• Molecules and Cells
• /
• v.25 no.2
• /
• pp.216-223
• /
• 2008

It has been reported that bone marrow (BM)-side population (SP) cells, with hematopoietic stem cell activity, can transdifferentiate into cardiomyocytes and contribute to myocardial repair. However, this has been questioned by recent studies showing that hematopoietic stem cells (HSCs) adopt a hematopoietic cell lineage in the ischemic myocardium. The present study was designed to investigate whether BM-SP cells can in fact transdifferentiate into functional cardiomyocytes. Phenotypically, BM-SP cells were $19.59%{\pm}9.00\;CD14^+$, $8.22%{\pm}2.72\;CD34^+$, $92.93%{\pm}2.68\;CD44^+$, $91.86%{\pm}4.07\;CD45^+$, $28.48%{\pm}2.24\;c-kit^+$, $71.09%{\pm}3.67\;Sca-1^+$. Expression of endothelial cell markers (CD31, Flk-1, Tie-2 and VEGF-A) was higher in BM-SP cells than whole BM cells. After five days of co-culture with neonatal cardiomyocytes, $7.2%{\pm}1.2$ of the BM-SP cells expressed sarcomeric ${\alpha}$-actinin as measured by flow cytometry. Moreover, BM-SP cells co-cultured on neonatal cardiomyocytes fixed to inhibit cell fusion also expressed sarcomeric ${\alpha}$-actinin. The co-cultured BM-SP cells showed neonatal cardiomyocyte-like action potentials of relatively long duration and shallow resting membrane potential. They also generated calcium transients with amplitude and duration similar to those of neonatal cardiomyocytes. These results show that BM-SP cells are capable of functional cardiomyogenic differentiation when co-cultured with neonatal cardiomyocytes.

• Restorative Dentistry and Endodontics
• /
• v.48 no.2
• /
• pp.18.1-18.10
• /
• 2023

Objectives: This study aimed to determine whether collagen triple helix repeat containing-1 (CTHRC1), which is involved in vascular remodeling and bone formation, can stimulate odontogenic differentiation and angiogenesis when administered to human dental pulp stem cells (hDPSCs). Materials and Methods: The viability of hDPSCs upon exposure to CTHRC1 was assessed with the WST-1 assay. CTHRC1 doses of 5, 10, and 20 ㎍/mL were administered to hDPSCs. Reverse-transcription polymerase reaction was used to detect dentin sialophosphoprotein, dentin matrix protein 1, vascular endothelial growth factor, and fibroblast growth factor 2. The formation of mineralization nodules was evaluated using Alizarin red. A scratch wound assay was conducted to evaluate the effect of CTHRC1 on cell migration. Data were analyzed using 1-way analysis of variance followed by the Tukey post hoc test. The threshold for statistical significance was set at p < 0.05. Results: CTHRC1 doses of 5, 10, and 20 ㎍/mL had no significant effect on the viability of hDPSCs. Mineralized nodules were formed and odontogenic markers were upregulated, indicating that CTHRC1 promoted odontogenic differentiation. Scratch wound assays demonstrated that CTHRC1 significantly enhanced the migration of hDPSCs. Conclusions: CTHRC1 promoted odontogenic differentiation and mineralization in hDPSCs.

• IMMUNE NETWORK
• /
• v.23 no.6
• /
• pp.45.1-45.22
• /
• 2023

Interstitial lung disease (ILD) involves persistent inflammation and fibrosis, leading to respiratory failure and even death. Adult tissue-derived mesenchymal stem cells (MSCs) show potential in ILD therapeutics but obtaining an adequate quantity of cells for drug application is difficult. Daewoong Pharmaceutical's MSCs (DW-MSCs) derived from embryonic stem cells sustain a high proliferative capacity following long-term culture and expansion. The aim of this study was to investigate the therapeutic potential of DW-MSCs in experimental mouse models of ILD. DW-MSCs were expanded up to 12 passages for in vivo application in bleomycin-induced pulmonary fibrosis and collagen-induced connective tissue disease-ILD mouse models. We assessed lung inflammation and fibrosis, lung tissue immune cells, fibrosis-related gene/protein expression, apoptosis and mitochondrial function of alveolar epithelial cells, and mitochondrial transfer ability. Intravenous administration of DWMSCs consistently improved lung fibrosis and reduced inflammatory and fibrotic markers expression in both models across various disease stages. The therapeutic effect of DW-MSCs was comparable to that following daily oral administration of nintedanib or pirfenidone. Mechanistically, DW-MSCs exhibited immunomodulatory effects by reducing the number of B cells during the early phase and increasing the ratio of Tregs to Th17 cells during the late phase of bleomycin-induced pulmonary fibrosis. Furthermore, DW-MSCs exhibited anti-apoptotic effects, increased cell viability, and improved mitochondrial respiration in alveolar epithelial cells by transferring their mitochondria to alveolar epithelial cells. Our findings indicate the strong potential of DW-MSCs in the treatment of ILD owing to their high efficacy and immunomodulatory and anti-apoptotic effects.

• Laboraroty Animal Research
• /
• v.34 no.4
• /
• pp.264-269
• /
• 2018

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors $p16^{Ink4a}$ (Cdkn2a, cyclin-dependent kinase inhibitor 2a), $p19^{Arf}$ (an alternative reading frame product of Cdkn2a,), and $p27^{Kip1}$ (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The $p16^{Ink4a}$ and $p19^{Arf}$ knockout mice were generated via transcription activator-like effector nucleases (TALENs), and $p27^{Kip1}$ knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

• Investigative Magnetic Resonance Imaging
• /
• v.16 no.1
• /
• pp.47-54
• /
• 2012

Purpose : This study was performed to evaluate the characteristics of rat mesenchymal stem cells (RMSCs) transduced with human ferritin gene and investigate $in$ $vitro$ MRI detectability of ferritin-transduced RMSCs. Materials and Methods: The RMSCs expressing both myc-tagged human ferritin heavy chain subunit (myc-FTH) and green fluorescence protein (GFP) were transduced with lentiviurs. Transduced cells were sorted by GFP expression using a fluorescence-activated cell sorter. Myc-FTH and GFP expression in transduced cells were detected by immunofluorescence staining. The cell proliferative ability and viability were assessed by MTT assay. The RMSC surface markers (CD29+/CD45-) were analyzed by flow cytometry. The intracellular iron amount was measured spectrophotometically and the presence of ferritin-iron accumulation was detected by Prussian blue staining. $In$ $vitro$ magnetic resonance imaging (MRI) study of cell phantoms was done on 9.4 T MR scanner to evaluate the feasibility of imaging the ferritin-transduced RMSCs. Results: The myc-FTH and GFP genes were stably transduced into RMSCs. No significant differences were observed in terms of biologic properties in transduced RMSCs compared with non-transduced RMSCs. Ferritin-transduced RMSCs exhibited increased iron accumulation ability and showed significantly lower $T_2$ relaxation time than non-transduced RMSCs. Conclusion: Ferritin gene as MR reporter gene could be used for non-invasive tracking and visualization of therapeutic mesenchymal stem cells by MRI.

• Molecules and Cells
• /
• v.24 no.3
• /
• pp.343-350
• /
• 2007

Mesenchymal stem/progenitor cells (MPCs) were isolated from porcine umbilical cord blood (UCB) and their morphology, proliferation, cell cycle status, cell-surface antigen profile and expression of hematopoietic cytokines were characterized. Their capacity to differentiate in vitro into osteocytes, adipocytes and chondrocytes was also evaluated. Primary cultures of adherent porcine MPCs (pMPCs) exhibited a typical fibroblast-like morphology with significant renewal capacity and proliferative ability. Subsequent robust cell growth was indicated by the high percentage of quiescent (G0/G1) cells. The cells expressed the mesenchymal surface markers, CD29, CD49b and CD105, but not the hematopoietic markers, CD45 and CD133 and synthesized hematopoietic cytokines. Over 21 days of induction, the cells differentiated into osteocytes adipocytes and chondrocytes. The expression of lineage specific genes was gradually upregulated during osteogenesis, adipogenesis and chondrogenesis. We conclude that porcine umbilical cord blood contains a population of MPCs capable of self-renewal and of differentiating in vitro into three classical mesenchymal lineages.

• BMB Reports
• /
• v.55 no.6
• /
• pp.281-286
• /
• 2022

Hepatocellular carcinoma is a major health burden, and though various treatments through much research are available, difficulties in early diagnosis and drug resistance to chemotherapy-based treatments render several ineffective. Cancer stem cell model has been used to explain formation of heterogeneous cell population within tumor mass, which is one of the underlying causes of high recurrence rate and acquired chemoresistance, highlighting the importance of CSC identification and understanding the molecular mechanisms of CSC drivers. Extracellular CSC-markers such as CD133, CD90 and EpCAM have been used successfully in CSC isolation, but studies have indicated that increasingly complex combinations are required for accurate identification. Pseudogene-derived long non-coding RNAs are useful candidates as intracellular CSC markers - factors that regulate pluripotency and self-renewal - given their cancer-specific expression and versatile regulation across several levels. Here, we present the use of microarray data to identify stemness-associated factors in liver cancer, and selection of sole pseudogene-derived lncRNA ZNF204P for experimental validation. ZNF204P knockdown impairs cell proliferation and migration/invasion. As the cytosolic ZNF204P shares miRNA binding sites with OCT4 and SOX2, well-known drivers of pluripotency and self-renewal, we propose that ZNF204P promotes tumorigenesis through the miRNA-145-5p/OCT4, SOX2 axis.

• Reproductive and Developmental Biology
• /
• v.35 no.1
• /
• pp.67-75
• /
• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.36 no.2
• /
• pp.87-93
• /
• 2010

Introduction: In our previous studies, we isolated porcine skin-derived mesenchymal stem cells (pSDMSCs) from the ears of adult miniature pigs and evaluated the pluripotency of these pSDMSCs based on expressions of transcription factors, such as Oct-4, Sox-2, and Nanog. Moreover, the characteristic of mesenchymal stem cells was revealed by the expression of various mesenchymal stem cell markers, including CD29, CD44, CD90, and vimentin. The aim of this study was to evaluate in vivo osteogenesis after maxillary sinus lift procedures with autogenous pSDMSCs and scaffold. Materials and Methods: The autogenous pSDMSCs were isolated from the 4 miniature pigs, and cultured to 3rd passage with same methods of our previous studies. After cell membranes were labeled using a PKH26, $1{\times}10^{7}$ cells/$100{\mu}L$ of autogenous pSDMSCs were grafted into the maxillary sinus with a demineralized bone matrix (DBM) and fibrin glue scaffold. In the contralateral control side, only a scaffold was grafted, without SDMSCs. After two animals each were euthanized at 2 and 4 weeks after grafting, the in vivo osteogenesis was evaluated with histolomorphometric and osteocalcin immunohistochemical studies. Results: In vivo PKH26 expression was detected in all specimens at 2 and 4 weeks after grafting. Trabecular bone formation and osteocalcin expression were more pronounced around the grafted materials in the autogenous pSDMSCs-grafted group compared to the control group. Newly generated bone was observed growing from the periphery to the center of the grafted material. Conclusion: The results of the present study suggest that autogenous skin-derived mesenchymal stem cells grafting with a DBM and fibrin glue scaffold can be a predictable method in the maxillary sinus floor elevation technique for implant surgery.

• Development and Reproduction
• /
• v.20 no.1
• /
• pp.63-71
• /
• 2016

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.