Although stem cells are used as important cell therapies in regenerative medicine, the electrophysiological problems that arise in the expansion of cells have not been known much. This study was conducted to investigate the functional expression of inward rectifying K+ current (IKir) using a patch-clamp technique, and the change in the resting membrane potential and the membrane capacitance were investigated in mesenchymal stem cells derived from human umbilical vein (hUC-MSC). The IKir plays an important role in regulating the resting membrane potential in many cells and is known to contribute to the maintenance of intracellular K+ concentration. In this study, electrophysiologically recorded current exhibited typical IKir characteristics. The current shifted along the K+ equilibrium potential (Ek) with the extracellular K+ concentration change. In addition, IKir was blocked by the divalent Ba2+ in a dose-dependent manner. The frequency of functional expression of IKir changed with number of passages (P2: 5.3% vs P8: 77.8% vs P12: 34.5%). There was no significant change in the resting membrane potential of hUC-MSC (P2: -21.0 mV, P8: -20.1 mV and P12: -21.9 mV). However, the capacitance of the cell membrane was significantly changed after P9 (P2: 8.9 pF vs P9: 16.9 pF) compared to P2. All the results suggest that changes in electrophysiological distribution of IKir as the passages increase may cause changes in K+ permeability even in cell proliferation and differentiation, suggesting a possible physiological role in maintaining cell homeostasis and resting membrane potential (RMP).

Human umbilical cord is easy to obtain because it is discarded after birth, so that ethical issues can be avoided. Chondrogenesis studies using MSCs from bone marrow, cord blood, and adipose have indicated that TGFβ3 and BMP6 stimulate chondrogenesis. Therefore, we investigated chondrogenesis of hUC-MSCs on TGFβ3, BMP6, and combination of the two growth factors. We initiated chondrogenesis of cells by application of physical forces to form 3D cell clusters. After initiation, we designated four experimental groups for differentiation of cells, as follows: control, 10 ng/mL TGFβ3, 100 ng/mL BMP6, and the combination of 5 ng/mL TGFβ3 and 50 ng/mL BMP6. For analysis of chondrogenesis, GAG contents, mRNA expression, histological analysis and immunohistochemistry (IHC) were performed. For analysis of GAG contents, GAG assay was performed and RT-PCR was performed for determination of chondrogenic markers. Histological analysis was performed through safranin O, alcian blue, and IHC was performed using collagen type I and II. GAG contents were increased 184% by TGFβ3, 147% by BMP6, and 189% by the combination of TGFβ3 and BMP6, compared to control. The growth factors improved collagen II and aggrecan expression; in particular, TGFβ3 and BMP6 showed a synergistic effect, compared to only TGFβ3 or BMP6 treated. The results of histological and IHC analysis indicated that chondrogenic differentiation in TGFβ3 and the combination of TGFβ3 and BMP6 showed more cartilage deposition. In conclusion, TGFβ3 and BMP6 differentiated hUC-MSCs into chondrogenic clusters of the combination treatment of the two growth factors showed more efficient chondrogenic ability.

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cord-derived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.

3BK21 program for Veterinary Science, College of Veterinary Medicine‘ Seoul National University. Seoul. Korea Human Co1'd blood has been used for the alternatives of bone marrow transplantation for more 10 years. Recently Mesenchymal s tem cell s , ES-like cells and endothelial stem cells has been successfuly isolated from huam co1'd blood Presentl y. it has been reported that a bout 70 incurable ans tractible di sease was possibly cured by umbili cal cord blood-deri ved s tem cells in the clinit;al test s‘ However‘ isolation and expansion of s tem cells from human umbilical cord blood(UCB) have been very difficult and an obstrucle for the clinical use This study showed that effi cient s iolat iona and expans ion of mesenchymal stem cells from UCB Full term UCB samples were obtained from the umbi lical vein after vaginal deli ve ry with the informed consent 0 1' the mothe1' approved by Borame Hospital Institutional Review Broad (IRB). And a lso. t his work was also a pproved by Seoul National University IRB. Recently, we isolated a population of s tem cells from human corcl bloocl (UCB)‘ which expressed embryo stage specific maker. SSEA-4. ancl the multi-potential stem cell marker‘ 。c t4 And we have sucessfully developed culture methods to expand ancl subculture these cells up to 1.000 billion from one single clone. Subsequently. we were a ble to transclifferente theses stem cells into insulin- producing is let- like structures. which co-express in sulin andC-pepticle, adipocyte, neuron‘ bone and cartilage. In acldition. the isola tion rate of MSC from UCB is about 70 % from the cord blood units. This isolation rate were not affected by maternal ages. the sex of baby, isolation time from the deli very. for example. 12 hrs. 24 hrs ‘ even 48 hrs from delivery Taken together. these findings might have a s ignificant potential to aclvance human UCB clerivecl stem- cell -basecl ther apeutics fOI' clinical use in near future

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.

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.

One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) in N2 medium and N2 supplement. The immunoreactive cells for -tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, -tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.

줄기세포를 임상에 적용하기 위해서는 체외에서 증식 과정이 필수적이다. 그러나 배아줄기세포와는 달리 성체줄기세포는 체외에서 증식할 경우 일정시간이 지나면 줄기세포의 특성을 잃기 때문에 임상사용에 있어 제한점을 가지고 있다. 이러한 문제점을 극복하기 위해 줄기세포의 특성을 잃지 않게 세포를 보존하는 방법이 필요하며, 본 연구에서는 탯줄 유래 줄기세포를 동결 보존한 후 해동시켜 줄기세포의 특성을 분석하였다. 사람의 탯줄 유래 세포를 분리하여 체외에서 배양한 후 2번째 또는 3번째 계대의 세포를 25% FBS와 10% DMSO가 첨가된 냉동배양액에 넣어 196℃에서 동결보존한 후, 6개월 뒤에 해동시켜 세포의 성장 속도와 유전자 및 단백질 발현을 살펴보았다. 냉동 보존한 후 세포를 해동시킨 결과74%의 생존율을 보였으며, 이 세포를 체외에서 배양하였을 경우, 냉동보존하기 전의 세포와 유사하게 방추사 모양의 섬유아세포의 형태를 나타냈다. 또한, 성장 속도 역시 냉동보존하기 전의 세포와 똑같이 10번째 계대까지 배양되었으며, 42번분열 능력을 나타냈다. RT-PCR 결과, 냉동 전후 세포 모두에서 Oct-4, nanog, SCF, NCAM, nestin, GATA4, BMP4, HLA-1 유전자는 모두 발현하였으며, Brachyury와 HLA-DR은 발현하지 않았다. 면역세포 화학 염색 결과, 배아줄기세포 단백질로 알려진 SSEA-3, -4, Oct-4 그리고 중간엽줄기세포 단백질인 Thy-1은 모두 발현하였으며, vimentin, fibronectin, HLA-1, HCAM, ICAM 모두 발현하였다. 그러나 SSEA-4과 Thy-1, vimentin, fibronectin, HLA-1는 냉동보존한 후 배양된 탯줄유래 세포에서 발현량이 증가하는 양상을 보였으며, CD44와 CD54는 감소하는 양상을 나타냈다. 또한, 조직적합성복합체 항원인 HLA-DR은 냉동보존 전후 탯줄 유래 세포에서 모두 발현하지 않았다. 이와 같이 유전자와 단백질의 발현은 냉동보존하기 전후의 탯줄 유래 세포에서 큰 차이가 없었다. 냉동 보존된 탯줄 유래 세포는 세포의 분열능력과 유전자 및 단백질의 발현이 냉동 보존 전 세포와 유사한 것으로 나타났다. 이러한 결과는 냉동보존법이 임상적으로 세포 치료 시 적절한 세포의 수나 시간을 맞추는데 효과적인 방법이 될 수 있을 것으로 기대된다.

인간 제대혈 세포는 조혈모세포, 중간엽 줄기세포와내피전구세포를 풍부하게 포함하고 있다. 인간 제대혈 속의 중간엽 줄기세포는 조혈모세포와는 달리 다능성 줄기세포이며 신경세포로 분화할 수 있는 잠재성을 가지고 있다. 본 연구에서는 세포배양을 통해 제대혈의 중간엽 줄기세포를 신경세포와 콜린성 신경세포로 분화를 유도하였다. 중간엽 줄기세포를 신경세포로 분화시키기 위해 배양액에 dimethyl sulphoxide(DMSO)와 butylated hydroxyani