Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

BACKGROUND: Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34(+) cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion. METHODOLOGY/PRINCIPAL FINDINGS: CB CD34(+) cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34(+) cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone. CONCLUSION/SIGNIFICANCE: Our data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34(+) cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts

Pharmacological Inhibition of Caspase and Calpain Proteases: A Novel Strategy to Enhance the Homing Responses of Cord Blood HSPCs during Expansion

Background: Expansion of hematopoietic stem/progenitor cells (HSPCs) is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB) derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion. Methodology/Principal Findings: CB derived CD34 + cells were expanded using a combination of growth factors with and without Caspase inhibitor-zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homingrelated molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors) caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice

Acellular Bone Marrow Extracts Significantly Enhance Engraftment Levels of Human Hematopoietic Stem Cells in Mouse Xeno-Transplantation Models

Hematopoietic stem cells (HSC) derived from cord blood (CB), bone marrow (BM), or mobilized peripheral blood (PBSC) can differentiate into multiple lineages such as lymphoid, myeloid, erythroid cells and platelets. The local microenvironment is critical to the differentiation of HSCs and to the preservation of their phenotype in vivo. This microenvironment comprises a physical support supplied by the organ matrix as well as tissue specific cytokines, chemokines and growth factors. We investigated the effects of acellular bovine bone marrow extracts (BME) on HSC in vitro and in vivo. We observed a significant increase in the number of myeloid and erythroid colonies in CB mononuclear cells (MNC) or CB CD34+ cells cultured in methylcellulose media supplemented with BME. Similarly, in xeno-transplantation experiments, pretreatment with BME during ex-vivo culture of HSCs induced a significant increase in HSC engraftment in vivo. Indeed, we observed both an increase in the number of differentiated myeloid, lymphoid and erythroid cells and an acceleration of engraftment. These results were obtained using CB MNCs, BM MNCs or CD34+ cells, transplanted in immuno-compromised mice (NOD/SCID or NSG). These findings establish the basis for exploring the use of BME in the expansion of CB HSC prior to HSC Transplantation. This study stresses the importance of the mechanical structure and soluble mediators present in the surrounding niche for the proper activity and differentiation of stem cells

The role and potential of umbilical cord blood in an era of new therapies: a review

In light of pioneering findings in the 1980s and an estimation of more than 130 million global annual births, umbilical cord blood (UCB) is considered to be the most plentiful reservoir of cells and to have regenerative potential for many clinical applications. Although UCB is used mainly against blood disorders, the spectrum of diseases for which it provides effective therapy has been expanded to include non-hematopoietic conditions; UCB has also been used as source for regenerative cell therapy and immune modulation. Thus, collection and banking of UCB-derived cells have become a popular option. However, there are questions regarding the cost versus the benefits of UCB banking, and it also raises complex ethical and legal issues. This review discusses many issues surrounding the conservation of UCB-derived cells and the great potential and current clinical applications of UCB in an era of new therapies. In particular, we describe the practical issues inherent in UCB collection, processing, and long-term storage as well as the different types of ‘stem’ or progenitor cells circulating in UCB and their uses in multiple clinical settings. Given these considerations, the trend toward UCB will continue to provide growing assistance to health care worldwide

Recipient-Specific Tolerance after HLA-Mismatched Umbilical Cord Blood Stem Cell Transplantation

Background: Lower incidence and severity of acute graft versus host disease (GVHD) has been observed in leukemia patients receiving HLA-mismatched umbilical cord (UCB) transplants. However, despite the increased use of UCB in stem cell transplantation, the mechanisms underlying these favorable outcomes are not well delineated.Methods: We analyzed antigen specific lymphocyte responses after transplant to determine whether the decreased allogeneic responsiveness of UCB lymphocytes is attributable to pan-unresponsiveness, lymphocyte repressive or recipient-specific tolerance.Results: Circulating lymphocytes collected early (3 months) after UCB transplant demonstrate a less naive phenotype compared with that in the infused graft. Additionally, after transplant, circulating peripheral blood UCB-derived lymphocytes produced normal levels of interferon-[gamma] and proliferated normally when stimulated with mitogen or third party alloantigen. In contrast, when stimulated with recipient antigen, circulating lymphocytes emerging posttransplant did not proliferate nor produce interferon-[gamma]. Moreover, analysis of interleukin-4 production revealed a Th2 response to recipient antigens. These data indicate early induction of immune tolerance of naive UCB graft lymphocytes with skewing of transplant recipient-specific immune response towards Th2 cytokine profile.Conclusions: UCB graft lymphocyte immune naivety and observed early tolerance induction may contribute to the observed favorable GVHD incidence, despite infusion of HLA mismatch grafts in the unrelated allogeneic setting

Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes

Cellular therapy of Epstein-Barr virus (EBV)+ posttransplantation lymphoproliferative diseases (PTLD) in cord blood transplant (CBT) recipients is limited by lack of donor access and the donor's naive neonatal immune system. We therefore used partially human leukocyte antigen–matched third-party in vitro expanded EBV-specific cytotoxic T lymphocytes (CTLs) to treat 2 CBT recipients with life-threatening, donor-derived monoclonal EBV+ diffuse large B-cell lymphomas with extranodal involvement developing in the context of graft-versus-host disease. Both patients had failed immunosuppression taper and Rituximab. After 5 and 9 infusions of 106 EBV-CTL/kg, respectively, each patient achieved a sustained complete remission without toxicity or graft-versus-host disease. Each is alive without recurrence at 20 and 15 months, respectively, post–EBV-PTLD diagnosis. This approach demonstrates the efficacy of using “off-the-shelf,” virus-specific third-party CTLs restricted by human leukocyte antigens expressed by the tumor to treat otherwise lethal EBV-PTLD. Such therapy may also be applicable to the treatment of other infections and residual or recurrent malignancy after CBT