Current Issue : April - June Volume : 2013 Issue Number : 2 Articles : 6 Articles
Introduction: Adipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including\r\nangiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration.\r\nTherefore, the aim of this study was to determine the effects ASC-secreted factors have in repairing chondral\r\ndefects.\r\nMethods: ASCs isolated from male Sprague Dawley rats were cultured in monolayer or alginate microbeads\r\nsupplemented with growth (GM) or chondrogenic medium (CM). Subsequent co-culture, conditioned media, and\r\nin vivo cartilage defect studies were performed.\r\nResults: ASC monolayers and microbeads cultured in CM had decreased FGF-2 gene expression and VEGF-A\r\nsecretion compared to ASCs cultured in GM. Chondrocytes co-cultured with GM-cultured ASCs for 7 days had\r\ndecreased mRNAs for col2, comp, and runx2. Chondrocytes treated for 12 or 24 hours with conditioned medium\r\nfrom GM-cultured ASCs had reduced sox9, acan, and col2 mRNAs; reduced proliferation and proteoglycan synthesis;\r\nand increased apoptosis. ASC-conditioned medium also increased endothelial cell tube lengthening whereas\r\nconditioned medium from CM-cultured ASCs had no effect. Treating ASCs with CM reduced or abolished these\r\ndeleterious effects while adding a neutralizing antibody for VEGF-A eliminated ASC-conditioned medium induced\r\nchondrocyte apoptosis and restored proteoglycan synthesis. FGF-2 also mitigated the deleterious effects VEGF-A\r\nhad on chondrocyte apoptosis and phenotype. When GM-grown ASC pellets were implanted in 1 mm non-critical\r\nhyaline cartilage defects in vivo, cartilage regeneration was inhibited as evaluated by radiographic and equilibrium\r\npartitioning of an ionic contrast agent via microCT imaging. Histology revealed that defects with GM-cultured ASCs\r\nhad no tissue ingrowth from the edges of the defect whereas empty defects and defects with CM-grown ASCs\r\nhad similar amounts of neocartilage formation.\r\nConclusions: ASCs must be treated to reduce the secretion of VEGF-A and other factors that inhibit cartilage\r\nregeneration, which can significantly influence how ASCs are used for repairing hyaline cartilage....
Embryonic stem cells have the ability to self-renew and can differentiate into all cell types of the three embryonic germ lineages. The undifferentiated embryonic cell Transcription Factor 1 (UTF1) gene is highly expressed in ES cells and we previously reported that UTF1 is tightly associated with chromatin and is required for differentiation of pluripotent mouse embryonic stem (ES) and embryonic carcinoma (EC) cells. In this study, we generated ES and EC cell lines constitutively expressing GFP-UTF1 to further investigate its role in differentiation. ES and EC cells constitutively expressing GFP-UTF1were suppressed in their proliferation and were still dependent on LIF for self-renewal. Embryoid body (EB) differentiation of GFP-UTF1 overexpressing ES cells showed both normal differentiation as well as a delayed or incomplete differentiation of a subset of cells. GFP-UTF1 was persistently expressed in undifferentiated cells whereas GFP-UTF1 expression was not detected in differentiated cells. Where GFP-UTF1 expressing ES cells differentiated normally in response to DMSO, EC cell differentiation was completely blocked. When ES and EC cells expressing GFP-UTF1 were treated with RA, differentiation markers were induced and endogenous UTF1 and GFP-UTF1 protein levels decreased. However, GFP-UTF1 and UTF1 (in ES cells) mRNA was still detected indicating that degradation of (GFP-)UTF1 protein preceded down regulation of (GFP-) UTF1 mRNA, suggesting that RA induced UTF1 degradation. Summarizing these data indicate that similar to UTF1 depletion, overexpression of GFP-UTF1 interfered with ES and EC cells differentiation....
Introduction: The influence of genetic background on the ability to generate induced pluripotent stem cells\r\n(iPSCs) has the potential to impact future applications, but has yet to be examined in detail. The purpose of this\r\nstudy was to determine if genetic background affects the efficiency of generating iPSCs during early reprograming\r\nas well as the pluripotent stability of the iPSCs during later stages of reprograming.\r\nMethods: Mouse embryonic fibroblasts (MEFs) were isolated from six strains of mice (NON/LtJ; C57BL/6J; DBA/2J;\r\nBALB/cJ; 129S1/SvlmJ; CAST/EiJ) that were selected based on genetic diversity and differences in ability to produce\r\nembryonic stem cell (ESC) lines. MEFs were reprogramed via doxycycline-inducible lentiviral transduction of murine\r\nOct4, Klf4, Sox2, and c-Myc. Differences in efficiency to generate iPSCs were assessed by comparing the total number of\r\ncolonies, the percentage of colonies positive for alkaline phosphatase staining and the percentage of cells positive for\r\nSSEA1. iPSC colonies were expanded to establish doxycycline-independent cell lines whose pluripotency was then\r\nevaluated via ability to form teratomas in NOD.CB17-Prkdcscid/J mice. Proliferation of non-transduced parent MEFs from\r\neach strain was also examined over ten days under conditions that simulated reprograming.\r\nResults: NON/LtJ and CAST/EiJ strains were more efficient than other strains in generating iPSCs for all parameters\r\nmeasured and parent MEFs from these strains were more proliferative than those from other strains. Doxycyclineindependent\r\niPSC lines were established using standard conditions for all strains except BALB/cJ, which required a\r\nhigher concentration (5x) of leukemia inhibitory factor (LIF). iPSCs from all strains were capable of producing\r\nteratomas in NOD.CB17-Prkdcscid/J mice.\r\nConclusions: The results of this study suggest that genetic background does affect iPSC generation and\r\npluripotent stability. In addition, our results demonstrate that strain differences in efficiency to generate iPSCs\r\nduring the early stages of reprograming are correlated with those observed in proliferation of parent MEFs. These\r\nfindings have important implications both for future iPSC applications as well as for future investigation into\r\ndetermining the genes responsible for reprograming efficiency and stability....
Introduction: Dental pulp stem cells (DPSCs) are an accessible cell source with therapeutic applicability in\r\nregeneration of damaged tissues. Current techniques for expansion of DPSCs require the use of Fetal Bovine Serum\r\n(FBS). However, animal-derived reagents stage safety issues in clinical therapy. By expanding DPSCs in serumfree/\r\nxenofree medium (SF/XF-M) or in medium containing human serum (HS-M), the problems can be eliminated.\r\nTherefore, the aim of our study was to identify suitable cell culture media alternatives for DPSCs.\r\nMethods: We studied the isolation, proliferation, morphology, cell surface markers (CD29, CD44, CD90,\r\nCD105, CD31, CD45 and CD146), stemness markers expression (Oct3/4, Sox2, Nanog and SSEA-4) and in vitro\r\nmultilineage differentiation of DPSCs in HS-M or SF/XF-M in comparison to FBS-M.\r\nResults: DPSCs expressed the cell surface and stemness markers in all studied conditions. The proliferation\r\nanalysis of cells cultured in different HS concentrations revealed that cells isolated in 20% HS-M and passaged in\r\n10% or 15% HS-M supported the cell growth. Direct isolation of cells in SF/XF-M did not support cell proliferation.\r\nTherefore, cells cultured in 20% HS-M were used for further SF/XF-M studies. However, proliferation of DPSCs\r\nwas significantly lower in SF/XF-M when compared with cells cultured in FBS-M and HS-M. In addition, proliferation\r\nof DPSCs in SF/XF-M could be enhanced by addition of 1% HS in cell culture medium. There were differences in\r\nosteogenic, chondrogenic and adipogenic differentiation efficacy between cells cultured in FBS, HS and SF/XF\r\ndifferentation media. More pronounced adipogenic and osteogenic differentiation was observed in HS differentiation\r\nmedium, however, in FBS-M cultured cells more effective chondrogenic differentiation was detected.\r\nConclusions: Our results indicate that HS is a suitable alternative to FBS for the expansion of DPSCs. The\r\ncomposition of SF/XF-M needs to be further optimized in terms of cell expandability and differentiation efficiency to\r\nreach clinical applicability....
Aims: Human adipose stem cells (hASCs) have been suggested as viable alternative for bone tissue engineering. However, the tissue response and osteogenic potential of BAG S53P4 or �Ÿ-TCP granules has not been studied in vivo when seeded with hASCs and/or co-incubated with BMP-2 and thus, was evaluated in the current study.\r\nMethods and results: Human ASCs were isolated, expanded and seeded on BAG and �Ÿ-TCP in vitro and, cell viability was assessed using Live/Dead staining. In a subcutaneous rodent implantation model, the cellular response and osteogenic potential of 1) plain, 2) hASC seeded, 3) BMP-2 co-incubated and 4) hASC seeded and BMP-2 co-incubated BAG and �Ÿ-TCP granules were investigated using computed tomography and semi-quantitative histologic scores after 4 and 8 weeks. Live/Dead staining confirmed good cell viability on both biomaterials prior to implantation. Overall, implantation of both biomaterials resulted in formation of well-vascularized granulation tissue without excessive inflammation, fibrosis or adverse reactions independent on group assignment and time point evaluated and thus, suggesting safety for prospective applications. However, our results also indicate that �Ÿ-TCP may temporarily stimulate foreign body giant cell formation after hASCs supplementation suggesting a resorptive response. Both biomaterials required supplementation of hASCs and/or BMP-2 to induce osteoblastic\r\nactivity. However, BAG induced calcification exclusively when seeded with BMP-2 activated hASCs, whereas �Ÿ-TCP required seeding with hASCs only.\r\nConclusion: BAG and �Ÿ-TCP granules can be safely implanted subcutaneously, induce a different cellular response and require hASC and/or BMP-2 supplementation to induce osteoblastic activity and calcification. A combination of �Ÿ-TCP and hASCs appeared to be a feasible way in enhancing osteoblastic activity resulting in early osteogenesis while minimizing safety and regulatory concerns in bone-tissue engineering....
Introduction: Transplantation of genetically modified human bone marrow-derived mesenchymal stem cells\r\n(hMSCs) with an accurate potential for chondrogenic differentiation may be a powerful means to enhance the\r\nhealing of articular cartilage lesions in patients. Here, we evaluated the benefits of delivering SOX9 (a key regulator\r\nof chondrocyte differentiation and cartilage formation) via safe, maintained, replication-defective recombinant\r\nadeno-associated virus (rAAV) vector on the capability of hMSCs to commit to an adequate chondrocyte\r\nphenotype compared with other mesenchymal lineages.\r\nMethods: The rAAV-FLAG-hSOX9 vector was provided to both undifferentiated and lineage-induced MSCs freshly\r\nisolated from patients to determine the effects of the candidate construct on the viability, biosynthetic activities,\r\nand ability of the cells to enter chondrogenic, osteogenic, and adipogenic differentiation programs compared with\r\ncontrol treatments (rAAV-lacZ or absence of vector administration).\r\nResults: Marked, prolonged expression of the transcription factor was noted in undifferentiated and\r\nchondrogenically differentiated cells transduced with rAAV-FLAG-hSOX9, leading to increased synthesis of major\r\nextracellular matrix components compared with control treatments, but without effect on proliferative activities.\r\nChondrogenic differentiation (SOX9, type II collagen, proteoglycan expression) was successfully achieved in all types\r\nof cells but strongly enhanced when the SOX9 vector was provided. Remarkably, rAAV-FLAG-hSOX9 delivery\r\nreduced the levels of markers of hypertrophy, terminal and osteogenic/adipogenic differentiation in hMSCs (type I\r\nand type X collagen, alkaline phosphatise (ALP), matrix metalloproteinase 13 (MMP13), and osteopontin (OP) with\r\ndiminished expression of the osteoblast-related transcription factor runt-related transcription factor 2 (RUNX2);\r\nlipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma 2 (PPARG2)), as well as their ability to\r\nundergo proper osteo-/adipogenic differentiation. These effects were accompanied with decreased levels of bcatenin\r\n(a mediator of the Wnt signaling pathway for osteoblast lineage differentiation) and enhanced parathyroid\r\nhormone-related protein (PTHrP) expression (an inhibitor of hypertrophic maturation, calcification, and bone\r\nformation) via SOX9 treatment\r\nConclusions: This study shows the potential benefits of rAAV-mediated SOX9 gene transfer to propagate hMSCs\r\nwith an advantageous chondrocyte differentiation potential for future, indirect therapeutic approaches that aim at\r\nrestoring articular cartilage defects in the human population....
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