Key Points
Delivery of ZFNs and donor templates results in high levels of gene correction in human CD34+ cells from multiple sources, including SCD BM. Modified CD34+ cells are capable of engrafting immunocompromised NSG mice and produce cells from multiple lineages.
The purpose of this study was to permit bone marrow mesenchymal stem cells (BMSCs) to reach their full potential in the treatment of chronic wounds. A biocompatible multifunctional crosslinker based temperature sensitive hydrogel was developed to deliver BMSCs, which improve the chronic inflammation microenvironments of wounds. A detailed in vitro investigation found that the hydrogel is suitable for BMSC encapsulation and can promote BMSC secretion of TGF-β1 and bFGF. In vivo, full-thickness skin defects were made on the backs of db/db mice to mimic diabetic ulcers. It was revealed that the hydrogel can inhibit pro-inflammatory M1 macrophage expression. After hydrogel association with BMSCs treated the wound, significantly greater wound contraction was observed in the hydrogel + BMSCs group. Histology and immunohistochemistry results confirmed that this treatment contributed to the rapid healing of diabetic skin wounds by promoting granulation tissue formation, angiogenesis, extracellular matrix secretion, wound contraction, and re-epithelialization. These results show that a hydrogel laden with BMSCs may be a promising therapeutic strategy for the management of diabetic ulcers.
To study functions of late embryogenesis abundant (LEA) proteins, which accumulate in plant cells under water deficit conditions, in vivo functional analyses were carried out using a yeast (Saccharomyces cerevisiae) heterologous expression system. Two lea genes, tomato le4 (group 2) and barley HVA1 (group 3), were expressed under the GAL1 promoter, and the gene products were detected using specific antisera. The growth of the transformants was scored and compared with a control strain to analyze the effect of these proteins on yeast cells under stress conditions. The yeast cells expressing HVA1 showed shorter lag period when transferred to a medium containing 1.2 M NaCl as compared to a control strain, while the cells expressing le4 did not show improved growth. Attenuated growth inhibition in a medium containing 1.2 M KCl was observed in the yeast cells expressing le4 and HVA1. No obvious growth improvement was observed in a high sorbitol medium in the cells expressing either le4 or HVA1. Increased freezing tolerance was observed in both lea-expressing cells, while no effect on heat tolerance was observed. These results support the hypothesis that different LEA proteins play a distinctive role in the protection against cellular dehydration.
Pericytes and other perivascular stem/stromal cells are of growing interest in the field of tissue engineering. A portion of perivascular cells are well recognized to have MSC (mesenchymal stem cell) characteristics, including multipotentiality, self-renewal, immunoregulatory functions, and diverse roles in tissue repair. Here, we investigate the differential but overlapping roles of two perivascular cell subsets in paracrine induction of bone repair. CD146+CD34−CD31−CD45−pericytes and CD34+CD146−CD31−CD45−adventitial cells were derived from human adipose tissue and applied alone or in combination to calvarial bone defects in mice. In vitro, osteogenic differentiation and tubulogenesis assays were performed using either fluorescence activated cell sorting-derived CD146+ pericytes or CD34+ adventitial cells. Results showed that CD146+ pericytes induced increased cord formation in vitro and angiogenesis in vivo in comparison with patient-matched CD34+ adventitial cells. In contrast, CD34+ adventitial cells demonstrated heightened paracrine-induced osteogenesis in vitro. When applied in a critical-size calvarial defect model in NOD/SCID mice, the combination treatment of CD146+ pericytes with CD34+ adventitial cells led to greater re-ossification than either cell type alone. In summary, adipose-derived CD146+ pericytes and CD34+ adventitial cells display functionally distinct yet overlapping and complementary roles in bone defect repair. Consequently, CD146+ pericytes and CD34+ adventitial cells may demonstrate synergistic bone healing when applied as a combination cellular therapy.
Mesenchymal stem cells (MSCs) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether SDF-1 transfection improve MSC viability and paracrine action in infarcted hearts. We found SDF-1-modified MSCs effectively expressed SDF-1 for at least 21 days after exposure to hypoxia. The apoptosis of Ad-SDF-1-MSCs was 42% of that seen in Ad-EGFP-MSCs and 53% of untreated MSCs. In the infarcted hearts, the number of DAPI-labeling cells in the Ad-SDF-1-MSC group was 5-fold that in the Ad-EGFP-MSC group. Importantly, expression of antifibrotic factor, HGF, was detected in cultured MSCs, and HGF expression levels were higher in Ad-SDF-MSC-treated hearts, compared with Ad-EGFP-MSC or control hearts. Compared with the control group, Ad-SDF-MSC transplantation significantly decreased the expression of collagens I and III and matrix metalloproteinase 2 and 9, but heart function was improved in d-SDF-MSC-treated animals. In conclusion, SDF-1-modified MSCs enhanced the tolerance of engrafted MSCs to hypoxic injury in vitro and improved their viability in infarcted hearts, thus helping preserve the contractile function and attenuate left ventricle (LV) remodeling, and this may be at least partly mediated by enhanced paracrine signaling from MSCs via antifibrotic factors such as HGF.
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