줄기세포 기반의 치주조직재생

박재욱,박주철 대한구강해부학회 2014 대한구강해부학회지 Vol.35 No.1

Periodontium is complex tissue composed of cementum, periodontal ligament and alveolar bone which holds the tooth in the bone. periodontitis is main cause of tooth loss leads to loss of attachment of connective tissue and irreversible bony destruction. So periodontitis has been one of the main concern to dentist, patient and oral health system. For recent years the main purpose of periodontics is regeneration of damaged peroidontium on shape, structure and function. In periodontal regeneration, new connective tissue fibers should be inserted in the cementum and bone, and construct the complex cementum-ligament-bone interfaces and provide a functional connection between a tooth and the surrounding jaw. Recently, many surgical, nonsurgical therapies and bone substitutes are using, but the clinical outcomes are still limiting. Bone transplantation or bone substitutes like guided bone regeneration, guided tissue regeneration don't have the capacity to regenerate destructed connective tissue. With cell based therapy, numerous growth factors and modulating agents have used but it made limited success. Stem cell based therapy is the most active researching field in medical and dental area. However, when diseased periodontal condition, tissue repair does not occur naturally because of the lack of sound stem cells. So exogenous regenerative tools such as ex vivo expanded/ manipulated stem cells will be needed to replenish the host cell niche and facililtate tissue regeneration. As the increasing success of regenerating other tissues(skin, cartilage, bone, cardiovascular component, pancreas), stem cell based periodontal regeneration with tissue engineering approach can be new field of treatment. Comparing stem cells from other sites of adult body, dental stem cells have advantage that is easy access to gaining site and have characteristics like proliferation, differentiation, and flexiblity. Some kind of reviewed stem cells, dental pulp stem cells (DPSCs), Stem cells from exfoliated deciduous teeth (SHED), PDL stem cells (PDLSCs), Stem cells from apical papilla, apical papilla stem cells (SCAP), Dental follicle cells (DFCs), and MSCs are most actively studied for stem cell based periodontal regeneration. However, safety problems are not completely examined, and the difficulty of ex vivo proliferation is still recognized to limiatation of stem cells. With effective stem cell delivery strategy, research to overcome these limitations should be continued. With recent advancement of stem cell based periodontal tissue engineering and periodontal regeneration, next step of research should be concentrated to clinical application of this advanced therapeutic method. Accordingly, further studies are required to develop new methods to identify and maintain multipotent stem cells in vitro and to determine the long term safety and efficacy of ex vivo expanded stem cells to repair periodontal defects in large animal models. With partial regeneration of tooth, whole tooth regeneration has been actively studied. When whole tooth regeneration succeed, regenerating periodontal ligament and contact of tooth-periodontium are needed to transplant regenerated tooth. So periodontal regeneration is essential step to achieve whole tooth regeneration and replantation.

Fast and Efficient Isolation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells by Using a Biocompatible Polymer

( Han Soo Kim ),( June Seok Heo ),( Jung Mok You ),( Tea Hoon Park ),( You Jeong Choi ),( Eunk Young Kim ),( Hyun Ok Kim ) 한국조직공학과 재생의학회 2010 조직공학과 재생의학 Vol.7 No.4

Mesenchymal stem cells (MSCs) differentiate into bone, fat, cartilage, tendon, and other organ progenitor cells. The rarity of MSCs in bone marrow necessitates fast and efficient isolation and/or in vitro expansion prior to clinical and biomedical applications. Previously, we reported that UV-exposed diphenylamino-s-triazine bridged p-phenylene vinylene (DTOPV-UV) with a hydrophilic and negative surface-containing carboxyl group is highly biocompatible and provides a substrate for efficient human bone marrow-derived MSC attachment. In this study, we applied this polymeric film to early adhesion and enrichment of MSCs from mouse bone marrow. With its high protein- binding capacity, DTOPV-UV film was more efficient in early capture of adherent bone marrow cells than conventional tissue culture polystyrene (TCPS). Cell binding to DTOPV-UV reached full capacity within 1 hr, whereas cell attachment to TCPS gradually increased over time. The isolated and culture-expanded MSCs from mouse bone marrow displayed typical morphology, phenotype, and differentiation into osteoblasts, adipocytes, and chondrocytes. Here, we demonstrate a novel method for isolating MSCs from mouse bone marrow using a biocompatible polymer. This method will aid the development of rapid and efficient isolation and in vitro expansion protocols for rare adherent cells.

Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells

GREEN DAVID WILLIAM,정한성,권혁재 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.3

Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC’s), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrowderived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC’s led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I-IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC’s.

Osteogenic and Angiogenic Potency of VEGF165-Transfected Canine Bone Marrow Mesenchymal Cells Combined with Coral Hydroxyapatite in Vitro

Zhang Quanyin,Zhang Jie,Chen Lin,Fan Yunjian,Long Jiazhen,Liu Shuguang 한국조직공학과 재생의학회 2021 조직공학과 재생의학 Vol.18 No.5

BACKGROUND: To explore the osteogenic and angiogenic potential of human vascular endothelial growth factor 165 (hVEGF165) gene-transfected canine bone marrow mesenchymal stem cells (BMSCs) combined with coral hydroxyapatite (CHA) scaffold. METHODS: We constructed a lentiviral vector and transfected canine BMSCs with the best multiplicity of infection. Osteogenesis was induced in the transfected groups (GFP-BMSCs group and hVEGF-BMSCs group) and non-transfected group (BMSCs group), followed by the evaluation of alkaline phosphatase (ALP) activity and alizarin red S staining. Cells from the three groups were co-cultured with CHA granules, respectively to obtain the tissue-engineered bone. MTT assay and fluorescence microscopy were employed to assess cell proliferation and adhesion. The expression of osteogenic and angiogenic related genes and proteins were evaluated at 7, 14, 21, and 28 days post osteoinduction in cell culture alone and cell co-culture with CHA, respectively using RT-PCR and ELISA. RESULTS: The hVEGF165 gene was transfected into BMSCs successfully. Higher ALP activity and more calcified nodules were found in the hVEGF-BMSCs group than in the control groups (p < 0.001). Cells attached and proliferated in CHA particles. Both cells cultured alone and cells co-culture with CHA expressed more osteogenic and angiogenic related genes and proteins in the hVEGF-BMSCs group compared to the GFP-BMSCs and BMSCs groups (p < 0.05). CONCLUSION: High expression of hVEGF165 in BMSCs potentially promote the osteogenic potential of BMSCs, and synergically drive the expression of other osteogenic and angiogenic factors. hVEGF-BMSCs co-cultured with CHA expressed more osteogenic and angiogenic related factors, creating a favorable microenvironment for osteogenesis and angiogenesis. Also, the findings have allowed for the construction of a CHA-hVEGF-BMSCs tissue-engineered bone. BACKGROUND: To explore the osteogenic and angiogenic potential of human vascular endothelial growth factor 165 (hVEGF165) gene-transfected canine bone marrow mesenchymal stem cells (BMSCs) combined with coral hydroxyapatite (CHA) scaffold. METHODS: We constructed a lentiviral vector and transfected canine BMSCs with the best multiplicity of infection. Osteogenesis was induced in the transfected groups (GFP-BMSCs group and hVEGF-BMSCs group) and non-transfected group (BMSCs group), followed by the evaluation of alkaline phosphatase (ALP) activity and alizarin red S staining. Cells from the three groups were co-cultured with CHA granules, respectively to obtain the tissue-engineered bone. MTT assay and fluorescence microscopy were employed to assess cell proliferation and adhesion. The expression of osteogenic and angiogenic related genes and proteins were evaluated at 7, 14, 21, and 28 days post osteoinduction in cell culture alone and cell co-culture with CHA, respectively using RT-PCR and ELISA. RESULTS: The hVEGF165 gene was transfected into BMSCs successfully. Higher ALP activity and more calcified nodules were found in the hVEGF-BMSCs group than in the control groups (p < 0.001). Cells attached and proliferated in CHA particles. Both cells cultured alone and cells co-culture with CHA expressed more osteogenic and angiogenic related genes and proteins in the hVEGF-BMSCs group compared to the GFP-BMSCs and BMSCs groups (p < 0.05). CONCLUSION: High expression of hVEGF165 in BMSCs potentially promote the osteogenic potential of BMSCs, and synergically drive the expression of other osteogenic and angiogenic factors. hVEGF-BMSCs co-cultured with CHA expressed more osteogenic and angiogenic related factors, creating a favorable microenvironment for osteogenesis and angiogenesis. Also, the findings have allowed for the construction of a CHA-hVEGF-BMSCs tissue-engineered bone.

Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells

Green, David W.,Kwon, Hyuk-Jae,Jung, Han-Sung Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.3

Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC's), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC's led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I-IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC's.

Profiling of Secretory Proteins from Human Bone Marrow- Derived Mesenchymal Stem Cells in Liver Repair by cDNA Microarray

( Heesun Hong ),( Sang Yeol Kim ),( Meegun Hong ),( Dong Joon Kim ),( Ki Tae Suk ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Background & Aim: Cirrhosis is the end stage of chronic liver disease, which may lead to severe hepatic dysfunction and even life-threatening conditions. The beneficial impact of mesenchymal stem cells (MSCs) transplantation on liver diseases has been confirmed on several studies which have shown the ability of MSCs to reduce liver fibrosis and improve liver function, although the secreted gene and/or proteins and molecular mechanisms behind it remain elusive. We aimed to identify secreted factors by undifferentiated bone marrow-derived mesenchymal stem cells (BM-MSCs) in order to describe related pathways potentially targeted gene by MSC in liver cirrhosis. Methods: Human bone marrow-derived mesenchymal stem cells (BM-MSCs) from normal and BM-MSCs transplanted patients who had liver cirrhosis and cultured specific medium condition for mesenchymal stem cells. We evaluated the differentiation potential to osteoblast and adipocytes, and morphological changes, cell proliferation and immunophenotyping assay were tested with trypan blue exclusion assay and CD34, CD45, CD45 and CD105. At passage 4-5 of BM-MSCs were used for cDNA microarrays to identify secreted genes and related pathway that differentially expressed in specific stem cell population in liver cirrhosis and identified by biomathmatical analysis. Results: BM MSCs secreted different factors normal and patients with liver cirrhosis. We found 2968 genomes of 15 maps in KEGG pathway, include Metabolic pathways, TGF-beta signaling pathway, Wnt signaling pathway, Cytokine-cytokine receptor interaction, HIF-1 signaling pathway, Ras signaling pathway, Natural killer cell mediated cytotoxicity and Antigen processing and presentation. Within these pathways, functionally up-regulated genes were 7 genes and down-regulated genes were 9 genes. In particular, we were able to identify potential specific genes for regulation of liver fibrosis (FBN2, P4HA1 and STC1), and KIR3DL2, which is gene for regulation of immune system process. Conclusion: Application of MSCs might target a widespread pattern of biological, cellular compositional and molecular functional event in the liver. MSC secreted genes and proteins can be differ depending on pathways and molecular mechanisms. Genes involved liver fibrosis are able to release hepatotropic factors from transplanted MSCs, also potentially supporting liver regenerations.

Neural Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells: Applicability for Inner Ear Therapy

Lee, Jae-Hong,Kang, Won Kyung,Seo, Jae-Hyun,Choi, Mi-Yung,Lee, Yang Hyun,Kim, Hyo Min,Park, Kyoung-Ho 대한청각학회 2012 Journal of Audiology & Otology Vol.16 No.2

<P><B>Background and Objectives</B></P><P>Regeneration or replacement of damaged hair cells and neurons in the cochlea might be an ideal treatment for sensorineural hearing loss (SNHL). The purpose of the present study was to investigate whether mesenchymal stem cells (MSCs), derived from the bone marrow of rats, could differentiate into auditory hair cells and neurons.</P><P><B>Materials and Methods</B></P><P>The centrifuge gradient method was used to isolate MSCs from the bone marrow of rats. To confirm whether bone marrow-derived MSCs can differentiate into neuronal cells, culture medium with glial cell-derived neurotrophic factor, brain-derived neurotrophic factor and neurotrophin-3 for 14 days. In addition, immunofluorescence staining and RT-PCR were performed for characterization of the neurospheres and differentiated cells from 7 and 14 day cultures.</P><P><B>Results</B></P><P>The results showed that MSCs could differentiate into neuron-positive and hair cell-positive cells, using different compositions of growth factors. And RT-PCR result was identified high or low of gene expression all these differentiated cells.</P><P><B>Conclusions</B></P><P>Rat bone marrow-derived MSCs differentiated into neuronal progenitor cells. These cells might be useful for the treatment of SNHL.</P>

Induction of the differentiation of porcine bone marrow mesenchymal stem cells into premature hepatocyte-like cells in an indirect coculture system with primary hepatocytes

Ullah Imran,서강민,위하연,김영민,이승훈,옥선아 한국통합생물학회 2020 Animal cells and systems Vol.24 No.5

Liver transplantation is currently the only option for patients with end-stage liver disease. Thus, other alternate therapeutic strategies are needed. Bone marrow mesenchymal stem cells (BMMSCs) are nonhematopoietic cells present in the bone marrow stroma that serve as precursors cells for various other cells. In this study, we evaluated the differentiation of porcine BM-MSCs into hepatocyte-like cells using three types of culture systems: hepatic induction medium (HIM), HIM/primary hepatocyte culture supernatant (HCS; 1:1 ratio), and a hepatocyte coculture system (HCCS; primary hepatocytes in the upper chamber, and BM-MSCs in the lower chamber). Primary hepatocytes were isolated from anesthetized healthy 1-month-old pigs by enzymatic digestion. Hepatic-specific marker expression (albumin [ALB], transferrin [TF], α-fetoprotein [AFP]), glycogen storage, low-density lipoprotein, and indocyanine green uptake were evaluated. Upregulation of hepatic-specific markers (ALB, TF, and AFP) was observed by real-time polymerase chain reaction in the HCCS group. Periodic acid-Schiff staining revealed enhanced glycogen storage in hepatocyte-like cells from the HCCS group compared with that from the HIM/HCS group. Furthermore, hepatocyte like-cells in the HCCS group showed improved LDL and ICG uptake than those in the other groups. Overall, our current study revealed that indirect coculture of primary hepatocytes and BM-MSCs enhanced the differentiation efficacy of BM-MSCs into hepatocyte-like cells by unknown useful soluble factors, including paracrine factors.

WT1 Is the Regulatory Gene in the Process of Hepatocyte-like Cells Differentiation from Bone Marrow Mesenchymal Stem Cells

( Jung Hoon Cha ),( Na Ri Park ),( Ho-shik Kim ),( Jong Young Choi ),( Seung Kew Yoon ),( Si Hyun Bae ) 대한간학회 2016 춘·추계 학술대회 (KASL) Vol.2016 No.1

Aims: Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) have been known to differentiate into multi-lineage cell types and used for differentiated hepatocyte-like cells. The mesenchymal- epithelial transition (MET) plays as a key of cellular transdifferentiation programs, including wound healing and tissue regeneration. Wilms’ tumor suppressor gene (WT1) controls transitions between the mesenchymal and epithelial state of cells. The purpose of this study is to clarify underlying differentiation mechanism and function of WT1 by screening the key factors in hepatic differentiation stem cells. Methods: To detect the regulatory gene of BM-hMSC into functional hepatocytes, protein/DNA array was performed in BM-MSCs before and after differentiation. Hepatic differentiation of BM-hMSCs was evaluated using RT-PCR, western blotting, periodic acid-schiff staining, and a urea synthesis assay. To determine the effect WT1, during induction of hepatic differentiation from BM-hMSCs which were transfected with WT1 siRNA and identified through the change of liver specific genes, transcription factors, and MET markers using RT-PCR and western blotting in WT1-knockdown BM-hMSCs. Results: Here, we demonstrate that WT1 increases during hepatic differentiation of BM-hMSCs. Differentiated hepatocyte-like cells changed in morphology, function and hepatic gene expression. Also, the expressions of epithelial markers were increased, while the expressions of mesenchymal markers were decreased. In contrast, downregulation of WT1 reduced hepatic differentiation. The mRNA expression of Albumin and TAT was decreased in the WT1-knockdown BM-hMSCs during hepatic differentiation. Furthermore, downregulation of WT1 increased the expression of mesenchymal markers but decreased the expression of epithelial markers. Also, during the hepatic differentiation, WT1-knockdown BM-hMSCs didn’t change in morphology, looked spindle or fusiform shape. Conclusions: In this study, we identified novel factors in the process of hepatic differentiation by MET. Our results demonstrate that BM-hMSCs may be a source of cells for liver regeneration and provide the mechanism of liver regeneration through MET process by the WT1.

Modulation of Bone Marrow Mesenchymal Stem Cells Using WT1 and EGR1 during Hepatocyte-Like Cells Differentiation Process

( Jung Hoon Cha ),( Na Ri Park ),( Sung Woo Cho ),( Jung-hee Kim ),( Wonhee Hur ),( Pil Soo Sung ),( Ho-shik Kim ),( Jong Young Choi ),( Seung Kew Yoon ),( Si Hyun Bae ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have been known to differentiate into multi-lineage cell types and used for differentiated hepatocyte-like cells. The mesenchymal-epithelial transition (MET) plays as a key of cellular transdifferentiation programs, including wound healing and tissue regeneration. The purpose of this study is to clarify underlying differentiation mechanism and function of Wilms’ tumor suppressor gene (WT1) and early growth response 1 (EGR1) by screening the key factors in hepatic differentiation stem cells. Methods: To detect the regulatory genes of hBM-MSC into functional hepatocytes, protein/DNA array was performed in hBM-MSCs before and after differentiation. To determine the effects WT1 and EGR1, knockdown and overexpression were obtained respectively by the use of siRNA and pcDNA3.1 (-)- mWT1 (B) or pcDNA3-mEGR1. The role of WT1 and EGR1 were identified through the change of liver-specific genes and MET markers using RT-PCR and western blotting. Results: We demonstrate that WT1 and EGR1 increase during hepatic differentiation of hBM-MSCs. Hepatic differentiated hBM-MSCs increased the expressions of epithelial markers but decreased the expressions of mesenchymal markers. However, downregulation of WT1 or EGR1 reduced hepatic differentiation and didn’t change in morphology, looked spindle or fusiform shape as compared control during hepatic differentiation of hBM-MSCs. Furthermore, the expression of mesenchymal markers was increased, while the expression of epithelial markers decreased in WT1 or EGR1 knockdown hBM-MSCs. In contrast, the expression of epithelial markers was increased, while the expression of mesenchymal markers decreased in WT1 or EGR1 overexpression hBM-MSCs. In addition, overexpressing of WT1 or EGR1 induced rapid maturation of hepatic differentiation. Conclusions: In this study, we identified novel factors in the process of hepatic differentiation by MET. Our results demonstrate that hBM-MSCs may be a source of cells for liver regeneration and provide the mechanism of liver regeneration through MET process by the WT1 and EGR1.