Human Placenta-Derived Mesenchymal Stem Cells Improve Neurological Function in Rats with Intrauterine Hypoxic-Ischaemic Encephalopathy by Reducing Apoptosis and Inflammatory Reactions.

BACKGROUND: Hypoxic-ischaemic encephalopathy (HIE) is a major cause of neonatal disability and mortality. Although hypothermia therapy offers some neuroprotection, the recovery of neurological function is limited. Therefore, new synergistic therapies are necessary to improve the prognosis. Mesenchymal stem cell-based therapy is emerging as a promising treatment option for HIE. In this study, we studied the therapeutic efficacy of human placenta-derived mesenchymal stem cells (PD-MSCs) in the HIE rat model and analyzed the underlying therapeutic mechanisms. METHODS: Rats were divided into 6 groups (n = 9 for each) as follows: control, HIE model, HIE + normal saline, and HIE + PD-MSC transplantation at days 7, 14 and 28 postpartum. Following PD-MSC transplantation, neurological behavior was evaluated using rotarod tests, traction tests, and the Morris water maze test. The degree of brain tissue damage was assessed by histological examination and Nissl staining. Expression levels of apoptosis-related proteins and inflammatory factors were quantified by Western blotting and enzyme-linked immunosorbent assays. Immunofluorescence was used to investigate the ability of PD-MSCs to repair the morphology and function of hippocampal neurons with hypoxic-ischaemic (HI) injury. RESULTS: PD-MSC transplantation enhanced motor coordination and muscle strength in HIE rats. This treatment also improved spatial memory ability by repairing pathological damage and preventing the loss of neurons in the cerebral cortex. The most effective treatment was observed in the HIE + PD-MSC transplantation at day 7 group. Expression levels of microtubule-associated protein-2 (MAP-2), B-cell lymphoma-2 (BCL-2), interleukin (IL)-10, and transforming growth factor (TGF -ß1) were significantly higher in the HIE + PD-MSC treatment groups compared to the HIE group, whereas the levels of BCL-2-associated X protein (BAX), BCL-2-associated agonist of cell death (BAD), IL-1ß and tumour necrosis factor α (TNF-α) were significantly lower. CONCLUSIONS: We demonstrated that intravenous injection of PD-MSC at 7, 14 and 28 days after intrauterine HI damage in a rat model could improve learning, memory, and motor function, possibly by inhibiting apoptosis and inflammatory damage. These findings indicate that autologous PD-MSC therapy could have potential application for the treatment of HIE.

Recent progress in mesenchymal stem cell-based therapy for acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening diseases in critically ill patients. Although pathophysiology of ALI/ARDS has been investigated in many studies, effective therapeutic strategies are still limited. Mesenchymal stem cell (MSC)-based therapy is emerging as a promising therapeutic intervention for patients with ALI. During the last two decades, researchers have focused on the efficacy and mechanism of MSC application in ALI animal models. MSC derived from variant resources exhibited therapeutic effects in preclinical studies of ALI with different mechanisms. Based on this, clinical studies on MSC treatment in ALI/ARDS has been tried recently, especially in COVID-19 caused lung injury. Emerging clinical trials of MSCs in treating COVID-19-related conditions have been registered in past two years. The advantages and potential of MSCs in the defense against COVID-19-related ALI or ARDS have been confirmed. This review provides a brief overview of recent research progress in MSC-based therapies in preclinical study and clinical trials in ALI treatment, as well as the underlying mechanisms.

Umbilical cord MSC-derived exosomes improve alveolar macrophage function and reduce LPS-induced acute lung injury.

Acute lung injury (ALI) is a severe condition that can progress to acute respiratory distress syndrome (ARDS), with a high mortality rate. Currently, no specific and compelling drug treatment plan exists. Mesenchymal stem cells (MSCs) have shown promising results in preclinical and clinical studies as a potential treatment for ALI and other lung-related conditions due to their immunomodulatory properties and ability to regenerate various cell types. The present study focuses on analyzing the role of umbilical cord MSC (UC-MSC))-derived exosomes in reducing lipopolysaccharide-induced ALI and investigating the mechanism involved. The study demonstrates that UC-MSC-derived exosomes effectively improved the metabolic function of alveolar macrophages and promoted their shift to an anti-inflammatory phenotype, leading to a reduction in ALI. The findings also suggest that creating three-dimensional microspheres from the MSCs first can enhance the effectiveness of the exosomes. Further research is needed to fully understand the mechanism of action and optimize the therapeutic potential of MSCs and their secretome in ALI and other lung-related conditions.

2α, 3α, 24-Thrihydroxyurs-12-en-24-ursolic acid enhances the cytotoxic effect of cisplatin on oral cancer cells by down-regulating autophagy.

This study aimed to investigate the effect and mechanism of 2α, 3α, 24-thrihydroxyurs-12-en-24-ursolic acid (TEOA) alone or in combination with cisplatin on oral cancer. TEOA, a pentacyclic triterpenoid compound isolated from the roots of Actinidia eriantha, has demonstrated antitumor activity in preclinical experiments. However, its role in oral cancer remains poorly understood. Our findings revealed that a low concentration of TEOA did not exhibit significant cytotoxicity against oral squamous cell carcinoma cells. However, when combined with cisplatin, TEOA showed a significant therapeutic effect. The combined treatments resulted in a significant inhibition of proliferation and migration and a significant increase in apoptosis of squamous cell carcinoma cells. Cisplatin exposure increased autophagy levels, which may contribute to chemoresistance. Of note, the presence of TEOA significantly inhibited cisplatin-induced autophagy, leading to improved chemotherapy efficacy. Our findings indicate that a mild low dosage of TEOA may enhance the cytotoxic effect of cisplatin by downregulating autophagy in oral cancer cells.

Luteolin suppresses oral carcinoma 3 (OC3) cell growth and migration via modulating polo-like kinase 1 (PLK1) expression and cellular energy metabolism. / æœ¨çŠ€è‰ç´ é€šè¿‡è°ƒèŠ‚PLK1è¡¨è¾¾å’Œç»†èƒžèƒ½é‡ä»£è°¢æŠ‘åˆ¶å£è ”ç™Œç»†èƒžOC3的生长和迁移.

Oral squamous cell carcinoma (OSCC) is a prevalent malignant tumor affecting the head and neck region (Leemans et al., 2018). It is often diagnosed at a later stage, leading to a poor prognosis (Muzaffar et al., 2021; Li et al., 2023). Despite advances in OSCC treatment, the overall 5-year survival rate of OSCC patients remains alarmingly low, falling below 50% (Jehn et al., 2019; Johnson et al., 2020). According to statistics, only 50% of patients with oral cancer can be treated with surgery. Once discovered, it is more frequently at an advanced stage. In addition, owing to the aggressively invasive and metastatic characteristics of OSCC, most patients die within one year of diagnosis. Hence, the pursuit of novel therapeutic drugs and treatments to improve the response of oral cancer to medication, along with a deeper understanding of their effects, remains crucial objectives in oral cancer research (Johnson et al., 2020; Bhat et al., 2021; Chen et al., 2023; Ruffin et al., 2023).

Repeat-dose toxicity study of human umbilical cord mesenchymal stem cells in cynomolgus monkeys by intravenous and subcutaneous injection.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are proposed for the treatment of acute lung injury and atopic dermatitis. To advance hUC-MSC entry into clinical trials, the effects of hUC-MSCs on the general toxicity, immune perturbation and toxicokinetic study of hUC-MSCs in cynomolgus monkeys were assessed. hUC-MSCs were administered to cynomolgus monkeys by intravenous infusion of 3.0 × 106 or 3.0 × 107cells/kg or by subcutaneous injection of 3.0 × 107cells/kg twice a week for 3 weeks followed by withdrawal and observation for 6 weeks. Toxicity was assessed by clinical observation, clinical pathology, ophthalmology, immunotoxicology and histopathology. Moreover, toxicokinetic study was performed using a validated qPCR method after the first and last dose. After 3rd or 4th dosing, one or three the monkeys in the intravenous high-dose group exhibited transient coma, which was eliminated by slow-speed infusion after 5th or 6th dosing. In all dose groups, hUC-MSCs significantly increased NEUT levels and decreased LYMPH and CD3+ levels, which are related to the immunosuppressive effect of hUC-MSCs. Subcutaneous nodules and granulomatous foci were found at the site of administration in all monkeys in the subcutaneous injection group. Other than above abnormalities, no obvious systemic toxicity was observed in any group. The hUC-MSCs was detectable in blood only within 1 h after intravenous and subcutaneous administration. The present study declared the preliminary safety of hUC-MSCs, but close monitoring of hUC-MSCs for adverse effects, such as coma induced by intravenous infusion, is warranted in future clinical trials.

A repeat-dose toxicity study of human umbilical cord mesenchymal stem cells in NOG mice by intravenous injection.

BACKGROUND: Stem cell-based therapies have demonstrated great potential in several clinical trials. However, safety data on stem cell application remain inadequate. This study evaluated the toxicity of human umbilical cord mesenchymal stem cells (hUC-MSCs) in NOD/Shi-scid/IL-2 Rγnull (NOG) mice. RESEARCH DESIGN AND METHODS: Mice were administered hUC-MSCs intravenously at doses of 3.5 × 106 cells/kg and 3.5 × 107 cells/kg. Toxicity was assessed by clinical observation, behavioral evaluation, pathology, organ weight, and histopathology. We determined the distribution of hUC-MSCs using a validated qPCR method and colonization using immunohistochemistry. RESULTS: No significant abnormal effects on clinical responses, body weight, or food intake were observed in the mice, except for two in the high-dose group that died during the last administration. Mouse activity in the high-dose group decreased 6 h after the first administration. Terminal examination revealed dose-dependent changes in hematology. The mice in the high-dose group displayed pulmonary artery wall plaques and mild alveolar wall microthrombi. hUC-MSCs colonized primarily the lung tissues and were largely distributed there 24 h after the final administration. CONCLUSIONS: The no observed adverse effect level for intravenous administration of hUC-MSCs in NOG mice over a period of 3 w was 3.5 × 106 cells/kg.

Umbilical cord mesenchymal stem cells relieve osteoarthritis in rats through immunoregulation and inhibition of chondrocyte apoptosis.

This study aims to investigate the effectiveness of umbilical cord mesenchymal stem cells (UCMSCs) in treating osteoarthritis (OA). Sprague-Dawley rats were used in in vivo experiments and divided into four groups: normal, OA model, saline, and UCMSC-treated groups (n = 6). An OA model was established by injecting iodoacetic acid into the joint cavity. The results indicate that UCMSC transplantation significantly reduced joint surface and articular cartilage damage, and the levels of IL-1ß, TNF-α, and MMP13 in the joint fluid were significantly reduced after UCMSC treatment. In vitro experiments showed that co-culturing UCMSCs and chondrocytes promoted the expression of aggrecan, COL2, SOX9, and BCL-2; downregulated the expression of BAX and BAD in chondrocytes; and promoted the expression of IL-10 and TGF-ß1 in UCMSCs. Additionally, the supernatant of UCMSCs inhibited the expression of IL-1ß and TNF-α in the articular cavity and promoted the expression of COL2 and aggrecan in vivo. These effects were impaired when IL-10 and TGF-ß1 were removed. Collectively, UCMSC transplantation appears to improve joint pathology, reduce inflammatory factors, and decrease chondrocyte apoptosis, likely through the involvement of IL-10 and TGF-ß1, thus providing a potential therapeutic option for patients with OA.

Mesenchymal stem/stromal cell-based cell-free therapy for the treatment of acute lung injury.

Acute lung injury (ALI) is a severe medical condition that causes inflammation and fluid buildup in the lung, resulting in respiratory distress. Moreover, ALI often occurs as a complication of other medical conditions or injuries, including the coronavirus disease of 2019. Mesenchymal stem/stromal cells (MSCs) are being studied extensively for their therapeutic potential in various diseases, including ALI. The results of recent studies suggest that the beneficial effects of MSCs may not be primarily due to the replacement of damaged cells but rather the release of extracellular vesicles (EVs) and other soluble factors through a paracrine mechanism. Furthermore, EVs derived from MSCs preserve the therapeutic action of the parent MSCs and this approach avoids the safety issues associated with live cell therapy. Thus, MSC-based cell-free therapy may be the focus of future clinical treatments.

Human placenta-derived mesenchymal stem cell administration protects against acute lung injury in a mouse model.

This study aims to investigate the effect of placenta-derived mesenchymal stem cells (PMSCs) administration on tissue repair following acute lung injury (ALI). PMSCs were transplanted intravenously to a mouse model of lipopolysaccharide-induced ALI. The therapeutic effects were determined by evaluating several indicators, including pathology; the wet/dry ratio of the lungs; blood gas analysis; the total protein content, cell numbers, and the activity of myeloperoxidase (MPO) in bronchial alveolar lavage fluid (BALF); and the levels of anti-inflammatory and proinflammatory cytokines in serum and BALF. To investigate the underlying mechanism, PMSC-derived exosomes were used for ALI treatment. Administration of PMSCs improved the degree of lung injury, reduced inflammation, increased the expression levels of anti-inflammatory cytokines, and protected lung function. As expected, the effects of PMSC-derived exosomes in the ALI model were similar to those of PMSCs, both in terms of improved lung function and reduced inflammation. These findings suggest that PMSCs have ameliorating effects on ALI that are potentially mediated via their secreted exosomes.

Strategy insight: Mechanical properties of biomaterials' influence on hydrogel-mesenchymal stromal cell combination for osteoarthritis therapy.

Osteoarthritis (OA) is a kind of degenerative joint disease usually found in older adults and those who have received meniscal surgery, bringing great suffering to a number of patients worldwide. One of the major pathological features of OA is retrograde changes in the articular cartilage. Mesenchymal stromal cells (MSCs) can differentiate into chondrocytes and promote cartilage regeneration, thus having great potential for the treatment of osteoarthritis. However, improving the therapeutic effect of MSCs in the joint cavity is still an open problem. Hydrogel made of different biomaterials has been recognized as an ideal carrier for MSCs in recent years. This review focuses on the influence of the mechanical properties of hydrogels on the efficacy of MSCs in OA treatment and compares artificial materials with articular cartilage, hoping to provide a reference for further development of modified hydrogels to improve the therapeutic effect of MSCs.

Effects of Sepsis Serum on the Fate of Adipose Tissue-Derived Stem Cells.

BACKGROUND: Adipose tissue-derived stem cells (ADSCs), a type of mesenchymal stem cell, have been used extensively in clinical trials for the treatment of multiple conditions, including sepsis. However, increasing evidence indicates that ADSCs vanish from tissues within days of administration. Consequently, it would be desirable to establish the mechanisms underlying the fate of ADSCs following transplantation. METHODS: In this study, sepsis serum from mouse models was used to mimic microenvironmental effects. Healthy donor-derived human ADSCs were cultured in vitro in the presence of mouse serum from normal or lipopolysaccharide (LPS)-induced sepsis models for the purposes of discriminant analysis. The effects of sepsis serum on ADSC surface markers and cell differentiation were analyzed by flow cytometry, and the proliferation of ADSCs was assessed using a Cell Counting Kit-8 (CCK-8) assay. Quantitative real-time PCR (qRT-PCR) was applied to assess the degree of ADSC differentiation. The effects of sepsis serum on the cytokine release and migration of ADSCs were determined based on ELISA and Transwell assays, respectively, and ADSC senescence was assessed by ß-galactosidase staining and western blotting. Furthermore, we performed metabolic profiling to determine the rates of extracellular acidification and oxidative phosphorylation and the production of adenosine triphosphate and reactive oxygen species. RESULTS: We found that sepsis serum enhanced the cytokine and growth factor secretion and migratory capacities of ADSCs. Moreover, the metabolic pattern of these cells was reprogrammed to a more activated oxidative phosphorylation stage, leading to an increase in osteoblastic differentiation capacity and reductions in adipogenesis and chondrogenesis. CONCLUSIONS: Our findings in this study reveal that a septic microenvironment can regulate the fate of ADSCs.

Estrogen upregulates DNA2 expression through the PI3K-AKT pathway in endometrial carcinoma. / é›Œæ¿€ç´ é€šè¿‡PI3K-AKTé€šè·¯ä¸Šè°ƒå­å®«å† è†œç™Œä¸­DNA2的表达.

Endometrial cancer is the most common gynecological malignancy, affecting up to 3% of women at some point during their lifetime (Morice et al., 2016; Li and Wang, 2021). Based on the pathogenesis and biological behavioral characteristics, endometrial cancer can be divided into estrogen-dependent (I) and non-estrogen-dependent (II) types (Ulrich, 2011). Type I accounts for approximately 80% of cases, of which the majority are endometrioid carcinomas, and the remaining are mucinous adenocarcinomas (Setiawan et al., 2013). It is generally recognized that long-term stimulation by high estrogen levels with the lack of progesterone antagonism is the most important risk factor; meanwhile, there is no definite conclusion on the specific pathogenesis. The incidence of endometrial cancer has been on the rise during the past two decades (Constantine et al., 2019; Gao et al., 2022; Luo et al., 2022). Moreover, the development of assisted reproductive technology and antiprogestin therapy following breast cancer surgery has elevated the risk of developing type I endometrial cancer to a certain extent (Vassard et al., 2019). Therefore, investigating the influence of estrogen in type I endometrial cancer may provide novel concepts for risk assessment and adjuvant therapy, and at the same time, provide a basis for research on new drugs to treat endometrial cancer.

DEHP mediates drug resistance by metabolic reprogramming in colorectal cancer cells.

Long-term exposure to diethylhexyl phthalate (DEHP), an endocrine-disrupting chemical (EDCs) and plasticizer widely used in consumer products, has been reported to be significantly positively correlated with increased risks of different human diseases, including various cancers, while the potential effect of DEHP on colorectal cancer progression was little studied. In the present study, we showed that DEHP could trigger the metabolic reprogramming of colorectal cancer cells, promote cell growth and decrease fluorouracil (5-FU) sensitivity. Mechanistic studies indicated that DEHP could reduce glycolysis activity and increase oxidative phosphorylation (OXPHOS) in SW620 cells. In addition, in vivo experiments showed that DEHP promoted tumorigenic progression and decreased survival time in mice. Collectively, our findings suggest that DEHP may be a potent risk factor for colorectal cancer development.

Targeting TRMT5 suppresses hepatocellular carcinoma progression via inhibiting the HIF-1α pathways. / 靶向TRMT5抑制HIF-1α信号通路调控肝癌进程.

Accumulating evidence has confirmed the links between transfer RNA (tRNA) modifications and tumor progression. The present study is the first to explore the role of tRNA methyltransferase 5 (TRMT5), which catalyzes the m1G37 modification of mitochondrial tRNAs in hepatocellular carcinoma (HCC) progression. Here, based on bioinformatics and clinical analyses, we identified that TRMT5 expression was upregulated in HCC, which correlated with poor prognosis. Silencing TRMT5 attenuated HCC proliferation and metastasis both in vivo and in vitro, which may be partially explained by declined extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Mechanistically, we discovered that knockdown of TRMT5 inactivated the hypoxia-inducible factor-1 (HIF-1) signaling pathway by preventing HIF-1α stability through the enhancement of cellular oxygen content. Moreover, our data indicated that inhibition of TRMT5 sensitized HCC to doxorubicin by adjusting HIF-|1α. In conclusion, our study revealed that targeting TRMT5 could inhibit HCC progression and increase the susceptibility of tumor cells to chemotherapy drugs. Thus, TRMT5 might be a carcinogenesis candidate gene that could serve as a potential target for HCC therapy.

CD4+ CTLs Act as a Key Effector Population for Allograft Rejection of MSCs in a Donor MHC-II Dependent Manner in Injured Liver.

Mesenchymal stromal/stem cells (MSCs) have been considered an attractive source of cytotherapy due to their promising effects on treating various diseases. Allogeneic MSCs (allo-MSCs) are extensively used in clinical trials due to their convenient preparation and credible performance. Traditionally, allo-MSCs are considered immunoprivileged with minimal immunogenicity and potent immunomodulatory capacity. However, growing evidence has suggested that allo-MSCs also induce immune response and cause rejection after transplantation, but the underlying cellular and molecular mechanisms remain to be elucidated. Here, we demonstrated that allografted MSCs upregulated MHC-II upon stimulation of IFN-γ in hepatic inflammatory environment by using mouse model of CCl4-induced liver injury. MHC-II upregulation enhanced the immunogenicity of allo-MSCs, leading to the activation of alloreactive T cells and rejection of allo-MSCs. However, MHC-II deficiency impaired the allogenic reactivity, thereby rescuing the loss of allo-MSCs. Mechanistically, CD4+ cytotoxic T lymphocytes (CTLs), rather than CD8+ CTLs, acted as the major effector for allo-MSC rejection. Under liver injury condition, the transplanted allo-MSCs upregulated CD80 and PD-L1, and CD8+ CTLs highly expressed CTLA-4 and PD-1, thereby inducing immune tolerance of CD8+ T cells to allo-MSCs. On the contrary, CD4+ CTLs minimally expressed CTLA-4 and PD-1; thus, they remain cytotoxic to allo-MSCs. Consequently, transplantation of MHC-II-deficient allo-MSCs substantially promoted their therapeutic effects in treating liver injury. This study revealed a novel mechanism of MSC allograft rejection mediated by CD4+ CTLs in injured liver, which provided new strategies for improving clinical performance of allo-MSCs in benefiting hepatic injury repair.

RNA-Sequencing approach for exploring the therapeutic effect of umbilical cord mesenchymal stem/stromal cells on lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) is significantly associated with morbidity and mortality in patients with critical diseases. In recent years, studies have identified that mesenchymal stem/stromal cells (MSCs) ameliorate ALI and pulmonary fibrosis. However, the mechanism underlying this outcome in ALI has not yet been investigated. In this study, RNA sequencing technology was used to analyze the gene expression profile of lung tissue in lipopolysaccharide (LPS)-induced ALI rats following treatment with human umbilical cord MSC (HUCMSC). Differential expression analyses, gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes enrichment, protein-protein interaction network identification, and hub gene analysis were also performed. HUCMSC treatment decreased inflammatory factor production and alveolar exudates, and attenuated lung damage in LPS-induced ALI rats. The RNA-Seq data indicated that HUCMSC treatment activated the IL-17, JAK-STAT, NF-κB, and TNF-α signaling pathways, increased oxygen transport, and decreased extracellular matrix organization. HUCMSC exert beneficial effects on ALI via these signaling pathways by reducing inflammation, inhibiting pulmonary fibrosis, and improving lung ventilation. Moreover, our study further revealed the hub genes (Tbx2, Nkx2-1, and Atf5) and signaling pathways involved in HUCMSC treatment, thus providing novel perspectives for future research into the molecular mechanisms underlying cell treatment of ALI. HUCMSC can regulate multiple genes and signaling pathways, which can prevent LPS-induced lung damage in an ALI rat model.

Targeting mitochondrial tyrosyl-tRNA synthetase YARS2 suppresses colorectal cancer progression.

Defects in tRNA expressions and modifications had been linked to various types of tumorigenesis and progression in recent studies, including colorectal cancer. In the present study, we evaluated transcript levels of mitochondrial tyrosyl-tRNA synthetase YARS2 in both colorectal cancer tissues and normal colorectal tissues using qRT-PCR. The results revealed that the mRNA expression level of YARS2 in colorectal cancer tissues was significantly higher than those in normal intestinal tissues. Knockdown of YARS2 in human colon cancer cell-line SW620 leads to significant inhibition of cell proliferation and migration. The steady-state level of tRNATyr, OCR, and ATP synthesis were decreased in the YARS2 knockdown cells. Moreover, our data indicated that inhibition of YARS2 is associated with increased reactive oxygen species levels which sensitize these cells to 5-FU treatment. In conclusion, our study revealed that targeting YARS2 could inhibit colorectal cancer progression. Thus, YARS2 might be a carcinogenesis candidate gene and can serve as a potential target for clinical therapy.

Establishment and characterization of IPS-OGC-C1: a novel induced pluripotent stem cell line from healthy human ovarian granulosa cells.

Ovarian granulosa cell (OGC) is a critical somatic component of the ovary, which provides physical support and the microenvironment required for the developing oocyte. Human OGCs are easy to obtain and culture as a by-product of follicular aspiration performed during in vitro fertilization (IVF) procedures. Therefore, OGCs offer a potent cell source to generate induced pluripotent stem cells (iPSCs). This study established a novel OGCs-derived iPSC cell line from the follicular fluid of a healthy female donor with a Chinese Han genetic background and named it IPS-OGC-C1. IPS-OGC-C1 was verified for embryonic stem cell morphology, cell marker expression, alkaline phosphatase (AP) activity, transcriptomic profile, and pluripotency capability in developing all three embryonic germ layers in vivo and in vitro.