Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs.

Tooth development and regeneration occur through reciprocal interactions between epithelial and ectodermal mesenchymal stem cells. However, the current studies on tooth development are limited, since epithelial stem cells are relatively difficult to obtain and maintain. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be alternative options for epithelial cell sources. To differentiate hESCs/hiPSCs into dental epithelial-like stem cells, this study investigated the hypothesis that direct interactions between pluripotent stem cells, such as hESCs or hiPSCs, and Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cell line may induce epithelial differentiation. Epithelial-like stem cells derived from hES (EPI-ES) and hiPSC (EPI-iPSC) had morphological and immunophenotypic characteristics of HERS/ERM cells, as well as similar gene expression. To overcome a rare population and insufficient expansion of primary cells, EPI-iPSC was immortalized with the SV40 large T antigen. The immortalized EPI-iPSC cell line had a normal karyotype, and a short tandem repeat (STR) analysis verified that it was derived from hiPSCs. The EPI-iPSC cell line co-cultured with dental pulp stem cells displayed increased amelogenic and odontogenic gene expression, exhibited higher dentin sialoprotein (DSPP) protein expression, and promoted mineralized nodule formation. These results indicated that the direct co-culture of hESCs/hiPSCs with HERS/ERM successfully established dental epithelial-like stem cells. Moreover, this differentiation protocol could help with understanding the functional roles of cell-to-cell communication and tissue engineering of teeth.

Decorin Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induces Macrophage Polarization via CD44 to Repair Hyperoxic Lung Injury.

Bronchopulmonary dysplasia (BPD), caused by hyperoxia in newborns and infants, results in lung damage and abnormal pulmonary function. However, the current treatments for BPD are steroidal and pharmacological therapies, which cause neurodevelopmental impairment. Treatment with umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) is an efficient alternative approach. To prevent pulmonary inflammation in BPD, this study investigated the hypothesis that a key regulator was secreted by MSCs to polarize inflammatory macrophages into anti-inflammatory macrophages at inflammation sites. Lipopolysaccharide-induced macrophages co-cultured with MSCs secreted low levels of the inflammatory cytokines, IL-8 and IL-6, but high levels of the anti-inflammatory cytokine, IL-10. Silencing decorin in MSCs suppressed the expression of CD44, which mediates anti-inflammatory activity in macrophages. The effects of MSCs were examined in a rat model of hyperoxic lung damage. Macrophage polarization differed depending on the levels of decorin secreted by MSCs. Moreover, intratracheal injection of decorin-silenced MSCs or MSCs secreting low levels of decorin confirmed impaired alveolarization of damaged lung tissues by down-regulation of decorin. In tissues, a decrease in the anti-inflammatory macrophage marker, CD163, was observed via CD44. Thus, we identified decorin as a key paracrine factor, inducing macrophage polarization via CD44, a master immunoregulator in mesenchymal stem cells.

WITHDRAWN: Ameloblast-like characteristics of human Hertwig's epithelial rest of Malassez/epithelial rest of Malassez cells via interaction with stem cells from human exfoliated deciduous teeth.

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Positively Correlated CD47 Activation and Autophagy in Umbilical Cord Blood-Derived Mesenchymal Stem Cells during Senescence.

Autophagy plays a critical role in stem cell maintenance and is related to cell growth and cellular senescence. It is important to find a quality-control marker for predicting senescent cells. This study verified that CD47 could be a candidate to select efficient mesenchymal stem cells (MSCs) to enhance the therapeutic effects of stem cell therapy by analyzing the antibody surface array. CD47 expression was significantly decreased during the expansion of MSCs in vitro (p < 0.01), with decreased CD47 expression correlated with accelerated senescence phenotype, which affected cell growth. UCB-MSCs transfected with CD47 siRNA significantly triggered the downregulation of pRB and upregulation of pp38, which are senescence-related markers. Additionally, autophagy-related markers, ATG5, ATG12, Beclin1, and LC3B, revealed significant downregulation with CD47 siRNA transfection. Furthermore, autophagy flux following treatment with an autophagy inducer, rapamycin, has shown that CD47 is a key player in autophagy and senescence to maintain and regulate the growth of MSCs, suggesting that CD47 may be a critical key marker for the selection of effective stem cells in cell therapy.

High Integrity and Fidelity of Long-Term Cryopreserved Umbilical Cord Blood for Transplantation.

Umbilical cord blood (UCB) is used as a source of donor cells for hematopoietic stem cell (HSC) transplantation. The success of transplantation is dependent on the quality of cord blood (CB) units for maximizing the chance of engraftment. Improved outcomes following transplantation are associated with certain factors of cryopreserved CB units: total volume and total nucleated cell (TNC) count, mononuclear cell (MNC) count, and CD34+ cell count. The role of the storage period of CB units in determining the viability and counts of cells is less clear and is related to the quality of cryopreserved CB units. Herein, we demonstrate the recovery of viable TNCs and CD34+ cells, as well as the MNC viability in 20-year-old cryopreserved CB units in a CB bank (MEDIPOST Co., Ltd., Seongnam-si, Gyeonggi-do, Korea). In addition, cell populations in CB units were evaluated for future clinical applications. The stable recovery rate of the viability of cryopreserved CB that had been stored for up to 20 years suggested the possibility of uses of the long-term cryopreservation of CB units. Similar relationships were observed in the recovery of TNCs and CD34+ cells in units of cryopreserved and fresh CB. The high-viability recovery of long-term cryopreserved CB suggests that successful hematopoietic stem cell (HSC) transplantation and other clinical applications, which are suitable for treating incurable diseases, may be performed regardless of long-term storage.

PTX-3 Secreted by Intra-Articular-Injected SMUP-Cells Reduces Pain in an Osteoarthritis Rat Model.

Mesenchymal stem cells (MSCs) are accessible, abundantly available, and capable of regenerating; they have the potential to be developed as therapeutic agents for diseases. However, concerns remain in their further application. In this study, we developed a SMall cell+Ultra Potent+Scale UP cell (SMUP-Cell) platform to improve whole-cell processing, including manufacturing bioreactors and xeno-free solutions for commercialization. To confirm the superiority of SMUP-Cell improvements, we demonstrated that a molecule secreted by SMUP-Cells is capable of polarizing inflammatory macrophages (M1) into their anti-inflammatory phenotype (M2) at the site of injury in a pain-associated osteoarthritis (OA) model. Lipopolysaccharide-stimulated macrophages co-cultured with SMUP-Cells expressed low levels of M1-phenotype markers (CD11b, tumor necrosis factor-α, interleukin-1α, and interleukin-6), but high levels of M2 markers (CD163 and arginase-1). To identify the paracrine action underlying the anti-inflammatory effect of SMUP-Cells, we employed a cytokine array and detected increased levels of pentraxin-related protein-3 (PTX-3). Additionally, PTX-3 mRNA silencing was applied to confirm PTX-3 function. PTX-3 silencing in SMUP-Cells significantly decreased their therapeutic effects against monosodium iodoacetate (MIA)-induced OA. Thus, PTX-3 expression in injected SMUP-Cells, applied as a therapeutic strategy, reduced pain in an OA model.

A Small-Sized Population of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Shows High Stemness Properties and Therapeutic Benefit.

Mesenchymal stem cells (MSCs) represent a promising means to promote tissue regeneration. However, the heterogeneity of MSCs impedes their use for regenerative medicine. Further investigation of this phenotype is required to develop cell therapies with improved clinical efficacy. Here, a small-sized population of human umbilical cord blood-derived MSCs (UCB-MSCs) was isolated using a filter and centrifuge system to analyze its stem cell characteristics. Consequently, this population showed higher cell growth and lower senescence. Additionally, it exhibited diverse stem cell properties including differentiation, stemness, and adhesion, as compared to those of the population before isolation. Using cell surface protein array or sorting analysis, both EGFR and CD49f were identified as markers associated with the small-sized population. Accordingly, suppression of these surface proteins abolished the superior characteristics of this population. Moreover, compared to that with large or nonisolated populations, the small-sized population showed greater therapeutic efficacy by promoting the engraftment potential of infused cells and reducing lung damage in an emphysema mouse model. Therefore, the isolation of this small-sized population of UCB-MSCs could be a simple and effective way to enhance the efficacy of cell therapy.

Primary Cilia Mediate Wnt5a/ß-catenin Signaling to Regulate Adipogenic Differentiation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Following Calcium Induction.

BACKGROUND: Regeneration of soft tissue defects is essential for adipose tissue pathologies and disease, trauma, or injury-induced damage. Here, we show that umbilical cord blood-derived mesenchymal stem cells could potentially be tailored and used for the reconstruction of specific damaged sites. Adipogenesis can be exploited in soft tissue reconstruction. Also, primary cilia play a role in the control of adipogenesis. METHODS: The adipogenic differentiation capacity of mesenchymal stem cells (MSCs) was shown to influence ciliogenesis. MSCs transfected with intraflagellar transport 88 (IFT88) small interfering RNA (siRNA), which blocks the assembly and maintenance of cilia, were examined to confirm the relationship between adipogenesis and ciliogenesis. Also, 1,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), calcium chelator, inhibited the ciliogenesis of MSCs in adipogenic differentiation. RESULTS: IFT88-knockdown led to decreased cilia formation and limitation of cilia elongation in adipogenesis. Additionally, intracellular calcium triggered cilia formation in MSCs adipogenesis. Interestingly, intracellular calcium cannot overcome the inhibition of adipogenesis caused by low numbers of cilia in MSCs. CONCLUSION: Our data suggested that ciliogenesis was negatively regulated by Wnt5a/ß-catenin signaling during adipogenesis. Thus, we suggest that calcium induction triggers adipogenesis and ciliogenesis.

Soluble PTX3 of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Attenuates Hyperoxic Lung Injury by Activating Macrophage Polarization in Neonatal Rat Model.

Therapeutic treatment of various inflammation-related diseases using mesenchymal stem cells (MSCs) has increased in recent years because of the paracrine action of these cells but shows several limitations. First, MSC-based therapies exhibit varying efficacies; thus, biomarkers should be determined to identify who may benefit from these candidate therapeutic agents. Second, the mechanism underlying the therapeutic effects is poorly understood. To evaluate the effects of human umbilical cord blood-derived MSCs (UCB-MSCs) on macrophages, the macrophage cell line NR8383 stimulated with lipopolysaccharide (LPS) was cocultured by UCB-MSCs. We found that UCB-MSCs mediated changes in macrophage polarization towards M2 from M1 macrophages. To identify the paracrine action underlying the anti-inflammation effect of UCB-MSCs, the secretion of UCB-MSCs exposed to LPS-stimulated NR8383 cells was tested using a biotin label-based 507 antibody array. Among the secreted proteins, we selected pentraxin-related protein PTX3/tumor necrosis factor-inducible gene 14 protein (PTX3) to investigate its association with UCB-MSCs in macrophage polarization. We found that human PTX3 was secreted from UCB-MSCs under inflammation condition and reinforced the M2 macrophage marker via the Dectin-1 receptor by activating MSK1/2 phosphorylation signaling in NR8383 cells. Accordingly, knockdown of PTX3 in UCB-MSCs significantly attenuated their therapeutic effects in a neonatal hyperoxic lung injury resulting in reduced survival, lung alveolarization, M2 marker expression, Dectin-1 levels, anti-inflammatory cytokines, and improved M1 marker expression and inflammatory cytokines compared to control MSC-injected rats. UCB-MSCs show therapeutic potential by controlling macrophage polarization. Interestingly, higher PTX3 levels in UCB-MSCs induced greater improvement in the therapeutic effects than lower PTX3 levels. Collectively, PTX3 is a potential marker with critical paracrine effects for predicting the therapeutic potential of MSC therapy in inflammatory diseases; quality control assessments using PTX3 may be useful for improving the therapeutic effects of UCB-MSCs.

Intracellular Calcium Determines the Adipogenic Differentiation Potential of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells via the Wnt5a/ß-Catenin Signaling Pathway.

Mesenchymal stem cells- (MSCs-) based therapies show different degrees of efficacies for the treatment of various diseases, including lipogenesis. We evaluated the adipogenic differentiation ability of human umbilical cord blood-derived MSCs (hUCB-MSCs) from different donors and examined the contribution of the intracellular calcium (Ca2+) level to this diversity. hUCB-MSCs treated with Ca2+ or the Ca2+ chelator BAPTA-AM increased and decreased adipogenic differentiation, respectively. Canonical Wnt5a/ß-catenin expression decreased during adipogenic differentiation of hUCB-MSCs. Treatment with Wnt5a blocked the adipogenic differentiation of hUCB-MSCs and activated the Wnt pathway, with a decrease in the adipogenesis markers PPARγ and leptin, and reduced lipid vacuole-associated Oil red O activity. In contrast, inhibition of the Wnt pathway with dickkopf-1 and ß-catenin small interfering RNA transfection promoted the adipogenic potential of hUCB-MSCs. Interestingly, the Ca2+-based system exhibited a synergic effect on adipogenic potential through the Wnt5a/ß-catenin pathway. Our data suggest that the variable adipogenic differentiation potential of hUCB-MSCs from different lots is due to variation in the intracellular Ca2+ level, which can be used as a marker to predict hUCB-MSCs selection for lipogenesis therapy. Overall, these results demonstrate that exogenous calcium treatment enhanced the adipogenic differentiation of hUCB-MSCs via negatively regulating the Wnt5a/ß-catenin signaling pathway.

Characterization of Primary Epithelial Cells Derived from Human Salivary Gland Contributing to in vivo Formation of Acini-like Structures.

Patients with head and neck cancer are treated with therapeutic irradiation, which can result in irreversible salivary gland dysfunction. Because there is no complete cure for such patients, stem cell therapy is an emerging alternative for functional restoration of salivary glands. In this study, we investigated in vitro characteristics of primarily isolated epithelial cells from human salivary gland (Epi-SGs) and in vivo formation of acini-like structures by Epi-SGs. Primarily isolated Epi-SGs showed typical epithelial cell-like morphology and expressed E-cadherin but not N-cadherin. Epi-SGs expressed epithelial stem cell (EpiSC) and embryonic stem cell (ESC) markers. During long-term culture, the expression of EpiSC and ESC markers was highly detected and maintained within the core population with small size and low cytoplasmic complexity. The core population expressed cytokeratin 7 and cytokeratin 14, known as duct markers indicating that Epi-SGs might be originated from the duct. When Epi-SGs were transplanted in vivo with Matrigel, acini-like structures were readily formed at 4 days after transplantation and they were maintained at 7 days after transplantation. Taken together, our data suggested that Epi-SGs might contain stem cells which were positive for EpiSC and ESC markers, and Epi-SGs might contribute to the regeneration of acini-like structures in vivo. We expect that Epi-SGs will be useful source for the functional restoration of damaged salivary gland.

Angiogenic Capacity of Dental Pulp Stem Cell Regulated by SDF-1α-CXCR4 Axis.

Previously, the perivascular characteristics of dental pulp stem cells (DPSCs) were reported, which suggested the potential application of DPSCs as perivascular cell source. In this study, we investigated whether DPSCs had angiogenic capacity by coinjection with human umbilical vein endothelial cells (HUVECs) in vivo; in addition, we determined the role of stromal cell-derived factor 1-α (SDF-1α) and C-X-C chemokine receptor type 4 (CXCR4) axis in the mutual interaction between DPSCs and HUVECs. Primarily isolated DPSCs showed mesenchymal stem cell- (MSC-) like characteristics. Moreover, DPSCs expressed perivascular markers such as NG2, α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor ß (PDGFRß), and CD146. In vivo angiogenic capacity of DPSCs was demonstrated by in vivo Matrigel plug assay. We could observe microvessel-like structures in the coinjection of DPSCs and HUVECs at 7 days postinjection. To block SDF-1α and CXCR4 axis between DPSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into Matrigel plug. No significant microvessel-like structures were observed at 7 days postinjection. In conclusion, DPSCs have perivascular characteristics that contribute to in vivo angiogenesis. The findings of this study have potential applications in neovascularization of engineered tissues and vascular diseases.

The Significance of SDF-1α-CXCR4 Axis in in vivo Angiogenic Ability of Human Periodontal Ligament Stem Cells.

Periodontal ligament stem cells (PDLSCs) are multipotent stem cells derived from periodontium and have mesenchymal stem cell (MSC)-like characteristics. Recently, the perivascular region was recognized as the developmental origin of MSCs, which suggests the in vivo angiogenic potential of PDLSCs. In this study, we investigated whether PDLSCs could be a potential source of perivascular cells, which could contribute to in vivo angiogenesis. PDLSCs exhibited typical MSC-like characteristics such as the expression pattern of surface markers (CD29, CD44, CD73, and CD105) and differentiation potentials (osteogenic and adipogenic differentiation). Moreover, PDLSCs expressed perivascular cell markers such as NG2, αsmooth muscle actin, platelet-derived growth factor receptor ß, and CD146. We conducted an in vivo Matrigel plug assay to confirm the in vivo angiogenic potential of PDLSCs. We could not observe significant vessel-like structures with PDLSCs alone or human umbilical vein endothelial cells (HU-VECs) alone at day 7 after injection. However, when PDLSCs and HUVECs were co-injected, there were vessel-like structures containing red blood cells in the lumens, which suggested that anastomosis occurred between newly formed vessels and host circulatory system. To block the SDF-1α and CXCR4 axis between PDLSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into the Matrigel plug. After day 3 and day 7 after injection, there were no significant vessel-like structures. In conclusion, we demonstrated the peri-vascular characteristics of PDLSCs and their contribution to in vivo angiogenesis, which might imply potential application of PDLSCs into the neovascularization of tissue engineering and vascular diseases.

In Vivo Angiogenic Capacity of Stem Cells from Human Exfoliated Deciduous Teeth with Human Umbilical Vein Endothelial Cells.

Dental pulp is a highly vascularized tissue requiring adequate blood supply for successful regeneration. In this study, we investigated the functional role of stem cells from human exfoliated deciduous teeth (SHEDs) as a perivascular source for in vivo formation of vessel-like structures. Primarily isolated SHEDs showed mesenchymal stem cell (MSC)-like characteristics including the expression of surface antigens and in vitro osteogenic and adipogenic differentiation potentials. Moreover, SHEDs were positive for NG2, α-smooth muscle actin (SMA), platelet-derived growth factor receptor beta (PDGFRß), and CD146 as pericyte markers. To prove feasibility of SHEDs as perivascular source, SHEDs were transplanted into immunodeficient mouse using Matrigel with or without human umbilical vein endothelial cells (HUVECs). Transplantation of SHEDs alone or HUVECs alone resulted in no formation of vessel-like structures with enough red blood cells. However, when SHEDs and HUVECs were transplanted together, extensive vessel-like structures were formed. The presence of murine erythrocytes within lumens suggested the formation of anastomoses between newly formed vessel-like structures in Matrigel plug and the host circulatory system. To understand underlying mechanisms of in vivo angiogenesis, the expression of angiogenic cytokine and chemokine, their receptors, and MMPs was compared between SHEDs and HUVECs. SHEDs showed higher expression of VEGF, SDF-1α, and PDGFRß than HUVECs. On the contrary, HUVECs showed higher expression of VEGF receptors, CXCR4, and PDGF-BB than SHEDs. This differential expression pattern suggested reciprocal interactions between SHEDs and HUVECs and their involvement during in vivo angiogenesis. In conclusion, SHEDs could be a feasible source of perivascular cells for in vivo angiogenesis.