Hee-Hoon Yoon

Effect of Ascorbic Acid on Bone Marrow-Derived Mesenchymal Stem Cell Proliferation and Differentiation

Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors, cytokines, and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover, ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study, we evaluated the effects of ascorbic acid on the proliferation, differentiation, extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks, following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover, the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration.

Hepatocyte transplantation for glycogen storage disease type Ib.

Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders with an incidence of 1 in 100,000. The two major subtypes are GSD-Ia, caused by a deficiency of glucose-6-phosphatase (G6Pase), and GSD-Ib, caused by a deficiency of glucose-6-phosphate transporter (G6PT). We report that a substantial improvement was achieved following several infusions of hepatocytes in a patient with GSD-Ib. Hepatocytes were isolated from the unused cadaveric whole livers of two donors. At the first transplantation, approximately 2 × 109 cells (2% of the estimated recipients total hepatocytes) were infused. Seven days later 1 × 109 (1% of liver mass) cryopreserved hepatocytes from the same donor were infused, and an additional 3 × 109 (3% of liver mass) cells from the second donor were infused 1 month after the second transplantation. After the hepatocyte transplantation, the patient showed no hypoglycemic symptoms despite the discontinuation of cornstarch meals. Liver biopsies on posttransplantation days 20 and 250 showed a normal level of glucose-6-phosphatase activity in presolubilization assay that was very low before transplantation. This was the first and successful clinical hepatocyte transplantation in Korea. In this study, hepatocyte transplantation allowed a normal diet in a patient with GSD-Ib, with substantial improvement in their quality of life. Hepatocyte transplantation might be an alternative to liver transplantation and dietary therapy in GSD-Ib.

Increase in Cell Migration and Angiogenesis in a Composite Silk Scaffold for Tissue-Engineered Ligaments

The purpose of this study was to evaluate the biocompatibility of silk and collagen‐hyaluronan (HA) in vitro by assessing anterior cruciate ligament (ACL) cell and T‐lymphocyte cultures on scaffolds. The use of composite scaffolds as artificial ligaments in ACL reconstruction and their effects on angiogenesis were evaluated in vivo. The silk scaffold was knitted by hand and dry coated with collagen‐HA, whereas the composite silk scaffold was made by covering a silk scaffold with a lyophilized collagen‐HA substrate. The initial attachment and proliferation of human ACL cells on the composite silk scaffold was superior to the attachment and proliferation observed on the silk scaffold. The immune response was higher in both scaffolds after 72 h (p < 0.05) compared with the control culture condition without scaffolding, as assessed by T‐lymphocyte cultures in vitro. There was no significant difference in the immune response in vitro between the silk and composite silk scaffolds. Silk and composite silk scaffolds were implanted as artificial ligaments in ACLs removed from the knees of dogs, and they were harvested 6 weeks after implantation. On gross examination, the onset of an inflammatory tissue reaction, such as synovitis, was seen in both the silk scaffold and the composite silk scaffold groups. An histological evaluation of the artificial ligament implants revealed the presence of monocytes in the silk composite scaffold and the absence of giant cells in all cases. MT staining in the composite silk scaffold‐grafted group showed granulation tissue consisting of fibroblasts, lymphocytes, monocytes, and collagen fibers. In addition, CD31 staining revealed the formation of new blood vessels. On the other hand, no reparative tissues, such as blood vessels, collagen, and cells, were observed in the silk scaffold‐grafted group. These results suggest that the lyophilized collagen‐HA substrate is biocompatible in vitro and enhances new blood vessel and cell migration in vivo.

Neural stimulation on human bone marrow-derived mesenchymal stem cells by extremely low frequency electromagnetic fields.

Adult stem cells are considered multipotent. Especially, human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have the potential to differentiate into nerve type cells. Electromagnetic fields (EMFs) are widely distributed in the environment, and recently there have been many reports on the biological effects of EMFs. hBM‐MSCs are weak and sensitive pluripotent stem cells, therefore extremely low frequency‐electromagnetic fields (ELF‐EMFs) could be affect the changes of biological functions within the cells. In our experiments, ELF‐EMFs inhibited the growth of hBM‐MSCs in 12 days exposure. Their gene level was changed and expression of the neural stem cell marker like nestin was decreased but the neural cell markers like MAP2, NEUROD1, NF‐L, and Tau were induced. In immunofluorescence study, we confirmed the expression of each protein of neural cells. And also both oligodendrocyte and astrocyte related proteins like O4 and GFAP were expressed by ELF‐EMFs. We suggest that EMFs can induce neural differentiation in BM‐MSCs without any chemicals or differentiation factors.

Application of mesenchymal stem cells derived from bone marrow and umbilical cord in human hair multiplication

BACKGROUND The methods currently used for treating alopecia have some limitations. The drug treatment is so temporary that medication discontinuance may progress depilation immediately. The number of hair transplantation restricts because total transplantable hair number is no increase. To overcome these problems, researchers have attempted the in vitro culturing of hair follicle cells and implanting these cells in the treatment area. OBJECTIVES In the present study, culture-expanded mesenchymal stem cells (MSCs) that do not possess aggregative activity were used to produce self-aggregated cell-aggregated spheroidal dermal papilla like tissues (DPLTs) with the aid of a special culture condition in vitro, and hair bulb structure inductive capacity pertinent to the aggregative activity was then evaluated. Then hair inducing activity of self-aggregated DPLTs employing MSCs was tested in athymic mice. METHODS We isolated and cultivated MSCs from bone marrow and umbilical cord in vitro. After propagated MSCs underwent preconditioning in dermal papilla forming medium (DPFM), then subcultured MSCs formed self-aggregated DPLTs. We compared real human scalp dermal papilla cells (hDPCs) with DPLTs employing DPCs, DPLTs employing hBM-MSCs and DPLTs employing hUC-MSCs. RESULTS Light microscopy and immunohistochemical staining were used to confirm that reconstructed DPLTs generated by this procedure had the size, shape, and expression of protein similar to actual DP. CONCLUSIONS The DPLTs have the same hair bulb structure inductive ability as natural DPLTs in vitro. Transplanted DPLTs can induce new hair follicle in athymic mice. As a result, UC-MSCs and BM-MSCs may be an applicable and novel cell source for the generation of human hair cell therapy.

Effect of Mechanical Stimulation on the Differentiation of Cord Stem Cells

In this study, we evaluated the effect of mechanical stimulation on the differentiation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) in osteogenic medium using a Flexcell system that imposed cyclic uniaxial mechanical stimulation at a strain of 0%, 5%, or 10% (5 s of stretch and 15 s of relaxation) for 10 days. The expression of MSC surface antigens (CD73, CD90, and CD105) was significantly decreased as strain increased. Mechanical stimulation inhibited the growth of UC-MSCs and slightly raised lactate dehydrogenase production. Mechanically stimulated groups produced more elastin and sulfated glycosaminoglycan than unstimulated groups and these increases were in proportion to the degree of strain. Reverse transcription-polymerase chain reaction analysis revealed that mechanical stimulation induced a significant increase in the mRNA expression of osteoblast differentiation markers. The mRNA levels of osteopontin, osteonectin, and type I collagen in the 5% and 10% strained groups were significantly higher than those in the 0% strained group. From the Western blot analysis, UC-MSCs produced bone sialoprotein and vimentin in a mechanical strain-dependent manner. Thus, cyclic mechanical loading was able to enhance the differentiation of human UC-MSCs into osteoblast-like cells as determined by osteogenic gene and protein expression. Furthermore, this finding has important implications for the use of the combination of mechanical and osteogenic differentiation media for UC-MSCs in tissue engineering and regenerative medicine.

Multipotent Progenitor Cells Derived From Human Umbilical Cord Blood Can Differentiate Into Hepatocyte-Like Cells in a Liver Injury Rat Model

Umbilical cord blood (UCB), a rich source of hematopoietic stem cells, offers practical and ethical advantages. It has been reported that various adult stem cells transplanted into a damaged liver show characteristics of a hepatic lineage. In a previous study, we reported on novel UCB-derived adult stem cells, termed umbilical cord blood-derived multipotent progenitor cells (UCB-MPCs). We demonstrated that these cells were capable of differentiating into hepatocyte- like cells in vitro. To assess the hepatic differentiation capacity of UCB-MPCs, rat models of hepatic injury were generated using carbon tetra-chloride (CCl(4)) with transplantation of cells into the liver. The transplanted cells successfully incorporated into the liver of the recipient animal differentiated into functional hepatocyte-like cells that expressed hepatocyte-specific markers, such as CK-18 and albumin. Moreover, human albumin was detected in the serum of the recipient rat model. These data indicated that UCB-MPCs were capable of displaying similar characteristics to those of functional hepatocytes in a recipient liver. UCB-MPCs may prove to be a useful, transplantable alternative for hepatic progenitor cells in both experimental and therapeutic applications.

Neural differentiation of umbilical cord mesenchymal stem cells by sub-sonic vibration.

AIMS Adult stem cells, such as umbilical cord-derived mesenchymal stem cells (UC-MSCs), have the potential to differentiate into various types of cells, including neurons. Research has shown that mechanical stimulation induces a response in MSCs, specifically, low and high intensity sub-sonic vibration (SSV) has been shown to facilitate wound healing. In this study, the effects of SSV were examined by assessing the proliferation and differentiation properties of MSCs. MAIN METHODS hUC-MSCs were isolated from Whartons jelly, including the smooth muscle layer of the umbilical cord. During subculture, the cells were passaged every 5-6 days using nonhematopoietic stem cell media. To measure the effect of sonic vibration, SSV was applied to these cells continuously for 5 days. KEY FINDINGS In this study, the morphology of hUC-MSCs was altered to resemble neurons by SSV. Further, the mRNA and protein levels of neuron-specific markers, including MAP2, NF-L, and NeuroD1, increased. In addition, other neural cell markers, such as GFAP and O4, were increased. These results suggest that hUC-MSCs differentiated into neural cells upon SSV nonselectively. In a mechanism study, the ERK level increased in a time-dependent manner upon SSV for 12 h. SIGNIFICANCE The results of this study suggest that SSV caused hUC-MSCs to differentiate into neural cells via ERK activation.

Effects of sub-sonic vibration on the proliferation and maturation of 3T3-L1 cells

AIMS Although low and high intensity sub-sonic vibrations (SSV) have been shown to facilitate wound healing, very few studies have investigated the effects of SSV on 3T3-L1 preadipocytes. Therefore, the present study was undertaken to investigate the influence of SSV on the proliferation and maturation of 3T3-L1 preadipocytes. MAIN METHODS To evaluate the effect of SSV on 3T3-L1 cell proliferation, the cells were maintained in an apparatus that administered SSV (0.5 V) for 3 days at a frequency of 10, 20, 30, or 40 Hz. In addition, to study the effect of SSV on 3T3-L1 cell maturation, the cells were stimulated with SSV for 6 days at a frequency of 10, 20, 30, or 45 Hz. KEY FINDINGS Sub-sonic vibrations inhibited the proliferation of 3T3-L1 preadipocytes at frequencies of 20 and 30 Hz. Triglyceride levels in cells subjected to SSV at frequencies ranging from 10 to 30 Hz increased compared with those measured in control cells. The expression of adipogenic genes, such as PPAR-γ and C/EBP-α, markedly increased in response to SSV at 20 Hz and 30 Hz during maturation. SIGNIFICANCE These results suggest that SSV affected adipogenic gene expression at 20 and 30 Hz.

Improved isolation of outer root sheath cells from human hair follicles and their proliferation behavior under serum-free condition

Hair follicle is a small but very complex and dynamic miniorgan of the human body. It is easy to isolate and culture mesenchymal cells but not epithelial cells of hair follicle. It is necessary for intact and healthy outer root sheath (ORS) cells to be isolated and cultured. In this study we developed an appropriate isolation method to yield 6.4±0.75×104 cells/hair follicle, which is about 9-fold comparing to our previous data. This yield was achieved by modifications such as different kinds of enzyme uses, fragmentation, and mechanical stimuli. Especially we detected that the different kinds of isolation enzyme could affect proliferation of ORS cells during primary culture. In addition, bovine pituitary extract (BPE) was needed for ORS cells to proliferate and to form colonies under serum-free, feeder layer-free culture condition, but type I collagen as a substratum did not have any positive effect. Moreover, ORS cells under BPE-added condition contained stem/progenitor cells expressing β1-integrin. CK19, and CD34. These results can provide useful cell culture information, not only in the study of hair biology but also in the field of tissue engineering and cell therapy for the treatment of alopecia.