Long-term quality of life and influencing factors in elderly patients after vertebroplasty based on the EQ-5D-3L Chinese utility scoring system: a retrospective study.

BACKGROUND: The quality of life of elderly patients after vertebroplasty is influenced by various factors. Although the EuroQol 5-Dimension 3-Level (EQ-5D-3L) scale has been widely used to assess quality of life, the factors affecting the long-term postoperative quality of life of elderly vertebroplasty patients in China have not been thoroughly studied. METHODS: This retrospective study included 519 patients aged 65 years and older who underwent elective vertebroplasty. We collected baseline data from these patients and conducted telephone follow-ups 12 months postoperation to evaluate their EQ-5D-3L health utility scores and EuroQol Visual Analogue Scale (EQ-VAS) scores. Univariate and multivariate linear regression models were used to analyse the factors affecting quality of life. RESULTS: Of the 519 patients, the majority were female (78.0%), aged 65 to 95 years, with an average age of 75.2 years. Twelve months postoperation, pain/discomfort was the most commonly reported issue for 68.4% of patients. The median EQ-5D-3L health utility score was 0.783, with a range between 0.450 and 0.887; the median EQ-VAS score was 75, ranging from 60 to 85. Multivariate linear regression analysis indicated that older age, hormone use, higher American Society of Anesthesiologists (ASA) grades, nondrinking habits, and low albumin levels were found to be independent risk factors affecting long-term quality of life in elderly patients after vertebroplasty. Additionally, a history of tumours, the number of vertebral compression fractures, and bone mineral density were also crucial influencing factors. CONCLUSIONS: Based on the use of the EQ-5D-3L Chinese utility scoring system, we evaluated the quality of life of patients aged 65 and above 12 months after vertebroplasty. This study identified several factors related to postoperative quality of life in elderly vertebroplasty patients, providing crucial evidence for further clinical decisions and patient education.

The evidence of a macrophage barrier in the xenotransplantation of human hematopoietic stem cells to severely immunodeficient rats.

BACKGROUND: The human-to-rat hematopoietic stem cell transplantation (HSCT) model is rare, unlike its human-to-mouse counterpart. The rat models are desired, especially in areas of physiology, toxicology, and pharmacology. In addition to lymphocytes, macrophages are also considered to be important for xenotransplantation. We generated a rat xenotransplantation model to prove the role of macrophages as a xenotransplantation barrier. METHODS: Immunodeficiency in SRG rats, which are Sprague-Dawley (SD) rats lacking Rag2 and Il2rg, was confirmed by flow cytometry and spleen immunostaining. Human umbilical cord blood was collected after scheduled cesarean section at the University of Tsukuba Hospital. Cord blood mononuclear cells (CB-MNCs) were transplanted into the SRG rats administered several injections of clodronate liposome (CL), which cause macrophage depletion. Survival of human cells was observed by flow cytometry. Rat macrophage phagocytosis assay was performed to check the species-specific effects of rat macrophages on injected human/rat blood cells. RESULTS: SRG rats were deficient in T/B/NK cells. Without CL pretreatment, human CB-MNCs were removed from SRG rats within 7 hours after transplantation. The rats pretreated with CL could survive after transplantation. Prolonged survival for more than 4 weeks was observed only following a one-time CL injection. Rat macrophages had a species-specific potential for the phagocytosis of human blood cells in vivo. CONCLUSION: In human-to-rat HSCT, the short period of early macrophage control, leading to macrophage immunotolerance, is important for engraftment. The generated model can be useful for the creation of future xenotransplantation models or other clinical research.

Key factor screening in mouse NASH model using single-cell sequencing combined with machine learning.

Aims: To identify and analyze genes closely related to the progression of nonalcoholic steatohepatitis (NASH) by employing a combination of single-cell RNA sequencing and machine-learning algorithms. Main methods: Single-cell RNA sequencing (scRNA-seq) analysis was performed to find the cell population with the most significant differences between the Chow and NASH groups. This approach was used to validate the developmental trajectory of this cell population and investigate changes in cellular communication and important signaling pathways among these cells. Subsequently, high dimensional Weighted Gene Co-expression Network Analysis (hdWGCNA) was used to find the key modules in NASH. Machine learning analyses were performed to further identify core genes. Deep learning techniques were applied to elucidate the correlation between core genes and immune cells. The accuracy of this correlation was further confirmed using deep learning techniques, specifically Convolutional Neural Networks. Key findings: By comparing scRNA-seq data between the Chow and NASH groups, we have observed a notable distinction existing in the Kupffer cell population. Signaling interactions between hepatic macrophages and other cells were significantly heightened in the NASH group. Through subsequent analysis of macrophage subtypes and key modules, we identified 150 genes tightly associated with NASH. Finally, we highlighted the 16 most significant core genes using multiple iterations of machine learning. Furthermore, we pointed out the close relationship between core genes and immune cells. Significances: Using scRNA-seq analysis and machine learning, we can distinguish NASH-related genes from large genetic datasets, providing theoretical support in finding potential targets for the development of novel therapies.

Developing Humanized Animal Models with Transplantable Human iPSC-Derived Cells.

Establishing reliable and reproducible animal models for disease modelling, drug screening and the understanding of disease susceptibility and pathogenesis is critical. However, traditional animal models differ significantly from humans in terms of physiology, immune response, and pathogenesis. As a result, it is difficult to translate laboratory findings into biomedical applications. Although several animal models with human chimeric genes, organs or systems have been developed in the past, their limited engraftment rate and physiological functions are a major obstacle to realize convincing models of humans. The lack of human transplantation resources and insufficient immune tolerance of recipient animals are the main challenges that need to be overcome to generate fully humanized animals. Recent advances in gene editing and pluripotent stem cell-based xenotransplantation technologies offer opportunities to create more accessible human-like models for biomedical research. In this article, we have combined our laboratory expertise to summarize humanized animal models, with a focus on hematopoietic/immune system and liver. We discuss their generation strategies and the potential donor cell sources, with particular attention given to human pluripotent stem cells. In particular, we discuss the advantages, limitations and emerging trends in their clinical and pharmaceutical applications. By providing insights into the current state of humanized animal models and their potential for biomedical applications, this article aims to advance the development of more accurate and reliable animal models for disease modeling and drug screening.

A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells.

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells (ECs) that play an important role in liver development and regeneration. Additionally, it is involved in various pathological processes, including steatosis, inflammation, fibrosis and hepatocellular carcinoma. However, the rapid dedifferentiation of LSECs after culture greatly limits their use in vitro modeling for biomedical applications. In this study, we developed a highly efficient protocol to induce LSEC-like cells from human induced pluripotent stem cells (hiPSCs) in only 8 days. Using single-cell transcriptomic analysis, we identified several novel LSEC-specific markers, such as EPAS1, LIFR, and NID1, as well as several previously revealed markers, such as CLEC4M, CLEC1B, CRHBP and FCN3. These LSEC markers are specifically expressed in our LSEC-like cells. Furthermore, hiPSC-derived cells expressed LSEC-specific proteins and exhibited LSEC-related functions, such as the uptake of acetylated low density lipoprotein (ac-LDL) and immune complex endocytosis. Overall, this study confirmed that our novel protocol allowed hiPSCs to rapidly acquire an LSEC-like phenotype and function in vitro. The ability to generate LSECs efficiently and rapidly may help to more precisely mimic liver development and disease progression in a liver-specific multicellular microenvironment, offering new insights into the development of novel therapeutic strategies.

Generation of human induced pluripotent stem cells carrying albumin-sfGFP reporter.

Current methodologies for hepatocyte induction from human induced pluripotent stem cells (hiPSCs) have limited efficacy due to lack of a functional hepatocyte reporter. To address this, we developed an endogenous albumin (ALB)-sfGFP reporter system in hiPSCs using homologous directed recombination (HDR)-mediated knock-in. The hiPSCs maintained the characteristic morphology, pluripotency, and normal karyotype while demonstrating successful differentiation into all three germ layers both in vitro and in vivo. Co-expression of EGFP and ALB was observed in the derived hepatocyte-like cells (HLCs). This reporter system holds promise for functional hepatocyte induction.

Downregulated CAV-1 in mouse spinal cord may alleviate bone cancer pain by inhibiting the ERK/CREB pathway.

BACKGROUND: This study aimed to assess the potential function of Caveolin-1 (CAV-1) in mice with bone cancer pain. METHOD: Using a mice bone cancer pain model we explored the contribution of CAV-1 expression to bone cancer pain on the 14th day after surgery, mice in the tumor group were randomized and treated with increasing doses of the CAV-1 inhibitor, methyl-beta-cyclodextrin. Pain was assessed by monitoring the number of spontaneous flinches (NSF) and paw withdrawal mechanical threshold (PMWT)mechanical withdrawal threshold (MWT). The localization and expression of CAV-1 in mouse neurons was also determined. Additionally, the protein levels of CAV-1, extracellular signal regulated kinase (ERK) 1/2, cAMP response element-binding protein (CREB) were monitored in mouse spinal cord tissues by western blotting. RESULTS: CAV-1 was remarkably upregulated in the spinal cord of the tumor group on the 4th day after surgery, then downregulated on day 10, and upregulated again at day 14. Such CAV-1 levels were maintained until day 28. In the tumor group, the expression of p-ERK1/2 and p-CERB were upregulated at day 14 after surgery. Intrathecal injection of methyl-beta-cyclodextrin (MCD) downregulated p-ERK1/2 and p-CERB expression which correlated with alleviation of pain. CONCLUSION: Inhibition of CAV-1 in the spinal cord alleviates bone cancer pain in mice which correlates with inhibition of the ERK/CREB pathway.

Effect of Perineum Block Anesthesia Combined with Unprotected Perineal Delivery on the Perineal Integrity Rate and Maternal-Infant Outcomes in Primiparas Taking Health Products Containing Traditional Chinese Medicine.

OBJECTIVE: The purpose of the study was to investigate the effect of perineum block anaesthesia combined with unprotected perineal delivery on the perineal integrity rate and maternal-infant outcomes in primiparas taking health products containing traditional Chinese medicine (TCM). METHODS: A total of 120 puerperae admitted to our hospital from July 2019 to July 2020 were selected as study subjects and divided into group A (n = 60) and group B (n = 60), according to the number table method. Both groups took health products containing TCM, and the puerperae in group A received perineum block anaesthesia combined with unprotected perineal delivery, while those in group B were treated with routine delivery combined with routine protected perineal delivery. After that, the effect of different delivery modes on the perineal integrity rate and maternal-infant outcomes in puerperae was analyzed by the comparison of delivery condition, perineal condition, and postpartum quality of life between the two groups. RESULTS: There were no significant differences in average age and other general data between the two groups (P > 0.05); the duration in first, second, and third stages of labor in group A was significantly lower than that in group B (P < 0.001); the Apgar score in group A was significantly higher than that in group B (P < 0.001); the number of puerperae with integrated perineum in group A was significantly higher than that in group B (P < 0.05), while the number of puerperae receiving episiotomy in group A was significantly lower than that in group B (P < 0.05); the quality of life score in group A was significantly higher than that in group B (P < 0.001); the incidence of maternal postpartum complications in group A was significantly lower than that in group B (P < 0.05). CONCLUSION: Perineum block anaesthesia combined with unprotected perineal delivery can effectively shorten maternal labor duration, improve perineal integrity rate, and reduce laceration of perineum, with a significant therapeutic effect, which is worthy of application and promotion.

Hepatitis B virus infection modeling using multi-cellular organoids derived from human induced pluripotent stem cells.

Chronic infection with hepatitis B virus (HBV) remains a global health concern despite the availability of vaccines. To date, the development of effective treatments has been severely hampered by the lack of reliable, reproducible, and scalable in vitro modeling systems that precisely recapitulate the virus life cycle and represent virus-host interactions. With the progressive understanding of liver organogenesis mechanisms, the development of human induced pluripotent stem cell (iPSC)-derived hepatic sources and stromal cellular compositions provides novel strategies for personalized modeling and treatment of liver disease. Further, advancements in three-dimensional culture of self-organized liver-like organoids considerably promote in vitro modeling of intact human liver tissue, in terms of both hepatic function and other physiological characteristics. Combined with our experiences in the investigation of HBV infections using liver organoids, we have summarized the advances in modeling reported thus far and discussed the limitations and ongoing challenges in the application of liver organoids, particularly those with multi-cellular components derived from human iPSCs. This review provides general guidelines for establishing clinical-grade iPSC-derived multi-cellular organoids in modeling personalized hepatitis virus infection and other liver diseases, as well as drug testing and transplantation therapy.

The NLRP3-related inflammasome modulates pain behavior in a rat model of trigeminal neuropathic pain.

AIMS: Nod-like receptor family pyrin domain containing 3 (NLRP3) may play an important role in neuropathic pain. Treatment for trigeminal neuropathic pain remains a challenge, as common drugs either do not demonstrate beneficial therapeutic effects or induce intolerance in patients. MAIN METHODS: In a rat model of trigeminal neuropathic pain, pain caused by the malpositioning of dental implants is similar to that experienced by humans. We used masculine Sprague-Dawley rats with inferior alveolar nerve damage as a model to investigate the differential regulation of NLRP3. First, we confirmed the level of NLRP3 in the medullary dorsal horn and variation of pain response behavior after silencing the expression of NLRP3 inflammasome bodies in rats with trigeminal neuropathic pain. Second, under localized anesthesia, we extracted the lower left second molar, implanted a micro-dental implant, and deliberately injured the inferior alveolar nerve. KEY FINDINGS: After nerve damage, the level of NLRP3-related inflammasomes was upregulated in microglia and the expression of a component of the inflammasome gradually increased during postoperative days 3-21. The suppression of adenovirus-shRNA-NLRP3 on postoperative day 1 markedly inhibited the expression of pro-inflammatory cytokines and the activation of the inflammasome and mechanical allodynia. Furthermore, it attenuated cell death in microglia, as evidenced by increased Bcl-2, Bcl-xL, Bax, and Bik expression. SIGNIFICANCE: The level of NLRP3 in the dorsal horn is a pivotal factor in trigeminal neuropathic pain, and inhibition of the early expression of NLRP3 might serve as a potential therapeutic approach.

Knockdown of VDAC1 alleviates the cognitive dysfunction secondary to sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a serious and diffuse cerebral dysregulation with a high morbidity and mortality caused by sepsis. Mitophagy plays an important role in SAE, and microglial phagocytosis of apoptotic cells (efferocytosis) is the core of the brain regenerative response. Voltage dependent anion channel (VDAC1) is an important regulator of mitophagy. However, it remains unknown whether VDAC1 influences SAE progression by regulating mitophagy and efferocytosis. Herein, we explored the mechanism where knockdown of VDAC1 alleviated the cognitive dysfunction caused by sepsis-associated encephalopathy and further elucidated the underlying molecular mechanisms. SAE model in mice was established through caecal ligation and puncture (CLP). The increased mitophagy and decreased efferocytosis were observed by the transmission electron microscope (TEM) in the SAE model. Besides, immunoblot tests showed an interaction between autophagy and efferocytosis. Further behavior tests and TEM results indicated that knockdown of VDAC1 alleviated the cognitive dysfunction by decreasing the autophagy and increasing the efferocytosis in a PINK1/Parkin-dependent manner. Based on these results, we conclude that knockdown of VDAC1 alleviates the cognitive dysfunction in the CLP-induced SAE mouse model.

Transcriptomic and Functional Evidence Show Similarities between Human Amniotic Epithelial Stem Cells and Keratinocytes.

Amniotic epithelial stem cells (AESCs) are considered as potential alternatives to keratinocytes (KCs) in tissue-engineered skin substitutes used for treating skin damage. However, their clinical application is limited since similarities and distinctions between AESCs and KCs remain unclear. Herein, a transcriptomics analysis and functional evaluation were used to understand the commonalities and differences between AESCs and KCs. RNA-sequencing revealed that AESCs are involved in multiple epidermis-associated biological processes shared by KCs and show more similarity to early stage immature KCs than to adult KCs. However, AESCs were observed to be heterogeneous, and some possessed hybrid mesenchymal and epithelial features distinct from KCs. A functional evaluation revealed that AESCs can phagocytose melanosomes transported by melanocytes in both 2D and 3D co-culture systems similar to KCs, which may help reconstitute pigmented skin. The overexpression of TP63 and activation of NOTCH signaling could promote AESC stemness and improve their differentiation features, respectively, bridging the gap between AESCs and KCs. These changes induced the convergence of AESC cell fate with KCs. In future, modified reprogramming strategies, such as the use of small molecules, may facilitate the further modulation human AESCs for use in skin regeneration.

Survival-Assured Liver Injury Preconditioning (SALIC) Enables Robust Expansion of Human Hepatocytes in Fah-/- Rag2-/- IL2rg-/- Rats.

Although liver-humanized animals are desirable tools for drug development and expansion of human hepatocytes in large quantities, their development is restricted to mice. In animals larger than mice, a precondition for efficient liver humanization remains preliminary because of different xeno-repopulation kinetics in livers of larger sizes. Since rats are ten times larger than mice and widely used in pharmacological studies, liver-humanized rats are more preferable. Here, Fah-/- Rag2-/- IL2rg-/- (FRG) rats are generated by CRISPR/Cas9, showing accelerated liver failure and lagged liver xeno-repopulation compared to FRG mice. A survival-assured liver injury preconditioning (SALIC) protocol, which consists of retrorsine pretreatment and cycling 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) administration by defined concentrations and time intervals, is developed to reduce the mortality of FRG rats and induce a regenerative microenvironment for xeno-repopulation. Human hepatocyte repopulation is boosted to 31 ± 4% in rat livers at 7 months after transplantation, equivalent to approximately a 1200-fold expansion. Human liver features of transcriptome and zonation are reproduced in humanized rats. Remarkably, they provide sufficient samples for the pharmacokinetic profiling of human-specific metabolites. This model is thus preferred for pharmacological studies and human hepatocyte production. SALIC may also be informative to hepatocyte transplantation in other large-sized species.

Hepatic stellate cells contribute to liver regeneration through galectins in hepatic stem cell niche.

BACKGROUND: As a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated. METHODS: To generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit-CD29+CD49f+/lowCD45-Ter-119- cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs. RESULTS: Following liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies. CONCLUSIONS: Our findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.

Metformin Inhibits Propofol-Induced Apoptosis of Mouse Hippocampal Neurons HT-22 Through Downregulating Cav-1.

OBJECTIVE: To elucidate the neuroprotective function of metformin in suppressing propofol-induced apoptosis of HT-22 cells. METHODS: HT-22 cells were treated with 0, 10 or 100 µmol/L propofol, followed by determination of their proliferative ability. Subsequently, changes in proliferation and apoptosis of propofol-treated HT-22 cells induced with metformin were assessed. Apoptosis-associated genes in HT-22 cells were detected by Western blot. At last, regulatory effects of Cav-1 on propofol and metformin-treated HT-22 cells were examined. RESULTS: Propofol treatment dose-dependently decreased proliferative ability and increased apoptosis ability in HT-22 cells, which were partially blocked by metformin administration. Upregulated Bcl-2 and downregulated Bax were observed in propofol-treated HT-22 cells following metformin administration. In addition, Cav-1 level in HT-22 cells was regulated by metformin treatment. Notably, metformin reversed propofol-induced apoptosis stimulation and proliferation decline in HT-22 cells via downregulating Cav-1. CONCLUSION: In our study, we found that propofol could induce apoptosis of HT-22 cells and metformin could rescue the apoptosis effect regulated by propofol. Then, we found that metformin protects propofol-induced neuronal apoptosis via downregulating Cav-1.

Impelling force and current challenges by chemicals in somatic cell reprogramming and expansion beyond hepatocytes.

In the field of regenerative medicine, generating numerous transplantable functional cells in the laboratory setting on a large scale is a major challenge. However, the in vitro maintenance and expansion of terminally differentiated cells are challenging because of the lack of specific environmental and intercellular signal stimulations, markedly hindering their therapeutic application. Remarkably, the generation of stem/progenitor cells or functional cells with effective proliferative potential is markedly in demand for disease modeling, cell-based transplantation, and drug discovery. Despite the potent genetic manipulation of transcription factors, integration-free chemically defined approaches for the conversion of somatic cell fate have garnered considerable attention in recent years. This review aims to summarize the progress thus far and discuss the advantages, limitations, and challenges of the impact of full chemicals on the stepwise reprogramming of pluripotency, direct lineage conversion, and direct lineage expansion on somatic cells. Owing to the current chemical-mediated induction, reprogrammed pluripotent stem cells with reproducibility difficulties, and direct lineage converted cells with marked functional deficiency, it is imperative to generate the desired cell types directly by chemically inducing their potent proliferation ability through a lineage-committed progenitor state, while upholding the maturation and engraftment capacity posttransplantation in vivo. Together with the comprehensive understanding of the mechanism of chemical drives, as well as the elucidation of specificity and commonalities, the precise manipulation of the expansion for diverse functional cell types could broaden the available cell sources and enhance the cellular function for clinical application in future.

Enhanced hepatic differentiation in the subpopulation of human amniotic stem cells under 3D multicellular microenvironment.

BACKGROUND: To solve the problem of liver transplantation donor insufficiency, an alternative cell transplantation therapy was investigated. We focused on amniotic epithelial cells (AECs) as a cell source because, unlike induced pluripotent stem cells, they are cost-effective and non-tumorigenic. The utilization of AECs in regenerative medicine, however, is in its infancy. A general profile for AECs has not been comprehensively analyzed. Moreover, no hepatic differentiation protocol for AECs has yet been established. To this end, we independently compiled human AEC libraries, purified amniotic stem cells (ASCs), and co-cultured them with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cell (HUVECs) in a 3D system which induces functional hepatic organoids. AIM: To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells. METHODS: AECs, MSCs, and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients. Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry, Alkaline phosphatase (AP) staining, and flow cytometry. An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay. AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics. The 2D and 3D culture were compared by relative gene expression using several differentiation protocols. ASCs, MSCs, and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging, periodic acid Schiff, and an indocyanine green (ICG) test. RESULTS: AECs have certain stemness markers such as EPCAM, SSEA4, and E-cadherin. One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers. Moreover, it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage. Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells. TJP1, associated with epidermal growth factor receptor, and MET, associated with hepatocyte growth factor receptor, were upregulated and may be important for hepatic differentiation. In conventional flat culture, the cells turned unviable and did not readily differentiate into hepatocytes. In 3D culture, however, hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol. Furthermore, the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity, hepatic-like glycogen storage, and ICG absorption/elimination. CONCLUSION: Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness. Under a 3D co-culture system, functional hepatic organoids were generated in a multicellular microenvironment.

[Expressions of B-cell-specific monoclonal leukemia virus insert site 1 and human telomerase reverse transcriptase genes in CD34+ cells during ex vivo expansion process].

OBJECTIVE: To investigate the relationship between the expressions of B-cell-specific monoclonal leukemia virus insert site 1 (Bmi1) and human telomerase reverse transcriptase (hTERT) genes and the proliferative capacity of stem cells. METHODS: Cord blood CD34+ cells were cultured in IMDM medium containing fetal bovine serum, stem cell growth factor, thrombopoietin, and Fms-like tyrosine kinase 3 ligand during a 28-day ex vivo expansion process, while the expansion fold, specific growth rate, and the colony-forming efficiency were monitored. Then the Bmi1 and hTERT mRNA expressions in CD34+ cells were detected by fluorescence quantitative PCR, and the relations between the expressions and the cell proliferative capacity were analyzed. RESULTS: CD34+ cells expanded (20.1 +/- 3.5) folds during the 28-day culture, while the proportion declined from 95.5% +/- 2.6% before culture to 2.1% +/- 0.4%. Both the specific growth rate and colony-forming efficiency of CD34+ cells declined significantly after 7 days, while the expression levels of Bmi1 and hTERT mRNA in CD34+ cells reached top at 7 days and declined gradually thereafter. CONCLUSION: The expressions of Bmi1 and hTERT genes may have a close relation to the proliferative capacity of CD34+ cells.