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No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34(+) Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice.

ABSTRACT: After cord blood (CB) transplantation, early platelet recovery in immune-deficient mice is obtained by expansion of CB CD34(+) cells with thrombopoietin (TPO) as single growth factor. Moreover, improvement of hematopoietic engraftment has been shown by cotransplantation of mesenchymal stem cells (MSC). We investigated whether a combination of both approaches would further enhance the outcome of CB transplantation in NOD SCID mice. NOD SCID mice were transplanted with either CB CD34(+) cells, CD34(+) cells with MSC, TPO-expanded CD34(+) cells or TPO-expanded CD34(+) cells with MSC. We analyzed human platelet recovery in the peripheral blood (PB) from day 4 after transplantation onward and human bone marrow (BM) engraftment at week 6. The different transplants were assessed in vitro for their migration capacity and expression of CXCR4. TPO expansion improved the early platelet recovery in the PB of the mice. Cotransplantation of MSC with CD34(+) cells improved BM engraftment and platelet levels in the PB 6 weeks after transplantation. Combining TPO expansion and MSC cotransplantation, however, neither resulted in a more efficient early platelet recovery, nor in a better BM engraftment, nor even very low or absent BM engraftment occurred. In vitro, MSC boosted the migration of CD34(+) cells, suggesting a possible mechanism for the increase in engraftment. Our results show that cotransplantation of MSC with TPO-expanded CD34(+) cells at most combines, but does not increase the separate advantages of these different strategies. A combination of both strategies even adds a risk of non engraftment.

SUBMITTER: van der Garde M

PROVIDER: S-EPMC4485368 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34(+) Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice.

van der Garde Mark M Brand Anneke A Slot Manon C MC de Graaf-Dijkstra Alice A Zwaginga Jaap Jan JJ van Hensbergen Yvette Y

Stem cells and development 20150313 12

After cord blood (CB) transplantation, early platelet recovery in immune-deficient mice is obtained by expansion of CB CD34(+) cells with thrombopoietin (TPO) as single growth factor. Moreover, improvement of hematopoietic engraftment has been shown by cotransplantation of mesenchymal stem cells (MSC). We investigated whether a combination of both approaches would further enhance the outcome of CB transplantation in NOD SCID mice. NOD SCID mice were transplanted with either CB CD34(+) cells, CD3 ...[more]

PMID: 25668618

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Project description:BackgroundCord 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 findingsCB 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/significanceOur 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.

Project description:BackgroundPoor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo.MethodsHuman MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice.ResultsWe did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively).ConclusionsCo-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.

Dataset Information

No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34(+) Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice.

Publications

No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34(+) Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice.

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