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

Intra-articular injection of autologous mesenchymal stem cells in six patients with knee osteoarthritis

Osteoarthritis [OA] is a progressive disorder of the joints caused by gradual loss of articular cartilage, which naturally possesses a limited regenerative capacity. In the present study, the potential of intra-articular injection of mesenchymal stem cells [MSCs] has been evaluated in six osteoarthritic patients. Six female volunteers, average age of 54.56 years, with radiologic evidence of knee OA that required joint replacement surgery were selected for this study. About 50 ml bone marrow was aspirated from each patient and taken to the cell laboratory, where MSCs were isolated and characterized in terms of some surface markers. About 20-24x106 passaged-2 cells were prepared and tested for microbial contamination prior to intra-articular injection. During a one-year follow-up period, we found no local or systemic adverse events. All patients were partly satisfied with the results of the study. Pain, functional status of the knee, and walking distance tended to be improved up to six months post-injection, after which pain appeared to be slightly increased and patients' walking abilities slightly decreased. Comparison of magnetic resonance images [MRI] at baseline and six months post-stem cell injection displayed an increase in cartilage thickness, extension of the repair tissue over the subchondral bone and a considerable decrease in the size of edematous subchondral patches in three out of six patients. The results indicated satisfactory effects of intra-articular injection of MSCs in patients with knee OA

Ex vivo expansion and differentiation of mesenchymal stem cells from goat bone marrow

Mesenchymal stem cells [MSCs] from large animals as goat which is genetically more closely related to human have rarely been gained attentions. The present study tried to isolate and characterize MSCs from goat bone marrow. Fibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures. Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineages to determine their MSC nature. Differentiations were determined by RT-PCR analysis of related gene expression. To identify the best culture conditions for propagation, passage-3 cells were plated either at varying cell densities or different fetal bovine serum [FBS] concentrations for a week, at the end of which the cultures were statistically compared with respect to the cell proliferation. In this study, we also determined goat MSC population doubling time [PDT] as the index of their in vitro expansion rate. Passage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in both specific staining as well as the specific gene expression profile. Moreover, there appeared to be more expansion when the cultures were initiated at 100 cells/cm[2] in a medium supplemented with 15% FBS. A relatively short PDT [24.94 +/- 2.67 hr] was a reflection of the goat MSC rapid rate of expansion. Taken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletal cell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBS concentration

Quantitative analysis of the proliferation and differentiation of rat articular chondrocytes in alginate 3D culture

While articular chondrocytes are among those appropriate candidates for cartilage regeneration, the cell dedifferentiation during monolayer culture has limited their application. Several investigations have indicated the usefulness of alginate, but the topic of proliferation and differentiation of chondrocytes in alginate culture has still remained controversial. Rat articular chondrocytes were released by enzymatic digestion, plated at 5 x 10[4] cells/cm[2] and culture-expanded. Passaged-5 cells were then cultivated in alginate as 2-mm beads for a period of two months during which the expansion rate and the level of cell differentiation were determined by [3-[A, 5- dimethylthiazolyl- 2-yl]-l, 5-diphenyl tetrazolium bromide] assay and real-time PCR analysis respectively and compared with those of chondrocytes in monolayer culture. Average population doubling time in alginate cultures [10.04 +/- 0.9 days] tended to be significantly [P<0.05] higher than that in monolayer cultures [2.94 +/- 0.3 days]. The period of alginate culture could be subdivided into expansion phase [Days 0-40]; during which proliferation appeared to be high and differentiation phase [Days 40-60] during which the expression of cartilage-specific genes including collagen II and aggrecan tended to be upregulated. During both the proliferation and differentiation phases, the expression of collagen I was low. At chondrocytes monolayer cultures, the proliferating cells appeared to have a very low expression level of cartilage-specific genes and a high expression level of collagen I gene during the entire culture period [P<0.05]. It seems that alginate provides conditions in which rat articular chondrocytes are able to undergo proliferation and differentiation in certain time point of cultivation period

Mesenchymal stem cell isolation from the removed medium of rat's bone marrow primary culture and their differentiation into skeletal cell lineages

In all protocols for isolation of mesenchymal stem cells [MSCs], a few days after culture initiation, the medium were discarded along with its contents of non-adherent cells and the adherent cell population kept and expanded as MSCs population. In the present study, attempt was made to expand the cells suspended in removed medium of primary culture and compare them with the adherent cell population. Four days after rat's bone marrow culture initiation, medium of the culture was collected and its suspended cells were culture-expanded in parallel with adherent cells till passage 3. During the culture period, the cells from either group were statistically compared with respect of the time required for cell confluency [the stage in which cells cover the entire surfaces] as an index of growth rate. At the end, the cells from both cultures were evaluated in terms of their differentiation potential. The primary culture of the cells from removed medium contained large colonies of spindle-shaped cells that reached into confluency after 5.36 +/- 0.5 days, while those from the adherent population possessed small colonies reaching into confluency in 8.09 +/- 0.70 days. According to the results, at all studied passages, the cells of removed medium were significantly [p<0.05] achieved confluency in shorter time than the adherent population. Moreover, the cells from either culture could easily differentiate into bone, cartilage and adipose cells. It seems that some cells from removed medium, usually discarded in medium substitution, are MSCs possessing more growth rate than the primarily adherent cell population