Placental mesenchymal stromal cells as an alternative tool for therapeutic angiogenesis.

Dysregulation of angiogenesis is a phenomenon observed in several disorders such as diabetic foot, critical limb ischemia and myocardial infarction. Mesenchymal stromal cells (MSCs) possess angiogenic potential and have recently emerged as a powerful tool for cell therapy to promote angiogenesis. Although bone marrow-derived MSCs are the primary cell of choice, obtaining them has become a challenge. The placenta has become a popular alternative as it is a highly vascular organ, easily available and ethically more favorable with a rich supply of MSCs. Comparatively, placenta-derived MSCs (PMSCs) are clinically promising due to their proliferative, migratory, clonogenic and immunomodulatory properties. PMSCs release a plethora of cytokines and chemokines key to angiogenic signaling and facilitate the possibility of delivering PMSC-derived exosomes as a targeted therapy to promote angiogenesis. However, there still remains the challenge of heterogeneity in the isolated populations, questions on the maternal or fetal origin of these cells and the diversity in previously reported isolation and culture conditions. Nonetheless, the growing rate of clinical trials using PMSCs clearly indicates a shift in favor of PMSCs. The overall aim of the review is to highlight the importance of this rather poorly understood cell type and emphasize the need for further investigations into their angiogenic potential as an alternative source for therapeutic angiogenesis.

Omega-3 polyunsaturated fatty acids promote angiogenesis in placenta derived mesenchymal stromal cells.

Enhancement of angiogenesis is solicited in wound repair and regeneration. Mesenchymal stromal cells derived from the placenta (P-MSCs) have an inherent angiogenic potential. Polyunsaturated fatty acids (PUFAs) in turn, specifically the omega-3 (N-3) are essential for growth and development. They are also recommended as dietary supplements during pregnancy. We therefore hypothesized that addition of N-3 PUFAs in P-MSC culture media may enhance their angiogenic potential. Hence, we treated P-MSCs with omega-3 (N-3) fatty acids -Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) at different concentrations and tested their angiogenic potential. We saw an upregulation of both bFGF and VEGFA. We also found enhanced in vitro tube formation ability of P-MSCs treated with DHA: EPA. We then looked at the influence of the conditioned medium (CM) collected from P-MSCs exposed to DHA: EPA on the key effector cells -HUVECs (Human Umbilical Vein derived endothelial cells and their functionality was further confirmed on chick yolk sac membrane. We found that the CM of P-MSCs exposed to DHA: EPA could enhance angiogenesis in both cases. These result were finally validated in an in vivo matrigel plug assay which revealed enhanced migration and vessel formation in CM treated with DHA: EPA. Our data thus reveals for the first time that supplementation with lower concentration of PUFA enhances the angiogenic potential of P-MSCs making them suitable for chronic wound healing applications.

Mesenchymal stromal cells isolated from gestationally diabetic human placenta exhibit insulin resistance, decreased clonogenicity and angiogenesis.

Pregnancy is known to be a diabetogenic state. With sedentary lifestyle and wrong dietary choices, gestational diabetes mellitus is on the rise. This raises a concern as placenta is becoming an acceptable choice, as a source of Mesenchymal Stromal Cells (MSCs). In our current study we questioned whether there exists a difference between MSCs isolated from normal and diabetic (Gd-P-MSCs) placenta, as the health of the cells used in therapy is of prime importance. We isolated and verified the Gd-P-MSCs based on their surface markers and differentiation potential. We looked at viability and proliferation and did not see a difference between the two. We analysed the glucose uptake potential of these cells by assessing the remnant glucose in the media, glucose within the cells by 2-NBDG and by glycogen storage. Despite only a slight downregulation of mRNA expression levels of glucose transporters, Gd-P-MSCs exhibited decreased glucose uptake even upon insulin stimulation and decreased glycogen storage, indicative of an insulin resistant state. We then assessed the colony forming ability of the cells and found a decreased clonogenicity in Gd-P-MSCs. We also examined the angiogenic potential of the cells by tube formation. Gd-P-MSCs showed decreased angiogenic potential when compared to normal cells. Thus we show for the first time, the effect of gestational diabetes on cells isolated from the chorionic villi of term placenta. Gd-P-MSCs are indeed insulin resistant, exhibit decreased clonogenicity and angiogenic potential. The present investigation is of relevance to the choice of sample for MSC isolation for therapeutic purposes.

Hypoxia primed placental mesenchymal stem cells for wound healing.

AIMS: To investigate how Placental Mesenchymal Stem Cells (P-MSCs) would adapt themselves and survive under hypoxic conditions which are prevalent in most injury sites. MAIN METHODS: P-MSCs were isolated from term placenta and characterised under normoxia and hypoxia (2-2.5% O2). Cells were examined for morphology and surface marker variations by flow cytometry analysis. Glucose stimulated insulin secretion was assayed by Insulin ELISA Kit. Gene expression levels were estimated using Real Time PCR for hypoxia inducible factor1 alpha, Insulin (INS), Glucose transporters (GLUT-1, GLUT-2 and GLUT-3), Adhesion Proteins- Integrins, Fibronectin1 (FN1), E-Cadherin (CDH1), and N-Cadherin (CDH2) and angiogenesis marker VEGFA. Immunofluorescence assay was done to confirm the presence of C-Peptide, GLUT 2, E-Cadherin and ITGB3. Adhesion was confirmed assessed on fibronectin binding. KEY FINDINGS: We show that insulin secretion is not hampered under hypoxia. We found an upregulation of glucose transporters under hypoxia indicating enhanced glucose uptake needed to cater to metabolic demands of proliferating cells. Up regulation of adhesion molecules was seen under hypoxia indicative of a favoured environment for retention of cells at the injury site. We also found increased level of angiogenesis of P-MSCs under hypoxia. SIGNIFICANCE: Our present study thus demonstrates for the first time that P-MSCs modulate themselves under hypoxic conditions by secreting insulin, up regulating glucose transporters and adhesion molecules and eventually exhibiting an increased angiogenic potential. We thus infer that priming P-MSCs under hypoxia, could make them more suitable for wound healing applications.

Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

Extracellular matrix plays an important role in regulating cell growth and differentiation. The biomimetic approach of cell-based tissue engineering is based on mirroring this in vivo micro environment for developing a functional tissue engineered construct. In this study, we treated normal tissue culture plates with selected extracellular matrix components consisting of glycosaminoglycans such as chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate, heparin and hyaluronic acid. Mesenchymal stem cells isolated from adult human bone marrow were cultured on the glycosaminoglycan treated culture plates to evaluate their regulatory role in cell growth and osteoblast differentiation. Although no significant improvement on human mesenchymal stem cell adhesion and proliferation was observed on the glycosaminoglycan-treated tissue culture plates, there was selective osteoblast differentiation, indicating its potential role in differentiation rather than proliferation. Osteoblast differentiation studies showed high osteogenic potential for all tested glycosaminoglycans except chondroitin-4-sulphate. Osteoblast differentiation-associated genes such as osterix, osteocalcin, integrin binding sialoprotein, osteonectin and collagen, type 1, alpha 1 showed significant upregulation. We identified osterix as the key transcription factor responsible for the enhanced bone matrix deposition observed on hyaluronic acid, heparin and chondroitin-6-sulphate. Hyaluronic acid provided the most favourable condition for osteoblast differentiation and bone matrix synthesis. Our results confirm and emphasise the significant role of extracellular matrix in regulating cell differentiation. To summarise, glycosaminoglycans of extracellular matrix played a significant role in regulating osteoblast differentiation and could be exploited in the biomimetic approach of fabricating or functionalizing scaffolds for stem cell based bone tissue engineering.

Modulation of physical environment makes placental mesenchymal stromal cells suitable for therapy.

Low level of oxygen at the site of injury is likely to affect the viability and proliferation of the transplanted mesenchymal stromal cells (MSCs). Hence there is a need to understand the effect of the physical environment on transplanted stromal cells. Therefore, we have studied the effect of the duration of hypoxic exposure alone or in combination with normoxia on placenta derived mesenchymal stem cell (PDMSCs). PDMSCs and bone marrow MSCs (BMMSCs) were analysed under four different culture conditions, exposure to direct normoxia (N), direct hypoxia (H) and intermittent normoxia followed by hypoxia (NH) and intermittent hypoxia followed by normoxia (HN). The effect on morphology, proliferation, metabolic activity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) and viability by 7AAD (7-amino-actinomycin D) were assayed, along with markers for MSCs and HLADR. No change in morphology, marker expression or HLADR was detected in N, H, NH or HN. An increase in proliferation rate, decrease in population doubling-time (PDT) and a relative increase in metabolic activity was strongly noted in the order: NH, N/HN and H. No significant difference was observed in the viability between N, H, NH or HN. A similar pattern was also observed in BMMSCS, indicating comparable suitability of PDMSCs in therapeutic applications. Thus we conclude that intermittent exposure to normoxia prior to hypoxic exposure is a better option than direct exposure to hypoxia. This may have clinical relevance in that they probably mirror the in vivo scenario of systemic delivery (NH) of cells as opposed to local delivery (H), thereby suggesting that systemic delivery is better than local delivery.