Thermostable Basic Fibroblast Growth Factor Enhances the Production and Activity of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Wharton's jelly-derived mesenchymal stem cell (WJ-MSC)-derived exosomes contain a diverse cargo and exhibit remarkable biological activity, rendering them suitable for regenerative and immune-modulating functions. However, the quantity of secretion is insufficient. A large body of prior work has investigated the use of various growth factors to enhance MSC-derived exosome production. In this study, we evaluated the utilization of thermostable basic fibroblast growth factor (TS-bFGF) with MSC culture and exosome production. MSCs cultured with TS-bFGF displayed superior proliferation, as evidenced by cell cycle analysis, compared with wild-type bFGF (WT-bFGF). Stemness was assessed through mRNA expression level and colony-forming unit (CFU) assays. Furthermore, nanoparticle tracking analysis (NTA) measurements revealed that MSCs cultured with TS-bFGF produced a greater quantity of exosomes, particularly under three-dimensional culture conditions. These produced exosomes demonstrated substantial anti-inflammatory and wound-healing effects, as confirmed by nitric oxide (NO) assays and scratch assays. Taken together, we demonstrate that utilization of TS-bFGF for WJ-MSC-derived exosome production not only increases exosome yield but also enhances the potential for various applications in inflammation regulation and wound healing.

Comparative study of nutritional composition and color traits of meats obtained from the horses and Korean native black pigs raised in Jeju Island.

OBJECTIVE: The present study aimed at comparing the nutritional composition and color traits between two meat types: Horse meat and pork from Korean native black pigs raised in Jeju Island. METHODS: After slaughter 24 h, the longissimus dorsi samples were taken from left side carcasses of the 32-mo-old Jeju female breed horses and the 6-mo-old Korean native black pigs (n = 10 each). The samples were then placed into cool boxes containing ice packs and transported to the Laboratory of Meat Science where all visual fats and connective tissues were trimmed off and then the samples were ground. All the samples were analyzed for nutritional composition (proximate composition, minerals, vitamins, fatty acids, and amino acids) and color traits. RESULTS: The horse meat contained significantly higher collagen, moisture and protein than the pork (p<0.05). The Jeju horse meat showed more desirable fatty acid profiles such as containing significantly lower saturated fatty acids (SFA), higher polyunsaturated fatty acids (PUFA) contents and PUFA/SFA ratios than the pork (p<0.05). Differences in concentrations of ten amino acids existed between the two meat types in which the horse meat had higher values for all these amino acids, total amino acids (20.33 g/100 g) and essential amino acids (10.06 g/100 g) than the pork (p<0.05). Also, the horse meat showed significantly higher concentrations of Fe (34.21 mg/100 g) and Cu (2.47 mg/100 g) than the pork (Fe, 17.42 mg/100 g and Cu, 1.51 mg/100 g) (p<0.05). All the vitamins detected showed statistical differences between the two meat types in which the horse meat had higher concentrations of vitamin B1 (25.19 mg/100 g), B2 (92.32 mg/100 g), B3 (2,115.51 mg/100 g), and B5 (67.13 mg/100 g) than the pork (p<0.05). CONCLUSION: Based on the results obtained in the study, it is concluded that the two meat types studied are rich in nutrients and the animal species strongly affected the nutritional values and color traits of the muscle tissues.

Assessment of frozen storage duration effect on quality characteristics of various horse muscles.

OBJECTIVE: The study aimed at assessing the effects of frozen storage duration on quality characteristics, lipid oxidation and sensory quality of various horse muscles. METHODS: Five representative muscles: longissimus dorsi (LD), gluteus medius (GM), semimembranosus (SM), biceps femoris (BF), and triceps brachii (TB) at 24 h post-mortem obtained from 28-mo-old Jeju female breed horses (n = 8) were used in the present investigation. The muscles were vacuum-packaged and frozen at -20°C for 120, 240, and 360 days. All the samples were analyzed for thawing and cooking losses, pH, Warner-Bratzler shear forces (WBSF), color traits, total volatile basic nitrogen (TVBN), thiobarbituric acid reactive substances (TBARS) and sensory traits. The muscle samples analyzed on day 0 of frozen storage (fresh, non-frozen) were used for comparison. RESULTS: Results revealed that thawing and cooking losses significantly (p<0.05) increased in all the muscles after 120 days and then remained unchanged up to 360 days of frozen storage. The TBARS and TVBN contents significantly increased as increasing frozen storage time up to 360 days (p<0.05). While, significant decreases in WBSF values were observed for all the muscles with increased frozen storage time (p<0.05). Frozen storage variously affected the color traits of the muscles for instance; the redness of LD, GM, and BF muscles showed a decreasing tendency during frozen storage while it was not changed in TB and SM muscles. Furthermore, the frozen storage did not produce detrimental effects on sensory quality as it did not cause flavor and juiciness defects whereas it partially improved the tenderness of all the muscles studied. CONCLUSION: Based on the results obtained from our work, it is concluded that frozen storage could be applied to increase the long-term shelf life of horsemeat while still retaining its sensory quality.

MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis.

Photomorphogenesis is an essential program in plant development. This process is effected by the balanced cooperation of many factors under light and dark conditions. In a previous study, we showed that MYB hypocotyl elongation-related (MYBH) is involved in cell elongation. To expand our understanding of MYBH function, we performed a yeast two-hybrid assay and identified an MYB-like Domain transcription factor (MYBD). In this study, we investigated the function of MYBD, which is an MYBH homolog involved in anthocyanin accumulation. MYBD expression increased in response to light or cytokinin, and MYBD enhanced anthocyanin biosynthesis via repression of MYBL2, which encodes a transcription factor that has a negative effect on this process. In addition, MYBD binding in vivo to the MYBL2 promoter and the lower level of histone H3K9 acetylation at the upstream region of MYBL2 in MYBD over-expressing plants in comparison with wild-type plants imply that MYBD represses MYBL2 expression via an epigenetic mechanism. HY5 directly binds to the MYBD promoter, which indicates that MYBD acts on HY5-downstream in light- or cytokinin-triggered signaling pathways, leading to anthocyanin accumulation. Our results suggest that, although MYBD and MYBH are homologs, they act in opposite ways during plant photomorphogenesis, and these functions should be examined in further studies.

The Differences in Chemical Composition, Physical Quality Traits and Nutritional Values of Horse Meat as Affected by Various Retail Cut Types.

The effects of retail cut type on chemical, quality and nutritional characteristics of horse meat were studied. Jeju female breed horses (n = 9) at 32-mo-old were slaughtered and the carcasses at 24 h post-mortem were fabricated into 10 retail cuts including: tender-loin, loin, strip-loin, shoulder-chuck-roll, shoulder-clod, top-round, outside-round, brisket, short-plate-brisket, and shank. The results revealed that all of parameters (chemical, meat quality and nutritional composition) examined significantly (p<0.05) differed between the cuts. The chemical composition range (minimum to maximum) of cuts was found as such: moisture 65.06% to 71.69%; protein 19.07% to 21.28%; collagen 1.40% to 2.45%; fat 2.56% to 12.14% and cholesterol 55.76 to 79.50 mg/100 g. Shoulder-chuck-roll had the highest pH and water-holding capacity, while top-round had the highest cooking loss. Shear force ranged between the cuts from 2.80 kg/cm(2) to 4.98 kg/cm(2). The Cu, Fe, and Zn contents ranged between the cuts from 1.52 mg/kg to 2.75 mg/kg, 21.25 mg/kg to 30.85 mg/kg, and 16.51 mg/kg to 40.42 mg/kg, respectively. Additionally, most of the cuts studied showed favorable polyunsaturated fatty acid/saturated fatty acid, n-3/n-6 and essential amino acid/non-essential amino acid ratios.

Regulatory role of BOTRYTIS INDUCED KINASE1 in ETHYLENE INSENSITIVE3-dependent gene expression in Arabidopsis.

KEY MESSAGE: Arabidopsis BIK1 negatively regulates EIN3-depedent gene expression as an immediate cellular response. BIK1 localizes to the plasma membrane and its autophosphorylation and kinase activity involves in EIN3 repression. BOTRYTIS INDUCED KINASE1 (BIK1) is a multifunctional receptor-like kinase that involves in ethylene-mediated plant defense signaling. The loss of function BIK1 becomes insensitive to ethylene, but it still accumulates a higher level of ETHYLENE INSENSITIVE3 (EIN3) that serves as the key transcription activator in ethylene signaling. To unequivocally elucidate BIK1 function on EIN3 regulation in ethylene signaling, we took a combined approach of transient expression assay and stable expression analysis of BIK1. In our cell-based functional assay BIK1 destabilized EIN3 and down-regulated EIN3-dependent transcription. Membrane localization and autophosphorylation of BIK1 were required for full repression of EIN3 function, but its kinase activity potential compromised such regulatory action. Consistently, the analysis of transgenic plants verified BIK1 function on EIN3 repression. Our findings have clarified that autophosphorylated BIK1 in the plasma membrane negatively regulates EIN3-dependent gene expression. Thus, ethylene insensitivity in bik1 appears to be an indirect or a feedback long-term response.

The Impact of Ripening Time on Technological Quality Traits, Chemical Change and Sensory Characteristics of Dry-cured Loin.

The effect of ripening time on the technological quality traits, fatty acid compositions and sensory characteristics of dry-cured loin was studied. Pork loins (n = 102) at 24 h post-mortem were used to produce dry-cured loins. The dry-cured loins were assessed at 30, 60, and 90 days of ripening for the aforementioned characteristics. Our results showed that the water activity (aw) decreased (p<0.05) up to 60 days and did not change thereafter. The lipid oxidation and weight loss levels significantly (p<0.05) increased with increased ripening time. The Commission Internationale de l'Eclairage (CIE) L* decreased for 90 days while CIE a* increased for 60 days and did not increase thereafter. More noticeably, the levels of most of unsaturated fatty acids and total polyunsaturated fatty acids significantly decreased as increasing ripening time up to 90 days. The 30 days-ripened loins had lower (p<0.05) color, flavor and overall acceptability scores than the loins ripened for 60 and 90 days, however, no differences in sensory traits occurred between the 60 and 90 day-ripened samples. Based on the results obtained in the present study, it is suggested that the ripening duration between 30 and 60 days could be more appropriate for producing dry-cured loin product with higher quality and economic benefits.

Inverse modulation of the energy sensor Snf1-related protein kinase 1 on hypoxia adaptation and salt stress tolerance in Arabidopsis thaliana.

Terrestrial plants are exposed to complex stresses of high salt-induced abscisic acid (ABA) and submergence-induced hypoxia when seawater floods fields. Many studies have investigated plant responses to individual stress conditions, but not so much for coupled or sequentially imposed stresses. We examined molecular regulatory mechanisms of gene expression underlying the cellular responses involved in crosstalk between salt and hypoxia stresses. Salt/ABA- and AtMYC2-dependent induction of a synthetic ABA-responsive element and the native RD22 promoters were utilized in our cell-based functional assays. Such promoter-based reporter induction was largely inhibited by hypoxia and hypoxia-inducible AKIN10 activity. Biochemical analyses showed that AKIN10 negatively modulates AtMYC2 protein accumulation via proteasome activity upon AKIN10 kinase activity-dependent protein modification. Further genetic analysis using transgenic plants expressing AKIN10 provided evidence that AKIN10 activity undermined AtMYC2-dependent salt tolerance. Our findings unravel a novel molecular interaction between the key signalling constituents leading crosstalk between salt and hypoxia stresses in Arabidopsis thaliana under the detrimental condition of submergence in saltwater.

Effect of Particular Breed on the Chemical Composition, Texture, Color, and Sensorial Characteristics of Dry-cured Ham.

The present study demonstrates the impact of specific breed on the characteristics of dry-cured ham. Eighty thighs from Korean native pig (KNP), crossbreed (Landrace×Yorkshire)♀×Duroc♂ (LYD), Berkshire (Ber), and Duroc (Du) pig breeds (n = 10 for each breed) were used for processing of dry-cured ham. The thighs were salted with 6% NaCl (w/w) and 100 ppm NaNO2, and total processing time was 413 days. The effects of breed on the physicochemical composition, texture, color and sensory characteristics were assessed on the biceps femoris muscle of the hams. The results revealed that the highest weight loss was found in the dry-cured ham of LYD breed and the lowest weight loss was found in Ber dry-cured ham. The KNP dry-cured ham contain higher intramuscular fat level than other breed hams (p<0.05). It was observed that the dry-cured ham made from KNP breed had the lowest water activity value and highest salt content, while the LYD dry-cure ham had higher total volatile basic nitrogen content than the Ber and Du hams (p<0.05). Zinc, iron and total monounsaturated fatty acids levels were higher in KNP ham while polyunsaturated fatty acids levels were higher in Du ham when compared to other breed hams (p<0.05). Additionally, the KNP dry-cured ham possessed higher Commission International de l'Eclairage (CIE) a* value, while the Du dry-cured ham had higher L*, CIE b* and hue angle values (p<0.05). Furthermore, breed significantly affected the sensory attributes of dry-cured hams with higher scores for color, aroma and taste found in KNP dry-cured ham as compared to other breed hams (p<0.05). The overall outcome of the study is that the breed has a potential effect on the specific chemical composition, texture, color and sensorial properties of dry-cured hams. These data could be useful for meat processors to select the suitable breeds for economical manufacturing of high quality dry-cured hams.

Efficient Treatment of Psoriasis Using Conditioned Media from Mesenchymal Stem Cell Spheroids Cultured to Produce Transforming Growth Factor-ß1-Enriched Small-Sized Extracellular Vesicles.

Psoriasis is a common chronic inflammatory disease in which keratinocytes proliferate abnormally due to excessive immune action. Psoriasis can be associated with various comorbidities and has a significant impact on health-related quality of life. Although many systemic treatments, including biologic agents, have been developed, topical treatment remains the main option for psoriasis management. Consequently, there is an urgent need to develop topical treatments with minimal side effects and high efficacy. Mesenchymal stem cells (MSCs) exhibit excellent immune regulation, anti-inflammatory activities, and therapeutic effects, and MSC-derived extracellular vesicles (EVs) can serve as crucial mediators of functional transfer from MSCs. Therefore, this study aimed to develop a safe and easy-to-use emulsion cream for treating psoriasis using MSC conditioned media (CM) containing EVs. We developed an enhanced Wharton's jelly MSC (WJ-MSC) culture method through a three-dimensional (3D) culture containing exogenous transforming growth factor-ß3. Using the 3D culture system, we obtained CM from WJ-MSCs, which yielded a higher EV production compared to that of conventional WJ-MSC culture methods, and investigated the effect of EV-enriched 3D-WJ-MSC-CM cream on psoriasis-related inflammation. Administration of the EV-enriched 3D-WJ-MSC-CM cream significantly reduced erythema, thickness, and scaling of skin lesions, alleviated imiquimod-induced psoriasiform lesions in mice, and ameliorated histopathological changes in mouse skin. The upregulated mRNA expression of inflammatory cytokines, including IL-17a, IL-22, IL-23, and IL-36, decreased in the lesions. In conclusion, we present here a new topical treatment for psoriasis using an MSC EV-enriched cream.

Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can differentiate into various tissues and are an essential source of various disease models and therapeutics. Various growth factors are required in order to culture pluripotent stem cells, among which basic fibroblast growth factor (bFGF) is essential for maintaining stem cell ability. However, bFGF has a short half-life (8 h) under normal mammalian cell culture conditions, and its activity decreases after 72 h, posing a serious problem in the production of high-quality stem cells. Here, we evaluated the various functions of pluripotent stem cells (PSCs) by utilizing an engineered thermostable bFGF (TS-bFGF) that is thermally stable and maintains activity longer under mammalian culture conditions. PSCs cultured with TS-bFGF showed better proliferation, stemness, morphology, and differentiation than cells cultured with wild-type bFGF. In light of the importance of stem cells in a wide range of applications in the medical and biotechnology fields, we anticipate that TS-bFGF, as a thermostable and long-acting bFGF, can play a key role in securing high-quality stem cells through various sets of stem cell culture processes.

Advanced 3D dynamic culture system with transforming growth factor-ß3 enhances production of potent extracellular vesicles with modified protein cargoes via upregulation of TGF-ß signaling.

INTRODUCTION: Mesenchymal stromal cells (MSCs) release extracellular vesicles (MSC-EVs) containing various cargoes. Although MSC-EVs show significant therapeutic effects, the low production of EVs in MSCs hinders MSC-EV-mediated therapeutic development. OBJECTIVES: Here, we developed an advanced three-dimensional (a3D) dynamic culture technique with exogenous transforming growth factor beta-3 (TGF-ß3) treatment (T-a3D) to produce potent MSC-EVs. METHODS: Our system enabled preparation of a highly concentrated EV-containing medium for efficient EV isolation and purification with higher yield and efficacy. RESULTS: MSC spheroids in T-a3D system (T-a3D spheroids) showed high expression of CD9 and TGF-ß3, which was dependent on TGF-ß signaling. Treatment with EVs produced under T-a3D conditions (T-a3D-EVs) led to significantly improved migration of dermal fibroblasts and wound closure in an excisional wound model. The relative total efficacy (relative yield of single-batch EVs (10-11-fold) × relative regeneration effect of EVs (2-3-fold)) of T-a3D-EVs was approximately up to 33-fold higher than that of 2D-EVs. Importantly the quantitative proteomic analyses of the T-a3D spheroids and T-a3D-EVs supported the improved EV production as well as the therapeutic potency of T-a3D-EVs. CONCLUSION: TGF-ß signalling differentially regulated by fluid shear stress produced in our system and exogenous TGF-ß3 addition was confirmed to play an important role in the enhanced production of EVs with modified protein cargoes. We suggest that the T-a3D system leads to the efficient production of MSC-EVs with high potential in therapies and clinical development.

Modulation of Osteogenic Differentiation of Adipose-Derived Stromal Cells by Co-Treatment with 3, 4'-Dihydroxyflavone, U0126, and N-Acetyl Cysteine.

Background and Objectives: Flavonoids form the largest group of plant phenols and have various biological and pharmacological activities. In this study, we investigated the effect of a flavonoid, 3, 4'-dihydroxyflavone (3, 4'-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs). Methods and Results: Treatment of 3, 4'-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteogenic differentiation of the 3, 4'-DHF-treated eADSCs, which may indicate that the effect of U0126 on bone morphogenetic protein signaling is involved in the regulation of 3, 4'-DHF in osteogenic differentiation of eADSCs. We revealed that 3, 4'-DHF could induce osteogenic differentiation of eADSCs by suppressing ROS generation and co-treatment of 3, 4'-DHF, U0126, and/or N-acetyl cysteine (NAC) resulted in the additive enhancement of osteogenic differentiation of eADSCs. Conclusions: Our results showed that co-treatment of 3, 4'-DHF, U0126, and/or NAC cumulatively regulated osteogenesis in eADSCs, suggesting that 3, 4'-DHF, a flavonoid, can provide a novel approach to the treatment of osteoporosis and can provide potential therapeutic applications in therapeutics and regenerative medicine for human and companion animals.

Rapid production method with increased yield of high-purity extracellular vesicles obtained using extended mitochondrial targeting domain peptide.

Extracellular vesicles (EVs) are nano-sized membranous structures involved in intercellular communication and various physiological and pathological processes. Here, we present a novel method for rapid (within 15 min), large-scale production of high-purity EVs using eMTDΔ4, a peptide derived from Noxa. The treatment of mesenchymal stem cells derived from human Wharton's jelly after trypsinization and subsequent eMTDΔ4 stimulation in a chemically defined sucrose buffer with orbital shaking led to a substantial increase (approximately 30-fold) in EV production with markedly high purity (approximately 45-fold). These EVs (TS-eEVs) showed higher regenerative and immunomodulatory potential than natural EVs obtained from the culture media after 48 h. The calcium chelator BAPTA-AM and calpain inhibitor ALLM, but not the natural EV biogenesis inhibitor GW4869, blocked the TS-eEV production induced by eMTDΔ4, indicating that the eMTDΔ4-mediated regulation of intracellular calcium levels and calpain activity are closely associated with the rapid, mass production of TS-eEVs. The present study may lead to considerable advances in EV-based drug development and production of stem cell-derived EVs for cell therapy.

Superior therapeutic activity of TGF-ß-induced extracellular vesicles against interstitial cystitis.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic disease characterized by incapacitating pelvic pain. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are considered key mediators of the paracrine action of MSCs and show better biological activities than the parent MSCs, especially in the bladder tissue, which may be unfavorable for MSC survival. Here, we produced MSC-EVs using advanced three-dimensional (a3D) culture with exogenous transforming growth factor-ß3 (TGF-ß3) (T-a3D-EVs). Treatment with T-a3D-EVs led to significantly enhanced wound healing and anti-inflammatory capacities. Moreover, submucosal layer injection of T-a3D-EVs in chronic IC/BPS animal model resulted in restoration of bladder function, superior anti-inflammatory activity, and recovery of damaged urothelium compared to MSCs. Interestingly, we detected increased TGF-ß1 level in T-a3D-EVs, which might be involved in the anti-inflammatory activity of these EVs. Taken together, we demonstrate the excellent immune-modulatory and regenerative abilities of T-a3D-EVs as observed by recovery from urothelial denudation and dysfunction, which could be a promising therapeutic strategy for IC/BPS.

The role of NUMB/NUMB isoforms in cancer stem cells.

Cancer stem cells (CSCs) are a subpopulation of cancer that can self-renew and differentiate into large tumor masses. Evidence accumulated to date shows that CSCs affect tumor proliferation, recurrence, and resistance to chemotherapy. Recent studies have shown that, like stem cells, CSCs maintain cells with self-renewal capacity by means of asymmetric division and promote cell proliferation by means of symmetric division. This cell division is regulated by fate determinants, such as the NUMB protein, which recently has also been confirmed as a tumor suppressor. Loss of NUMB expression leads to uncontrolled proliferation and amplification of the CSC pool, which promotes the Notch signaling pathway and reduces the expression of the p53 protein. NUMB genes are alternatively spliced to produce six functionally distinct isoforms. An interesting recent discovery is that the protein NUMB isoform produced by alternative splicing of NUMB plays an important role in promoting carcinogenesis. In this review, we summarize the known functions of NUMB and NUMB isoforms related to the proliferation and generation of CSCs. [BMB Reports 2021; 54(7): 335-343].

GPR50 Promotes Hepatocellular Carcinoma Progression via the Notch Signaling Pathway through Direct Interaction with ADAM17.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, and it is thus critical to identify novel molecular biomarkers of HCC prognosis and elucidate the molecular mechanisms underlying HCC progression. Here, we show that G-protein-coupled receptor 50 (GPR50) in HCC is overexpressed and that GPR50 knockdown may downregulate cancer cell progression through attenuation of the Notch signaling pathway. GPR50 knockdown was found to reduce HCC progression by inactivating Notch signaling in a ligand-independent manner through a disintegrin and metalloproteinase metallopeptidase domain 17 (ADAM17), a proteolytic enzyme that cleaves the Notch receptor, which was corroborated by GPR50 overexpression in hepatocytes. GPR50 silencing also downregulated transcription and translation of ADAM17 through the AKT/specificity protein-1 (SP1) signaling axis. Notably, GPR50 was found to directly interact with ADAM17. Overall, we demonstrate a novel GPR50-mediated regulation of the ADAM17-Notch signaling pathway, which can provide insights into HCC progression and prognosis and development of Notch-based HCC treatment strategies.

3,2'-Dihydroxyflavone Improves the Proliferation and Survival of Human Pluripotent Stem Cells and Their Differentiation into Hematopoietic Progenitor Cells.

Efficient maintenance of the undifferentiated status of human pluripotent stem cells (hiPSCs) is crucial for producing cells with improved proliferation, survival and differentiation, which can be successfully used for stem cell research and therapy. Here, we generated iPSCs from healthy donor peripheral blood mononuclear cells (PBMCs) and analyzed the proliferation and differentiation capacities of the generated iPSCs using single cell NGS-based 24-chromosome aneuploidy screening and RNA sequencing. In addition, we screened various natural compounds for molecules that could enhance the proliferation and differentiation potential of hiPSCs. Among the tested compounds, 3,2'-dihydroxyflavone (3,2'-DHF) significantly increased cell proliferation and expression of naïve stemness markers and decreased the dissociation-induced apoptosis of hiPSCs. Of note, 3,2'-DHF-treated hiPSCs showed upregulation of intracellular glutathione (GSH) and an increase in the percentage of GSH-high cells in an analysis with a FreSHtracer system. Interestingly, culture of the 3,2'-DHF-treated hiPSCs in differentiation media enhanced their mesodermal differentiation and differentiation into CD34+ CD45+ hematopoietic progenitor cells (HPC) and natural killer cells (NK) cells. Taken together, our results demonstrate that the natural compound 3,2'-DHF can improve the proliferation and differentiation capacities of hiPSCs and increase the efficiency of HPC and NK cell production from hiPSCs.

Improved Isolation and Culture of Urine-Derived Stem Cells (USCs) and Enhanced Production of Immune Cells from the USC-Derived Induced Pluripotent Stem Cells.

The availability of autologous adult stem cells is one of the essential prerequisites for human stem cell therapy. Urine-derived stem cells (USCs) are considered as desirable cell sources for cell therapy because donor-specific USCs are easily and non-invasively obtained from urine. Efficient isolation, expansion, and differentiation methods of USCs are necessary to increase their availability. Here, we developed a method for efficient isolation and expansion of USCs using Matrigel, and the rho-associated protein kinase (ROCK) inhibitor, Y-27632. The prepared USCs showed significantly enhanced migration, colony forming capacity, and differentiation into osteogenic or chondrogenic lineage. The USCs were successfully reprogramed into induced pluripotent stem cells (USC-iPSCs) and further differentiated into kidney organoid and hematopoietic progenitor cells (HPCs). Using flavonoid molecules, the isolation efficiency of USCs and the production of HPCs from the USC-iPSCs was increased. Taken together, we present an improved isolation method of USCs utilizing Matrigel, a ROCK inhibitor and flavonoids, and enhanced differentiation of USC-iPSC to HPC by flavonoids. These novel findings could significantly enhance the use of USCs and USC-iPSCs for stem cell research and further application in regenerative stem cell-based therapies.

The immobilization of fibronectin- and fibroblast growth factor 2-derived peptides on a culture plate supports the attachment and proliferation of human pluripotent stem cells.

Pluripotent stem cells (PSCs) offer a promising tool for regenerative medicine. The clinical application of PSCs inevitably requires a large-scale culture in a highly defined environment. The present study aimed to devise defined coating materials for the efficient adhesion and proliferation of human PSCs (hPSCs). We tested the activity of seven fibronectin-derived peptides and three laminin-derived peptides for the attachment and proliferation of hPSCs through their immobilization on the bottom of culture dishes by creating a fusion protein with the mussel adhesion protein. Among the extracellular matrix (ECM) mimetics tested, one fibronectin-derived peptide, PHSRN-GRGDSP, significantly promoted adhesion, enhanced alkaline phosphatase activity, and increased pluripotency-related gene expression in hPSCs compared to Matrigel. Furthermore, co-immobilization of a particular canofin peptide derived from fibroblast growth factor 2 increased pluripotency marker expression, which may offer the possibility of culture without growth factor supplementation. Our findings afford a novel defined condition for the efficient culture of hPSCs and may be utilized in future clinical applications.