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Stromal Progenitor Ligands Promote Cardiac Stem-Progenitor Grafts

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Review of "Priming with Ligands Secreted by Human Stromal Progenitor Cells Promotes Grafts of Cardiac Stem/Progenitor Cells After Myocardial Infarction" from Stem Cells by Stuart P. Atkinson

Poor cellular engraftment remains a serious problem for the therapeutic utility of stem cells and their derivatives [1, 2], although multiple studies have shown that the paracrine action of co-transplanted mesenchymal stem cells (MSCs) can improve integration levels [3] . The laboratory of Jeffrey L. Spees at the University of Vermont, USA now report on their work, studying the effects of ligands produced by MSCs on the proliferation and survival of adult rat cardiac stem cells (CSCs) and  their effects on cellular engraftment [4].

The group first treated CSCs with conditioned medium (CM) collected from human MSCs, p75-sorted MSCs (p75 - low-affinity nerve growth factor receptor – this population of MSCs has a reported increased regenerative capacity [3, 5]) and from dermal fibroblasts. 10×-concentrated CM (10×CM) from either MSC population induced proliferation, compared to a control non-conditioned serum-free media (SFM), mediating increased cell number, BrdU incorporation, and Ki67 expression. MSC-CM also induced nuclear STAT3-phosphorylation (p-STAT3), which is linked to the self-renewing state in multiple stem cell types. Phospho-Akt was induced in response to CM from all sources, but also by the SFM control. Inhibition of either the phosphorylation of STAT3 or Akt by pharmacological inhibition led to a reduction in CM-mediated proliferative effects, while inhibition of both led to a cumulative repressive effect. Additional studies found that CSCs did not proliferate in the absence of Insulin, even in the presence of other mitogenic components, suggesting that CM from the MSCs contains Insulin and/or Insulin-like growth factor 1 (IGF-1). CM could also protect CSCs from hypoxia, as would be encountered in the heart following myocardial infarction (MI), increasing cell survival from around 48% in the SFM control to over 80% with MSC-CM, with this increase abolished by STAT3 inhibition.

In vivo studies next studied endogenous CSCs after the infusion of CM one day after MI in mice. Analysis after a further day found that CM reduced cardiac cell apoptosis/necrosis, although biochemical assays found that similar size infarcts at 1 week in CM- and SFM-treated mice. Rare putative cKit+ CSCs were apparent after CM treatment although appearance and number was highly variable.  These findings led the researchers to ask whether priming of CSCs with p75MSC-CM and subepicardial injection in rat hearts after MI could improve graft success better than simple infusion. At 1 week after control (CSC+SFM) injection, no CSCs (labelled with GFP) were apparent in two animals, while the highest engraftment was very low (~40 cells). However, CSCs primed with p75MSC-CM led to the grafting of several thousand CSCs into subepicardial locations, as well as proliferation, migration into zones with infarction, integration into blood vessel walls as CD31-positive endothelial cells and the generation of smooth muscle cells and myofibroblasts. At one month the researchers detected CSCs in infarct and border zones in multiple tissue sections and cells which had differentiated into CD31-positive endothelial cells.

To understand the important factors within p75MSC-CM, the researchers utilised Affymetrix chip gene expression profiles [5] and antibody blocking/neutralization studies using 10×p75MSC-CM and CSCs under hypoxic conditions. These studies identified human Connective Tissue Growth Factor (CTGF) and Insulin as being vitally important. Blocking CTGF reduced p-STAT3 in CSCs, while CTGF addition to SFM boosted p-STAT3 levels. Meanwhile blockage of Insulin led to a reduction in the protective abilities of CM on CSCs, while addition of Insulin boosted p-AKT levels and CSC survival and proliferation under hypoxic conditions. Experimentation with recombinant peptides discovered that the c-terminal domain of CTGF (CTGF-D4), which is associated with cell adhesion and proliferation [6, 7], when added to CSCs alongside Insulin, was sufficient to promote survival and proliferation under hypoxia. Priming of CSCs ex vivo with CTGF-D4 and Insulin and injection after MI led to levels of engraftment similar to that of p75MSC-CM priming with a similar distribution, migration and differentiation of CSCs.

Previously published wide-ranging preconditioning strategies have been shown to modestly increase the survival of adult stem/progenitor cells, ESCs, and ESC-derivatives after transplantation to the heart (see original paper for extensive references). This study identifies a simple and seemingly effective method for improving grafting of cardiac stem/progenitor cells, which may prove to be more successful than previous strategies. Additionally, the tangential injection method to deliver CSCs to the subepicardial space lying outside and adjacent to the injured myocardium, may also prove to be more efficacious than intravenous, intra-arterial, or intramuscular cell injection. Looking ahead, the authors also hope that CM or CTGF-D4/Insulin may prove successful in improving graft success in other tissues following injury.

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