Isolation, characterization and osteogenic potential of mouse digit tip blastema cells in comparison with bone marrow-derived mesenchymal stem cells in vitro

Objective: limb regeneration mediated by blastema cells [BlCs] in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells [BM-MSCs] as an alternative accessible cell source to BlCs for regeneration of appendages

Materials and Methods: in this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction [qRT-PCR] and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase [ALP] activity, calcium content, and osteogenic gene expression were evaluated in both BMMSCs and BlCs cultures at days 7, 14, and 21

Results: qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs [P<0.05]. Alizarin red staining [ARS], calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points

Conclusion: characteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages

Umbilical cord blood platelet lysate as serum substitute in expansion of human mesenchymal stem cells

Objective: The diverse clinical applications for human mesenchymal stem cells [hM- SCs] in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate [UCB-PL] as a standard substitute for fetal bovine serum [FBS] and human peripheral blood-PL [PB-PL]

Materials and Methods: In this experimental study, platelet concentrates [PC] from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing [viral and microbial], total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS

Results: UCB-PL contained high levels of protein content, platelet-derived growth factor-AB [PDGF-AB], and transforming growth factor [TGF] compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70?C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages

Conclusion: PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy

Comparative in vitro evaluation of human dental pulp and follicle stem cell commitment

Objective: Pulp and periodontal tissues are well-known sources of mesenchymal stem cells [MSCs] that provide a promising place in tissue engineering and regenerative medicine. The molecular mechanisms underlying commitment and differentiation of dental stem cells that originate from different dental tissues are not fully understood. In this study, we have compared the expression levels of pluripotency factors along with immunological and developmentally-related markers in the culture of human dental pulp stem cells [hDPSCs], human dental follicle stem cells [hDFSCs], and human embryonic stem cells [hESCs]

Materials and Methods: In this experimental study, isolated human dental stem cells were investigated using quantitative polymerase chain reaction [qPCR], immunostaining, and fluorescence-activated cell sorting [FACS]. Additionally, we conducted gene ontology [GO] analysis of differentially expressed genes and compared them between dental stem cells and pluripotent stem cells

Results: The results demonstrated that pluripotency [OCT4 and SOX2] and immunological [IL-6 and TLR4] factors had higher expressions in hDFSCs, with the exception of the JAGGED-1/NOTCH1 ratio, c-MYC and NESTIN which expressed more in hDPSCs. Immunostaining of OCT4, SOX2 and c-MYC showed cytoplasmic and nucleus localization in both groups at similar passages. GO analysis showed that the majority of hDFSCs and hDPSCs populations were in the synthesis [S] and mitosis [M] phases of the cell cycle, respectively

Conclusion: This study showed different status of heterogeneous hDPSCs and hDFSCs in terms of stemness, differentiation fate, and cell cycle phases. Therefore, the different behaviors of dental stem cells should be considered based on clinical treatment variations

Percutaneous autologous bone marrow-derived mesenchymal stromal cell implantation is safe for reconstruction of human lower limb long bone atrophic nonunion

Objective: Nonunion is defined as a minimum of a 9-month period of time since an injury with no visibly progressive signs of healing for 3 months. Recent studies show that application of mesenchymal stromal cells [MSCs] in the laboratory setting is effective for bone regeneration. Animal studies have shown that MSCs can be used to treat nonunions. For the first time in an Iranian population, the present study investigated the safety of MSC implantation to treat human lower limb long bone nonunion

Materials and Methods: It is a prospective clinical trial for evaluating the safety of using autologus bone marrow derived mesenchymal stromal cells for treating nonunion. Orthopedic surgeons evaluated 12 patients with lower limb long bone nonunion for participation in this study. From these, 5 complied with the eligibility criteria and received MSCs. Under fluoroscopic guidance, patients received a one-time implantation of 20-50x106 MSCs into the nonunion site. All patients were followed by anterior-posterior and lateral X-rays from the affected limb, in addition to hematological, biochemical, and serological laboratory tests obtained before and 1, 3, 6, and 12 months after the implantation. Possible adverse effects that included local or systemic, serious or non-serious, and related or unrelated effects were recorded during this time period

Results: From a safety perspective, all patients tolerated the MSCs implantation during the 12 months of the trial. Three patients had evidence of bony union based on the after implantation X- rays

Conclusion: The results have suggested that implantation of bone marrow-derived MSCs is a safe treatment for nonunion. A double-blind, controlled clinical trial is required to assess the efficacy of this treatment

Effect of chitosan grafted polyethylenimine nanoparticles as a gene carrier on mesenchymal stem cells viability

This study discusses the effect of complexes of chitosan grafted polyethylenimine[Ch-PEI] with plasmid DNA on viability of mesenchymal stem cells[MSCs] derived from human marrow. Ch-PEI/pDNA nanoparticles were synthesized through the complex coacervation method using pIRES plasmid containing Green Fluorescent Protein [GFP] gene. To confirm the complexation, samples were run through an agarose gel. Human bone marrow mesenchymal stem cells were studied for the cytotoxicity of the nanoparticles by MTT assay. MTT results indicated Ch-PEI does not have any significant cytotoxicity compared with PEI and Lipofectamine2000 leading to 40% cytotoxicity. According to the results it seems that grafting chitosan with PEI improves the MSCs viability