigated in this protocol. Biomimetic scaffolds-dependent co-cultures of CD34+ cells and hAMSCs can effectively promote the expansion of CD34+ cells; meanwhile, indirect contact is superior to direct contact. The combination of biomimetic scaffolds and hAMSCs represents a new strategy for achieving clinical-scale ex vivo expansion of CD34+ cells.

432. Valproic Acid Treatment Enhances Hematopoietic Stem and Progenitor Cell Multipotency Ex Vivo for Enhanced Long-Term Engraftment of Gene-Modified Cells

The promising field of genome editing in hematopoietic stem and progenitor (HSPC) for use in autologous and allogeneic transplantation therapies relies on being able to engraft the edited cells into the bone marrow and to have those engrafted cells produce all the hematopoietic lineages necessary for proper immune and red blood cell function. Depending on the cell source to be used in the editing process, the fraction of CD34+ HSPCs can be quite low - approximately 0.0005%, 0.01%, or 0.1% for mobilized peripheral blood (mPB), bone marrow aspirate (BM), or cord blood, respectively. The fraction of long-term repopulating true stem cells (LT-HSCs) within these CD34+ cell populations, capable of long-term reconstitution of the entire hematopoietic lineage after transplantation, is even lower (<1%). In addition, it has recently been shown that LT-HSCs derived from cord blood can be much less permissive to homology-directed repair (HDR)-driven gene correction which may be essential for some types of therapeutic genome editing. Several groups have discovered small molecules that promote expansion of cord blood-derived CD34+ HSPCs ex vivo while maintaining the stemness of the HSPCs, however these effects have not been reported in HSPCs from mPB or BM. Here we show that the small-molecule epigenetic modifier valproic acid (VPA) improves HDR-mediated targeted integration (TI) in mPB and BM HSPCs. In addition to increasing the fraction of LT-HSCs with TI by up to 100-fold, VPA also dramatically increases the overall number of cells expressing LT-HSC markers (CD34+CD133+CD90+CD49f+ or CD34+CD38-CD45RA-CD90+CD49f+) by up to 500-fold. VPA-treated gene-edited HSCs differentiate normally in vitro and retain consistent levels of TI in both erythroid and myeloid lineages. These results further the development of genome-edited mPB and BM-derived HSPC therapies.

Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34(+) Cells.

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+ cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

Encapsulated feeder cells within alginate beads for ex vivo expansion of cord blood-derived CD34(+) cells.

A co-culture system based on encapsulated feeder cells within alginate beads was developed through optimizing the detailed aspects of the cell culture system to expand CD34-positive (CD34(+)) cells ex vivo. Mesenchymal stem cells isolated from different sources (human amniotic (hAMSCs) and umbilical cord (UCMSCs)), and human fibroblast cells (HFs) have been respectively chosen as feeder cells and the results showed that the hAMSCs were superior to UCMSCs and HFs in conventional two-dimensional (2D) co-cultures regarding the promotion of total nucleated cell (TNCs) expansion and the maintenance of the CD34(+) phenotype. Alginate beads were employed to limit the growth of hAMSCs, which could effectively restrict the proliferation of the encapsulated hAMSCs, while the cell viability of hAMSCs was still highly maintained. Intriguingly, only a few hAMSCs migrated out of the alginate beads, whereas secreted growth factors and cytokines could be easily released. Furthermore, the alginate beads supported CD34(+) cells/hAMSCs 2D indirect co-culture exhibited increased TNCs expansion, higher percentages of CD34(+) and CD34(+)CD38(-) cells, and better cell vitality when compared to the 2D co-culture. Therefore, the co-culture system based on encapsulated hAMSCs within alginate beads can effectively promote CD34(+) cells to expand ex vivo.

Towards the Development of a Closed, Nanofiber-Based Culture System for Clinical Expansion of Cord Blood-Derived CD34+ Cells

AbstractAbstract 4411Interest in ex vivo hematopoietic stem and progenitor cell (HSPC) expansion has increased in recent years due to the growing importance of these cells in the treatment of a variety of both malignant and non-malignant diseases. Ex vivo expansion of cord blood-derived cells has been particularly investigated because cord is a valuable and readily available source of HSPCs, yet contains limited numbers of cells in each unit. Despite these efforts, most attempts to use expanded cord blood HSPCs in the clinic have been unsuccessful due to the generation of insufficient numbers of cells with the appropriate phenotype and the ability to function in vivo.In many ex vivo culture systems, HSPCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSPCs with a substrate. In the natural bone marrow microenvironment, HSPCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D nanofiber-based cell culture substrate (NANEX™). The functionalized NANEX™ substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSPC proliferation while minimizing differentiation.Here, we present our recent work towards developing a closed, NANEX™-based platform for large-scale clinical expansions of cord blood-derived CD34+ cells. We demonstrate that NANEX™ expands CD34+ cells from cord an average of more than 150-fold in 10 day culture, which is at least 2-fold higher than that obtained in standard tissue culture plates. Additionally, we show an approximately 1.5-fold higher proliferation of colony forming cells and a significantly higher engraftment rate in NSG mice for NANEX™-expanded cells compared to cells cultured in tissue culture plates. Furthermore, we demonstrate that the NANEX™ scaffold maintains its HSPC growth promoting characteristics after processing into a closed culture system and offers significant advantages over other culture platforms typically used for HSPC expansions in the clinic (culture bags and T-flasks). Our data indicates that NANEX™ technology provides a robust ex vivo expansion of cord blood HSPCs and, with further development, offers great potential for clinical applications requiring large numbers of functional cells. Disclosures:No relevant conflicts of interest to declare.

The Effect of Human Bone Marrow Stroma-Derived Heparan Sulfate on the Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells

In order to address cell dose limitations associated with the use of cord blood hematopoietic stem cell (HSC) transplantation, we explored the effect of bone marrow stroma-derived heparan sulfate (HS) on the ex vivo expansion of HSCs. Heparan sulfate was isolated and purified from the conditioned media of human bone marrow stromal cells and used for the expansion of cord blood-derived CD34(+) cells in the presence of a cocktail of cytokines. The number of myeloid lineage-committed progenitor cells was increased at low dosage of HS as illustrated by an increase in the total number of colony-forming cells (CFC) and colonies of erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) precursors. Notably, the stroma-derived HS did not alter the growth of CD34(+) HSCs or negatively affect the levels of various HSC phenotypic markers after expansion. This study shows that HS secreted into solution by stromal cells has the capacity to support hematopoietic cytokines in the maintenance and expansion of HSCs. The incorporation of stroma-derived HS as a reagent may improve the efficacy of cord blood HSC transplantation by enhancing the number of committed cells and accelerating the rate of engraftment.

Linear polyamine copper chelator tetraethylenepentamine augments long-term ex vivo expansion of cord blood-derived CD34+ cells and increases their engraftment potential in NOD/SCID mice

ObjectiveWe previously demonstrated that cellular copper is involved in the regulation of proliferation and differentiation of hematopoietic progenitor cells. Modulation of cellular copper was achieved by supplementing the culture with a copper chelator that reduces cell copper content, or copper salts, which elevate the level of cellular copper. In the present study, we evaluated the effect of short-term (3-week) treatment with the copper chelator tetraethylenepentamine (TEPA) on short- and long-term (up to 11 weeks) ex vivo expansion of hematopoietic progenitors, as well as on their SCID engraftment potential. Materials and methodsCord blood-derived purified CD34+ cells were grown in liquid medium supplemented with the cytokines stem cell factor, thrombopoietin, Flt3 ligand, and IL-6, and the chelator TEPA for the first 3 weeks and then for up to 11 weeks with cytokines alone. Control cultures were supplemented with cytokines alone for the entire culture duration. Cultured cells were characterized by immunophenotyping and cloning (CFUc). Transplantability was assayed by injection of repurified CD34+ cells into NOD/SCID mice. ResultsIn the short term, TEPA supported increased percentages of early progenitors over control cultures incubated with cytokines alone (CD34+CD38−, p=0.001 and CD34+Lin−, p=0.016). In the long term, TEPA pretreated cultures showed prolonged expansion of CD34+ cells (p=0.01) and CFUc (p=0.002) compared with that of untreated cultures. The SCID engraftment potential of CD34+ cells repurified from the TEPA-treated cultures was higher compared with that of the control, i.e., only cytokine-treated cultures (p=0.03). ConclusionTEPA enabled preferential proliferation of early progenitor cells with the phenotype CD34+CD38− and CD34+CD38− Lin− during the first weeks of culture, resulting in the observed increased long-term ex vivo expansion and engraftment capabilities.