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Increasing the Survival of ASCs to Promote Regeneration

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Enhancing In Vivo Survival of Adipose-Derived Stromal Cells Through Bcl-2 Overexpression Using a Minicircle Vector

From Stem Cells Translational Medicine

Survival of therapeutically relevant multipotent stem cells, such as human adipose-derived stromal cells (hASCs), is currently a significant hurdle to stem cell therapy.   This barrier is primarily caused by environmental stresses on transplanted cells by the often refractory cellular environment on the transplant site (Allen et alBauer et al and Broughton et al).   This often leads to apoptosis (Zhang et al) and poor engraftment (Wu et al), and therefore it has been postulated that modulating the apoptosis pathway may enhance the therapeutic worth of stem cells.   In a study published in Stem Cells TM, the group of Michael T. Longaker at the Stanford University School of Medicine, USA have studied the overexpression of the Bcl-2 gene, known to imbue survival advantages (Domen and Domen and Weissman), in hASCs and demonstrated an increased level of tissue regeneration which can be translated to a clinical setting (Hyun et al).

hASCs adenoviral transduced with Bcl-2 had significantly higher Bcl-2 mRNA and protein than control transduced hASCs or hASCs transduced with a short hairpin against Bcl2 (shBcl-2) to promote silencing which was further confirmed through histological analysis of ASCs transplanted into a calvarial defect.   Furthermore, apoptosis was lower in vitro in Bcl-2 ASCs while proliferation was only affected by overexpression/silencing slightly suggesting that any Bcl-2 survival effects were not due to increased proliferation.   Further apoptosis analysis using a protein array to assess 35 apoptosis-related proteins demonstrated that increased Bcl-2 levels mediated an increase in the prosurvival X-linked inhibitor of apoptosis (XIAP) while levels of the apoptotic mediators cytochrome c, second mitochondria-derived activator of caspases (Smac/DIABLO) and Caspase 3 were significantly decreased.   mRNA analysis in vitro confirmed the increase in XIAP in response to Bcl-2 overexpression and also demonstrated a lower level for Bcl-2-associated X protein (BAX), which also promotes mitochondrial apoptosis.   Decreased Smac/DIABLO transcripts were also confirmed, alongside the decrease of other apoptotic mediators (APAF-1, Caspase 3 and Caspase 7), while at the protein level the reduction of BAX, APAF-1, cytochrome C, Smac/DAIBLO, Caspase 3 and Caspase 7 in response to Bcl-2 overexpression was confirmed.

The group then found that this increased viability and reduction in apoptosis through Bcl-2 expression led to an increase in the early survival of the ASCs after implantation with a scaffold in vivo.   When placed in a calvarial defect, Bcl-2 ASCs survived better up to 2 weeks and, excitingly, allowed for greater osteogenic regeneration at 2 and 4 weeks, due in part to an observed decrease in apoptosis and increase in survival with in vitro RNA and protein findings confirmed.   These in vivo findings were then further confirmed in a mouse excisional soft tissue wound healing model, where Bcl-2 overexpression again mediated higher cell survival and correlated with accelerated wound healing rates leading to a complete wound closure at day 12 compared to day 14 for control implanted cells.  Finally, a non-integrating minicircle vector that expressed Bcl-2 was tested through a double transfection strategy.  This gave a 25-35% efficiency of transfection with mRNA and protein analysis demonstrating a significant increase in BCL-2 levels in ASCs and ultimately allowed for increased skeletal regeneration at 1, 2 and 4 weeks.

In conclusion, this study suggests that the apoptosis in therapeutically relevant transplanted cells, which occurs in part due to the refractory nature of the wound environment, plays a significant role in the failure of regenerative therapies, as inhibition of apoptosis allows for better therapeutic benefits.   This has been shown before for hematopoietic stem cells (Imai et al) and neural stem cells (Micci et al), but the authors apply this to ASCs, a good source of adult stem cells seen as, by many, as one of the most clinically relevant stem cell types.    The incorporation of a non-integrating minicircle vector further enhances the clinical relevance of this study, although certain safety aspects must be addressed first.   Cells which apoptose are generally dysfunctional but also potentially tumourigenic, and the inhibition of apoptosis could potentially allow for the outgrowth of harmful cell populations (Madyd et al).   The self-limiting nature of non-integrating means of gene expression should avoid tumourigenic potential but this still has to be fully assessed.

 

References

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  • Domen J, Weissman IL (2003) Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpression bcl-2. Exp Hematol 31:631–639
  • Imai Y et al (2010) Caspase inhibitor ZVAD-fmk facilitates engraftment of donor hematopoietic stem cells in intra-bone marrow-bone marrow transplantation. Stem Cells Dev 19:461–468
  • Madjd Z et al. (2009) CD44+ cancer cells express higher levels of the anti-apoptotic protein Bcl-2 in breast tumors. Cancer Immun 9:4
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  • Wu Y et al (2007) Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 25:2648–2659.
  • Zhang M et al. (2001) Cardiomyocyte grafting for cardiac repair: Graft cell death and anti-death strategies. J Mol Cell Cardiol 33:907–921

 

 Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.