Yuqing Jin

Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells

BackgroundBone marrow-derived mesenchymal stem cells (BMSCs) are a widely researched adult stem cell population capable of differentiation into various lineages. Because many promising applications of tissue engineering require cell expansion following harvest and involve the treatment of diseases and conditions found in an aging population, the effect of donor age and ex vivo handling must be understood in order to develop clinical techniques and therapeutics based on these cells. Furthermore, there currently exists little understanding as to how these two factors may be influenced by one another.ResultsDifferences in the adipogenic, chondrogenic, and osteogenic differentiation capacity of murine MSCs harvested from donor animals of different age and number of passages of these cells were observed. Cells from younger donors adhered to tissue culture polystyrene better and proliferated in greater number than those from older animals. Chondrogenic and osteogenic potential decreased with age for each group, and adipogenic differentiation decreased only in cells from the oldest donors. Significant decreases in differentiation potentials due to passage were observed as well for osteogenesis of BMSCs from the youngest donors and chondrogenesis of the cells from the oldest donors.ConclusionBoth increasing age and the number of passages have lineage dependent effects on BMSC differentiation potential. Furthermore, there is an obvious interplay between donor age and cell passage that in the future must be accounted for when developing cell-based therapies for clinical use.

A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1.

Bone-marrow-derived mesenchymal stem cells (MSCs) have been shown to possess immunosuppressive properties, e.g., by inhibiting T cell proliferation. Activated T cells can also enhance the immunosuppression ability of MSCs. The precise mechanisms underlying MSC-mediated immunosuppression remain largely undefined, although both cell-cell contact and soluble factors have been implicated; nor is it clear how the immunosuppressive property of MSCs is modulated by T cells. Using MSCs isolated from mouse bone marrow, we show here that interferon gamma (IFNγ), a well-known proinflammatory cytokine produced by activated T cells, plays an important role in priming the immunosuppressive property of MSCs. Mechanistically, IFNγ acts directly on MSCs and leads to up-regulation of B7-H1, an inhibitory surface molecule in these stem cells. MSCs primed by activated T cells derived from IFNγ−/− mouse exhibited dramatically reduced ability to suppress T cell proliferation, a defect that can be rescued by supplying exogenous IFNγ. Moreover, siRNA-mediated knockdown of B7-H1 in MSCs abolished immunosuppression by these cells. Taken together, our results suggest that IFNγ plays a critical role in triggering the immunosuppresion by MSCs through up-regulating B7-H1 in these cells, and provide evidence supporting the cell-cell contact mechanism in MSC-mediated immunosuppression.

Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment.

Schwann cell purification is usually difficult due to the contamination of fibroblasts, which often become a predominant cell type in Schwann cell culture in vitro. We have developed a novel and efficient method to enrich Schwann cells by differential detachment of two types of cells. In the culture, cells were treated with a multiplex collagenase and the Schwann cells were found to detach faster than fibroblasts and thus Schwann cells could be easily isolated. Within 5 days, Schwann cell purity could reach above 99%, which was confirmed by immunostaining characterization and flow cytometric analysis. In addition, this efficient method can reach a high cell yield after two rounds of differential detachment procedures and does not require antimitotic treatment or special equipment as often needed by other reported methods.

Cryopreservation of tissue-engineered epithelial sheets in trehalose.

Tissue-engineered epidermal membranes are useful for clinical wound healing. To facilitate these products in the clinic, optimized storage methods need to be developed. We studied the efficiency of extracellular trehalose at various concentrations for cryopreserving human tissue-engineered epidermal membranes compared with that of dimethyl-sulfoxide (DMSO) used by most organ banks for cryopreserving skin grafts and artificial skin substitutes. Keratinocyte (KC) viability, proliferation and marker expression following cryopreservation in trehalose were examined with similar results to those using DMSO. Trehalose concentration (0.4m) was optimized according to the described cellular activities following cryopreservation. Artificial epidermal substitutes were then cryopreserved in trehalose at the optimized concentration. Cell viability, growth factor secretion and wound healing properties of cryopreserved artificial epidermal substitutes using nude mice were examined and compared with those of DMSO cryopreservation. Cryopreservation with trehalose enhanced human KC viability in suspension and artificial skin substitutes. In addition, trehalose cryopreservation provided fast recovery of EGF and TGF-β1 secretion by KCs after thawing. When transplanted into nude mice, trehalose-cryopreserved artificial skin repaired skin defects in a similar manner to that of a non-cryopreserved control. Moreover, trehalose-cryopreserved artificial skin resulted in engraftment and wound closure that was significantly enhanced compared with that of DMSO-cryopreserved epidermal membranes. The results indicate that the use of trehalose improves cryopreservation of tissue-engineered epithelial sheets.

Specific Marker Expression and Cell State of Schwann Cells during Culture In Vitro

Schwann cells (SCs) in animals exist in different developmental stages or wound repair phases, distinguished mainly by the expression of SC-specific markers. No study has yet determined SC state under in vitro culture conditions, and the specific markers expressed in SC are obscure as well. In this study, we harvested sciatic nerves from newborn mice and isolated SCs by an enzyme-digestion method, then we examined the expression profiles of ten markers (S100, p75NTR, Sox10, Sox2, GAP43, NCAM, Krox20, Oct6, MBP, and MPZ) at both the RNA and protein levels in in vitro mouse SCs and speculated their relation with in vivo SC stages. We assayed RNA and protein levels of SC specific markers by immunofluorescence, Western Blot, and real-time quantitative RT-PCR. The results show that the expression of most markers (S100, p75NTR, GAP43, NCAM, Krox20, Oct6, MBP and MPZ) was not detectable in all of early stage cultured SCs. The expression of transcription factors Sox10 and Sox2 was, however, detectable in all SCs. After 8 days, the positive expression rate of all markers except GAP43 and Oct6 was almost 100%.These results indicates Sox10 is a necessary marker for SC identification, while S100 is not reliable. SCs cultured in vitro express Sox2, P75NTR, NCAM, GAP43, Oct6, and MPZ, suggesting that they are similar to in vivo undifferentiated iSCs or dedifferentiated iSCs after nerve injury.

MRI of iron oxide nanoparticle-labeled ADSCs in a model of hindlimb ischemia.

Adipose-derived stem cells (ADSCs) exhibit tremendous potential for repair of ischemic diseases. However, studies on the fate, migration, differentiation, and body distribution of the labeled ADSCs are rarely reported. In this study, magnetic iron oxide nanoparticles were designed, synthesized, and coated with meso-2,3-dimercaptosuccinic acid (DMSA) to produce DMSA nanoparticles (DMSA-NPs). The properties, size distribution, and characterization of DMSA-NPs were evaluated. Green fluorescent protein expressing ADSCs (GFP-ADSCs) were obtained and labeled with DMSA-NPs. The viability, cytotoxicity and multi-differentiation capacity of labeled GFP-ADSCs were evaluated in vitro. Labeled and non-labeled GFP-ADSCs were injected into a mouse model of hindlimb ischemia, and 3T magnetic resonance imaging (MRI) was acquired. The synthesized DMSA-NPs efficiently labeled the GFP-ADSCs in vitro and in vivo without affecting cell viability, proliferation, cell cycle, and multi-differentiation capacity. The MRI showed hypointense spots in the labeled GFP-ADSCs that lasted up to 8 weeks. Prussian blue staining and immunofluorescence assay at 4 and 8 weeks indicated that the labeled GFP-ADSCs were in and around the ischemic sites and some differentiated into capillaries. This observation is identical to that seen for transplants of unlabeled cells. Labeled cells were also identified mainly in the liver and spleen, with significantly smaller amounts in the lungs, intestines, heart, and kidney. Developed DMSA-NPs were shown to exhibit a considerable potential for use as nanoprobes for MRI of stem cells, which will enhance our understanding of cell-based therapeutic strategies for ischemic diseases.

Peripheral nerve repair with epimysium conduit

Autologous tissues such as skeletal muscle have high biocampatibility and can effectively promote nerve regeneration compared to other biological and artificial materials; however, the reasonable and effective application of skeletal muscle requires further study. The purpose of this investigation was to assess the possibility of preparing a hollow nerve conduit, termed the epimysium conduit (EMC), using thin crimps of epimysium with skeletal muscle fibers and evaluate its effectiveness in repairing peripheral nerve defects. We prepared nerve conduits containing lumen with the external oblique muscle of the CAG-EFGP transgenic mice using microsurgical techniques for bridge repair of a 5-mm long sciatic nerve defect in wild-type mice. Systematic histological and functional assessments of the regenerated nerves were performed 8 and 12 weeks after surgery. EMC was found to effectively repair the sciatic nerve defect with significantly greater effectiveness than artificial conduits; however, the repair effect of EMC was lower than that of autologous nerve grafting for some parameters. In addition, our findings showed that some EMC-derived cell components migrated into the region of the regenerated nerves and contributed to reconstruction. Based on these findings, we conclude that a hollow conduit prepared with epimysium and a few skeletal muscle fibers is ideal for repairing peripheral nerve defects, and the cell components in the grafts contribute to nerve regeneration and structural remodeling, which provides an alternative option for the emergency primary repair of peripheral nerve defects in clinical practice.

Purification of Schwann cells from adult rats by differential detachment

Differential detachment by collagenase treatment is a new and efficient method for Schwann cell (SC) purification. As its effect on adult animals remains unclear, we have investigated the possibility of SC purification from adult rats. To avoid any systematic bias, Schwann cell purity before and after purification were compared by morphology, immunostaining of P75NTR and S100 and flow cytometric analysis. The final SC purities reached 99% as confirmed by three independent analyses SC purity and the cell yields were above 106 cells after two rounds of purification. The method of differential detachment is also suitable for SC purification in adult rats and could be useful for research and clinical applications.

Human eyelid adipose tissue-derived Schwann cells promote regeneration of a transected sciatic nerve

Schwann cells (SCs) can promote the regeneration of injured peripheral nerves while the clinical application is limited by donor site complications and the inability to generate an ample amount of cells. In this study, we have isolated human eyelid adipose-derived Schwann cells (hE-SCs) from human eyelid adipose tissue and identified the cell phenotype and function. Using immunofluorescence and H & E staining, we detected subtle nerve fibers and SCs in human eyelid adipose tissue. Immunofluorescence staining indicated that hE-SCs expressed glial markers, such as S100, p75NTR GFAP, Sox10 and Krox20. To explore whether hE-SCs promote the regeneration of injured peripheral nerves in vivo, a Balb/c-nu mice model was used in the study, and mice were randomly assigned to five groups: Matrigel; hE-SCs/P0; hE-SCs/P2; hE-FLCs/P2; and Autograft. After 12 weeks, functional and histological assessments of the regenerated nerves showed that sciatic nerve defect was more effectively repaired in the hE-SCs/P2 group which achieved 66.1 ± 6.5% purity, than the other three groups and recovered to similar level to the Autograft group. These results indicated that hE-SCs can promote the regeneration of injured peripheral nerve and the abundant, easily accessible supply of adipose tissue might be a promising source of SCs for peripheral nerve repair.

The effect of co-transplantation of nerve fibroblasts and Schwann cells on peripheral nerve repair

Combinations of fibroblasts (Fbs) and corresponding epithelial cells have been widely used in many tissues, such as the skin and breast tissues, to augment tissue repair and remodeling. Recently, a large amount of new data has indicated that nerve Fbs play critical roles in Schwann cells (SCs) and axons in vitro. However, little is known regarding the effects of co-transplanting nerve Fbs and SCs on peripheral nerve repair in vivo. The aim of this study was to investigate the effect of co-transplanting sciatic nerve Fbs (SN-Fbs) and sciatic nerve SCs (SN-SCs) on nerve regeneration. We developed a 5 mm nerve-defect model in mice using a polyurethane (PUR) catheter and then injected one of four different mixtures of cells into the catheters to form the following four groups: pure Matrigel (Control group), SN-Fbs alone (SN-Fb group), SN-Fbs combined with SN-SCs at a ratio of 1:2 (Fb&SC group) and SN-SCs alone (SN-SC group). Histological and functional analyses were performed 3 months later. The results indicated that in vitro, the expression levels of NGF, BDNF and GDNF were significantly higher, and in vivo, a more moderate amount of extracellular matrix was produced in the Fb&SC group than in the SN-SC group. Compared to the other groups, co-transplanting SN-Fbs with SCs at a 1:2 ratio had significantly positive effects on nerve regeneration and functional recovery.