Biological resources including proteins, cells, and tissues were confronted with both safe and stable preservation for practical use in biotechnological industry. Particularly, cell therapy for regenerative engineering is needed to restricted regulation and accurate preservation. Therefore, this study was investigated improved conditions of mesenchymal stem cells from human umbilical cord (hUCs) or aspirated adipose tissues (hATs) for clinical cell banks. Both cells were isolated according to standard operation procedure of Hurim BioCell Inc. and analyzed the inherent characteristics in passage 4. To compare the ability of experimental groups after cryopreservation, proliferation ability using calculated values and cytomorphological patterns of each experimental step were analyzed. Also proteins such as ice-binding protein or caspase inhibitor were applied to add the preservation medium of hUCs or hATs. Result of preservation solution with 20% serum was considered a positive group. Recovery rate and expansion results showed specific dosage and cell type-dependent differences in the experimental group. Chromosomal stability and multipotency of hUCs or hATs were expressed stable pattern after cryopreservation using advanced medium. As a result, these additives could be substituted for xenogenic sources in banking of hUCs or hATs.

One of the most effective and safe therapeutic methods for treating vitiligo, mixed autologous keratinocytes (KCs) and melanocytes (MCs) cultures have been used for autologous cell transplantation. However, the present transplantation method is faced with a problem that may require a large amount of skin tissue and keratinocytes have limited culture potency. We have found previously that human adipose derived stromal cells (hASCs) from aspirated fat tissue could be used in place of KCs and sufficient amounts of hASCs for transplantation could be obtained by small amount of aspirated fat tissue. The present investigation was determined the effect of ASCs on ex vivo expansion MCs for transplantation. In addition, we examined for a preservation conditions of MCs which have reported low recovery rates and a slowdown in growth after cryopreservation. Various conditions including ASCs ratio, incubation period, and additive materials for MCs cultivation was determined to improve the expansion ability of MCs. The growth rate of MCs colony was elevated 6.85 folds compared the previous conditions. These MCs showed a specific expression of immature melanocyte protein, Trp-2, but did not express the mature melanocyte proteins and markers (c-kit, CD133, and etc.) of mesenchymal stem cells that represents in ASCs feeder. Results in cryopreservation experiments were determined a preservation medium for MCs showing an increased recovery rates after thawing. The characteristics of MCs after cryopreservation using a designed medium were indicated consistent morphology and immunophenotype. In conclusion, ASCs as a feeder could be used in place of keratinocytes for ex vivo expansion of MCs. For clinical trial for vitiligo patients, efficiency experiments in preclinical state should be followed.

Recently, human mesenchymal stem cells (MSCs) are attracting attention as a useful source for regenerative therapy. Controlled production of cell therapy requires the establishment and management of an accurate isolation, characterization and monitoring for quality assurance of developing MSCs mediated. In this study, we were confirmed maintenance of potency of isolated and cultured human umbilical cord (hUC)-MSCs during ex vivo expansion or after cryopreservation. Expression of their cell specific marker was analyzed by flow cytometry and the differentiation potency was confirmed by guided differentiation of adipocyte, osteocyte, chondrocyte and hepatocyte after expanding over 15 doublings in vitro. Safe production of developing a cell therapy was proved by testing for microbial, mycoplasma, endotoxin, and adventitious agents. Also stability of cells in cultivation, preservation and/or differentiation was determined chromosomal assay. In developing using hUC-MSCs, cells showed an accurate isolation and stable expansion in ex vivo condition. The results of several management assay showed that the stem cell marker expression of CD31, CD34 and CD45 were under 10%, however CD90 was over 90% by FACS analysis. Any contamination and mutation in all tests weren't detected in specific points for safe or stable production of hMC-MSCs. Also the proliferation and differentiation potency maintains during in vitro culture and after cryopreservation of hUC-MSCs. These results could be used as standard methods of maintenance of hUC-MSCs for cell therapy products and clinical application.

Human mesenchymal stem cells are known that multipotent stromal cells have the ability to divide asymmetrically, differentiate into many tissue types, and modulate cellular fate or function. Previous reports have proved that direct or indirect effects of mesenchymal stem cells in damaged cells or tissue were able to contribute to regenerative remodeling. One of incurable diseases, vitiligo is a depigmenting skin disorder resulting from the loss of melanocytes in the epidermis. Although vitiligo is a common disorder with a frequency of 0.1~2% in population, it still remains incurable and recurrent. Up to now, various treatment methods has been available for vitiligo therapy. Especially, transplantation of melanocytes (MCs) cultured with keratinocytes (KCs) is well-known therapy in clinic. We have recently reported functional role of adipose-derived stromal cells (ASCs) could assist MCs growth and maintenance of immature MCs. Therefore, the present study investigated whether the influence of ASCs may be elevated a transplantation yield of MCs in vivo. Transplantation was accomplished by syringe injection or grafting after dermabrasion. The procedure of dermabrasion is a mechanically invasive skin planning method and may be to help settle adequate location of transplanted cells to therapy. To improve an efficacy of cell transplantation, various additives or conditions of ratio were compared in vivo. These data was concluded that mixture of MCs and ASCs in the determined condition was improved engraftments of melanocytes for patients with vitiligo.

Mesenchymal stem cells (MSCs) has been reported as multipotent progenitor cells that can be expanded rapidly in vitro and differentiated into multiple mesodermal cell type. Human MSCs have been reported to be associated with neural differentiation especially in the cholinergic phenotype in several neural system. In this study, We investigated the ability of MSCs derived human aipose tissue to differentiation into neural cells expressing Islet-1 and further differentiates into cholinergic neurons in cholinergic differentiation media. Immunocytochemistry was performed to detect the expression of Islet-1 and demonstrate characteristic of neurons and cholinergic neurons. Islet-1 was massively detected in the induction stage. Following cholinergic differentiation from Islet-1-expressing MSCs, Cholinergic neuron marker ChAT was higly expressed. Also we examined the neuroprotective effects and neural differentiation of transplanted human adipose tissue-derived mesenchymal stem cells (AT-MSCs) in ischemic stroke. For transplantation, after 3days after MCAO. animal were divided into 2 group: Group A : injected phosphate buffered saline (PBS;5 ㎕ n=10), Group B: transplanted AT-MSCs (5×105 cells, n=10). Each animal received an injection into the right penumbra region (from bregma : AP;－1.3 ㎜, ML;－4.0 ㎜, DV;－5.9 ㎜). In all animals, behavior test were performed at 1, 3, 6, 9, 12, 15 days after MCAO, that was conducted by investigators who were blined to the experimental groups. mNSS test demonstrated that motor, sensory, and balance behavior were impaired after MCAO ischemic insult. Ischemic rats that received AT-MSCs exhibited significantly improved functional performance compared with PBS injected animals and histological analysis revealed that transplanted AT-MSCs expressed marker for neuron. These results suggest that AT-MSCs can be differentiated into neuron especially in cholinergic neuron and may be a potential source of treatment for neurodegenerative disease such as stroke.

Mesenchymal stem cells constitute an potential cellular source to promote brain regeneration with Parkinson's disease. Mesenchymal stem cells have significant advantages over other stem cell types and greater potential for immediate clinical application. The purpose of this study was to investigate whether hMSCs from the human adipose tissue could be induced to differentiate into dopaminergic cells and to assess the developmental potential of hMSC for selectively replacing the midbrain dopamine neurons lost in Parkinson's disease in vitro and in vivo. MSCs were cultured under conditions that promote differentiation of dopaminergic neuron. Using media that include SHH, FGF8, and GDNF. the MSCs were induced in vitro to become dopaminergic neurons. The expressions of the LIM homeobox transcription factor 1, alpha (Lmx1a), tyrosine hydroxylase(TH) proteins were determined by immunofluorescence. Lmx1a has been shown sufficient to confer neurogenic activity on mesencephalic floor plate cells and to determine a mesencephalic dopaminergic neurons fate. This result suggests that hMSCs have the ability to differfentiate into dopaminergic neurons. hMSCs were then transplanted into the striatal in a rat model of Parkinson's disease. The rats were unilaterally lesioned in the substantia nigra with 6-hydroxydopamine and were tested for rotational apomorphine-induced behavior. Following differentiation of dopaminergic neuron, cells displayed dopaminergic morphology and that they expressed dopaminergic marks genes. Finally transplantation of hMSCs into the striatal of Parkinsonian rats resulted in improvement of their behavioral deficits by apomorphine-induced rotational behavior. The hMSCs transplanted rats were proved to be better than compared with the transplantation of PBS. Immunohistochemical analysis of grafted brains revealed that abundant hMSCs survived from the grafts and some of them displayed dopaminergic marks. Our results indicate that hMSC may serve as a good cell source for the treatment of neurodegenerative diseases and have high potential for being used in multiple applications. This cellular approach might become a restorative therapy in Parkinson's disease.