CD34+/M-cadherin+ Bone Marrow Progenitor Cells Promote Arteriogenesis in Ischemic Hindlimbs of ApoE−/− Mice

BACKGROUND: Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34⁺/M-cad⁺ BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34⁺/M-cad⁺ BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. METHODS AND FINDINGS: Colony-forming cell assays and flow cytometry analysis showed that CD34⁺/M-cad⁺ BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE⁻/⁻ mice, CD34⁺/M-cad⁺ BMCs alleviated ischemia and significantly improved blood flow compared with CD34⁺/M-cad⁻ BMCs, CD34⁻/M-cad⁺ BMCs, or unselected BMCs. Significantly more arterioles were seen in CD34⁺/M-cad⁺ cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore, histologic assessment and morphometric analyses of hindlimbs treated with GFP⁺ CD34⁺/M-cad⁺ cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP⁺ CD34⁺/M-cad⁺ cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34⁺/M-cad⁺ progenitor cells. A cytokine antibody array revealed that CD34⁺/M-cad⁺ cell-conditioned medium contained higher levels of cytokines in a unique pattern, including bFGF, CRG-2, EGF, Flt-3 ligand, IGF-1, SDF-1, and VEGFR-3, than did CD34⁺/M-cad⁻ cell-conditioned medium. The proangiogenic cytokines secreted by CD34⁺/M-cad⁺ cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34⁺/M-cad⁻ cells during hypoxia. CONCLUSION: CD34⁺/M-cad⁺ BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE⁻/⁻ mice by consistently improving blood flow and promoting arteriogenesis. Additionally, CD34⁺/M-cad⁺ BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors

Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation

[Background] Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes.[Results] We used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 °C. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1Thr108) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins.[Conclusions] In this study we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.This work has been financially supported by grants AGL2013-47300-C3-3-R and PROMETEOII/2014/042 from the Spanish government and the Generalitat Valenciana, respectively, awarded to JMG. MLM and EGR also want to thank the Spanish government for her FPI grants. Analysis of yeast genomic sequences was made possible through the NIH P41 GM103533 grant funding the Yeast Resource Center. MJD is a Rita Allen Scholar and Fellow in the Genetic Networks program at the Canadian Institute for Advanced Research. MS was supported by NSF fellowship DGE-1256082