Optimization of Genetic Engineering and Homologous Recombination of Collagen Type I Genes in Rat Bone Marrow Mesenchymal Stem Cells (MSC)

Tarnowski, Maciej; Szydło, Anna; Anioł, Jacek; Koryciak-Komarska, Halina; Lesiak, Marta; Gutmajster, Ewa; Sieroń, Aleksander; Kusz, Damian

doi:10.1089/cell.2009.0084

Cited by 11 publications

(9 citation statements)

References 35 publications

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“…This observation that stiffer, uninduced MSCs exhibit limited potency and osteogenic commitment is consistent with other related works that showed the tendency of attached and undifferentiated stem cells to become stiffer during differentiation (36) (50), low mechanical stiffness (E lo ), and high relative nuclear fluctuation (NF hi ) (color-coded green). In contrast, subpopulations that did not meet this criterion (i.e., those that were either large or of high mechanical stiffness or of low relative nuclear fluctuation) were only bipotent (color-coded red).…”

Section: Si Appendixsupporting

confidence: 92%

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

Lee

Shi

Poon

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

154

153

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The capacity to produce therapeutically relevant quantities of multipotent mesenchymal stromal cells (MSCs) via in vitro culture is a common prerequisite for stem cell-based therapies. Although culture expanded MSCs are widely studied and considered for therapeutic applications, it has remained challenging to identify a unique set of characteristics that enables robust identification and isolation of the multipotent stem cells. New means to describe and separate this rare cell type and its downstream progenitor cells within heterogeneous cell populations will contribute significantly to basic biological understanding and can potentially improve efficacy of stem and progenitor cell-based therapies. Here, we use multivariate biophysical analysis of culture-expanded, bone marrow-derived MSCs, correlating these quantitative measures with biomolecular markers and in vitro and in vivo functionality. We find that, although no single biophysical property robustly predicts stem cell multipotency, there exists a unique and minimal set of three biophysical markers that together are predictive of multipotent subpopulations, in vitro and in vivo. Subpopulations of culture-expanded stromal cells from both adult and fetal bone marrow that exhibit sufficiently small cell diameter, low cell stiffness, and high nuclear membrane fluctuations are highly clonogenic and also exhibit gene, protein, and functional signatures of multipotency. Further, we show that high-throughput inertial microfluidics enables efficient sorting of committed osteoprogenitor cells, as distinct from these mesenchymal stem cells, in adult bone marrow. Together, these results demonstrate novel methods and markers of stemness that facilitate physical isolation, study, and therapeutic use of culture-expanded, stromal cell subpopulations.

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Section: Si Appendixsupporting

confidence: 92%

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

Lee

Shi

Poon

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

154

153

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“…However, the expression decreased with a prolonged time after selection. Other studies also established a method for transfecting the human VEGF 165 gene into MSCs in which the target gene and protein could be expressed effectively (Guo-ping et al 2010;Tarnowski et al 2010).…”

Section: Discussionmentioning

confidence: 99%

VEGF165 expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro

Yan

Xiao

et al. 2012

Cytotechnology

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A short half-life and low levels of growth factors in an injured microenvironment necessitates the sustainable delivery of growth factors and stem cells to augment the regeneration of injured tissues. Our aim was to investigate the ability of VEGF 165 expressing bone marrow mesenchymal stem cells (BMMSCs) to differentiate into hepatocytes when cultured with hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in vitro. We isolated, cultured and identified rabbit BMMSCs, then electroporated the BMMSCs with VEGF 165 -pCMV6-AC-GFP plasmid. G418 was used to select transfected cells and the efficiency was up to 70%. The groups were then divided as follows: Group A was electroporated with pCMV6-AC-GFP plasmid ? HGF ? EGF and Group B was electroporated with VEGF 165 -pCMV6-AC-GFP plasmid ?HGF ? EGF. After 14 days, BMMSCs were induced into short spindle and polygonal cells. Alpha-fetoprotein (AFP) was positive and albumin (ALB) was negative in Group A, while both AFP and ALB were positive in group B on day 10. AFP and ALB in both groups were positive on day 20, but the quantity of AFP in group B decreased with prolonged time and was about 43.5% less than group A. The quantity of the ALB gene was increased with prolonged time in both groups. However, there was no significant difference between group A and B on day 10 and 20. Our results demonstrated that VEGF 165 -pCMV6-AC-GFP plasmid modified BMMSCs still had the ability to differentiate into hepatocytes. The VEGF 165 gene promoted BMMSCs to differentiate into hepatocyte-like cells under the induction of HGF and EGF, and reduced the differentiation time. These results have implications for cell therapies.

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“…Bone marrow (BM) from the femur cavity was flushed using α-MEM medium (Invitrogen Corporation, Paisley, UK) containing 10% FCS (Hyclone Laboratories, Perbio Science, Cheshire, UK), 1% L-glutamine and 1% penicillin/streptomycin [21]. The cell suspension was centrifuged ( 350 g, seven minutes), and cells were plated in culture flasks ( 200,000 cells/cm 2 ).…”

Section: Methodsmentioning

confidence: 99%

Mesenchymal stem cells with overexpression of midkine enhance cell survival and attenuate cardiac dysfunction in a rat model of myocardial infarction

Zhao

Zhang

et al. 2014

Stem Cell Res Ther

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IntroductionElevated midkine (MK) expression may contribute to ventricular remodeling and ameliorate cardiac dysfunction after myocardial infarction (MI). Ex vivo modification of signaling mechanisms in mesenchymal stem cells (MSCs) with MK overexpression may improve the efficacy of cell-based therapy. This study sought to assess the safety and efficacy of MSCs with MK overexpression transplantation in a rat model of MI.MethodsA pLenO-DCE vector lentivirus encoding MK was constructed and infected in MSCs. MSC migration activity and cytoprotection was examined in hypoxia-induced H9C2 cells using transwell insert in vitro. Rats were randomized into five groups: sham, MI plus injection of phosphate buffered saline (PBS), MSCs, MSCs-green fluorescent protein (MSCs-GFP) and MSCs-MK, respectively. Survival rates were compared among groups using log-rank test and left ventricular function was measured by echocardiography at baseline, 4, 8 and 12 weeks.ResultsOverexpression of MK partially prevented hypoxia-induced MSC apoptosis and exerted MSC cytoprotection to anoxia induced H9C2 cells. The underlying mechanisms may be associated with the increased mRNA and protein levels of vascular endothelial growth factor (VEGF), transformation growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1) and stromal cell-derived factor 1 (SDF-1a) in MSCs-MK compared with isolated MSCs and MSCs-GFP. Consistent with the qPCR results, the culture supernatant of MSCs-MK had more SDF-1a (9.23 ng/ml), VEGF (8.34 ng/ml) and TGF-β1 (17.88 ng/ml) expression. In vivo, a greater proportion of cell survival was observed in the MSCs-MK group than in the MSCs-GFP group. Moreover, MSCs-MK administration was related to a significant improvement of cardiac function compared with other control groups at 12 weeks.ConclusionsTherapies employing MSCs with MK overexpression may represent an effective treatment for improving cardiac dysfunction and survival rate after MI.

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Optimization of Genetic Engineering and Homologous Recombination of Collagen Type I Genes in Rat Bone Marrow Mesenchymal Stem Cells (MSC)

Cited by 11 publications

References 35 publications

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

VEGF165 expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro

Mesenchymal stem cells with overexpression of midkine enhance cell survival and attenuate cardiac dysfunction in a rat model of myocardial infarction

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