Ryoung-Hoon Jeon

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor.

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy.

Tissue-engineered bone formation using periosteal-derived cells and polydioxanone/pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells.

The aim of this study was to generate tissue-engineered bone formation using periosteal-derived cells seeded into a polydioxanone/pluronic F127 (PDO/Pluronic F127) scaffold with adipose tissue-derived CD146 positive endothelial-like cells. Considering the hematopoietic and mesenchymal phenotypes of adipose tissue-derived cells cultured in EBM-2 medium, CD146 positive adipose tissue-derived cells was sorted to purify more endothelial cells in characterization. These sorted cells were referred to as adipose tissue-derived CD146 positive endothelial-like cells. Periosteum is a good source of osteogenic cells for tissue-engineered bone formation. Periosteal-derived cells were found to have good osteogenic capacity in a PDO/Pluronic F127 scaffold, which could provide a suitable environment for the osteoblastic differentiation of these cells. Through the investigation of capillary-like tube formation on matrigel and the cellular proliferation of adipose tissue-derived CD146 positive endothelial-like cells cultured in different media conditions, we examined these cells could be cultured in EBM-2 with osteogenic induction factors. We also observed that the osteogenic activity of periosteal-derived cells could be good in EBM-2 with osteogenic induction factors, in the early period of culture. The experimental results obtained in the miniature pig model suggest that tissue-engineered bone formation using periosteal-derived cells and PDO/Pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells can be used to restore the bony defects of the maxillofacial region when used in clinics.

Comparison of Immunomodulation Properties of Porcine Mesenchymal Stromal/Stem Cells Derived from the Bone Marrow, Adipose Tissue, and Dermal Skin Tissue

Mesenchymal stromal/stem cells (MSCs) demonstrate immunomodulation capacity that has been implicated in the reduction of graft-versus-host disease. Accordingly, we herein investigated the capacity of MSCs derived from several tissue sources to modulate both proinflammatory (interferon [IFN] γ and tumor necrosis factor [TNF] α) and immunosuppressive cytokines (transforming growth factor [TGF] β and interleukin [IL] 10) employing xenogeneic human MSC-mixed lymphocyte reaction (MLR) test. Bone marrow-derived MSCs showed higher self-renewal capacity with relatively slow proliferation rate in contrast to adipose-derived MSCs which displayed higher proliferation rate. Except for the lipoprotein gene, there were no marked changes in osteogenesis- and adipogenesis-related genes following in vitro differentiation; however, the histological marker analysis revealed that adipose MSCs could be differentiated into both adipose and bone tissue. TGFβ and IL10 were detected in adipose MSCs and bone marrow MSCs, respectively. However, skin-derived MSCs expressed both IFNγ and IL10, which may render them sensitive to immunomodulation. The xenogeneic human MLR test revealed that MSCs had a partial immunomodulation capacity, as proliferation of activated and resting peripheral blood mononuclear cells was not affected, but this did not differ among MSC sources. MSCs were not tumorigenic when introduced into immunodeficient mice. We concluded that the characteristics of MSCs are tissue source-dependent and their in vivo application requires more in-depth investigation regarding their precise immunomodulation capacities.

Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo.

BackgroundRecent findings have revealed that the female gonad may have regenerative activity with having germ line stem cells in juveniles and adults. Application of these germ line stem cells could be an alternative therapy for reproductive disorders in regenerative medicine.MethodsTo enhance the potency of differentiation into oocyte-like cells (OLCs) and folliculogenesis, we overexpressed Oct4 in ovarian stem/stromal cell (OvSCs) and examined the cellular properties related to stemness and self-renewal ability and finally demonstrated the ability of in vitro differentiation and folliculogenesis.ResultsOvarian cortex included putative stem cells in terms of AP activity, cell cycle status, cell proliferation, expression of mesenchymal lineage surface markers and pluripotent transcriptional markers. Further, Oct4 transfected OvSCs (Oct4-OvSCs) were enhanced their AP activity and cell proliferation compared to OvSCs. The potential on in vitro differentiation into OLCs and in vivo folliculogenesis was also evaluated in OvSCs and Oct4-OvSCs, respectively. Oct4-OvSCs possessed higher oogenesis potential in vitro than OvSCs, in terms of expression of germ cell markers by RT-PCR and the number of OLCs. When OvSCs and Oct4-OvSCs were xeno-transplanted into infertile mice ovaries, the OvSCs transplantation induced new primary follicle formation and hormonal levels of estradiol and FSH remained similar to that of normal mice. However, Oct4-OvSCs possessed higher ability for folliculogenesis based on inducing developing follicles with thecal layer and granulosa cells and more similar estradiol level to normal mice.ConclusionsThese findings demonstrated that putative stem cells were present in ovarian cortex and exhibited differentiation ability into OLCs and folliculogenesis in vivo, and Oct4-overexpression enhanced these ability, suggesting their cellular models based on gene therapy in understanding the mechanisms of oogenesis and folliculogenesis, and finally in view of reproductive cell therapy.

Generation of osteogenic construct using periosteal‐derived osteoblasts and polydioxanone/pluronic F127 scaffold with periosteal‐derived CD146 positive endothelial‐like cells

The purpose of this study was to generate tissue-engineered bone using human periosteal-derived osteoblasts (PO) and polydioxanone/pluronic F127 (PDO/pluronic F127) scaffold with preseeded human periosteal-derived CD146 positive endothelial-like cells (PE). PE were purified from the periosteal cell population by cell sorting. One of the important factors to consider in generating tissue-engineered bone using osteoprecursor and endothelial cells and a specific scaffold is whether the function of osteoprecursor and endothelial cells can be maintained in originally different culture medium conditions. After human PE were preseeded into PDO/pluronic F127 scaffold and cultured in endothelial cell basal medium-2 for 7 days, human PO were seeded into the PDO/pluronic F127 scaffold with PE, and then, this cell-scaffold construct was cultured in endothelial cell basal medium-2 with osteogenic induction factors, including ascorbic acid, dexamethasone, and β-glycerophosphate, for a further 7 days. Then, this 2-week cultured construct was grafted into the mandibular defect of miniature pig. Twelve weeks after implantation, the animal was sacrificed. Clinical examination revealed that newly formed bone was seen more clearly in the defect with human PO and PDO/pluronic F127 scaffold with preseeded human PE. The experimental results suggest that tissue-engineered bone formation using human PO and PDO/pluronic F127 scaffold with preseeded human PE can be used to restore skeletal integrity to various bony defects when used in clinics.

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages.

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearsons correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearsons correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs.

Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos

To study gene expression and to determine distinctive characteristics of embryos produced by different methods, normalisation of the gene(s) of interest against reference gene(s) has commonly been employed. Therefore, the present study aimed to assess which reference genes tend to express more stably in single porcine blastocysts produced in vivo (IVO) or by parthenogenetic activation (PA), in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) using different analysis programs, namely geNorm, Normfinder and Bestkeeper. Commonly used reference genes including 18S rRNA (18S), H2A histone family, member Z (H2A), hypoxanthine phosphoribosyltransferase1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein 4 (RPL4), peptidylprolyl isomerase A (PPIA), beta actin (ACTB), succinate dehydrogenase complex, subunit A (SDHA) and hydroxymethylbilane synthase (HMBS2) were analysed; most of them resulted in significantly (P<0.05) different cycle threshold (CT) values in porcine embryos except for SDHA and H2A. In evaluation of stable reference genes across in vivo and in vitro porcine blastocysts, three kinds of programs showed slightly different results; however, there were similar patterns about the rankings of more or less stability overall. In conclusion, SDHA and H2A were determined as the most appropriate reference genes for reliable normalisation in order to find the comparative gene expression in porcine blastocysts produced by different methods, whereas 18S was regarded as a less-stable reference gene. The present study has evaluated the stability of commonly used reference genes for accurate normalisation in porcine embryos to obtain reliable results.

The Effect of Preferable Enrichments in the Laboratory Minipigs

Miniature pig (minipig) has been considered as an important laboratory animal in the developmental biotechnology researches with respect to xenotransplantation, stem cell, somatic cell nuclear transfer and embryo transfer. Given that the laboratory minipigs are normally housed at an indoor facility, they pass the time with lying or sleeping unless it is feeding time. Therefore, it is necessary to provide environmental enrichments to satisfy their innate needs and to lessen atypical behaviors caused by stress, on the purpose of welfare. We quantitatively investigated the type of preferable enrichment for the laboratory minipigs as well as its effect on their daily life. They presented a great interest to the pliable pail but a rapid loss of attraction to non-preferable enrichments. When the daily life of the single housed minipigs was quantified based on duration of playing or resting, they were more actively engaged in lively activities in the presence of enrichments. In addition, the provision of enrichments could effectively alleviate the conflicts during group housing when new pen mate was introduced, resulting in reduction of wound cases. We believe the considerations of animal welfare are essential to the conduct of better research because animals in the non-stressful environment will be more physiologically stable and provide more reliable results in the animal experiments.