The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.
Adenosine Monophosphate/therapeutic use* ; Alanine/therapeutic use* ; Alveolar Epithelial Cells/virology* ; Antibodies, Neutralizing/therapeutic use* ; COVID-19/virology* ; Down-Regulation ; Drug Discovery ; Human Embryonic Stem Cells/metabolism* ; Humans ; Immunity ; Lipid Metabolism ; Lung/virology* ; RNA, Viral/metabolism* ; SARS-CoV-2/physiology* ; Virus Replication/drug effects*

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.
Animals ; Cell Culture Techniques ; Embryonic Stem Cells ; Extracellular Matrix ; Feeder Cells ; Human Embryonic Stem Cells ; Humans ; Induced Pluripotent Stem Cells ; Pluripotent Stem Cells ; Stem Cells

No abstract available.
Extracellular Matrix ; Glucose Tolerance Test ; Human Embryonic Stem Cells ; Machine Learning ; Memory, Short-Term ; Neural Networks (Computer) ; Nocturia ; Regenerative Medicine ; Wearable Electronic Devices ; Algorithms

OBJECTIVETo explore the role of bromodomain and extra terminal (BET) bromodomain in hematopoietic differentiation from human enbryonic stem cells (hESC).

METHODSThe effect of BET hematopoietic inhibitor I-BET151 on hematopoietic differentiation from hESC was detected by using a monolayer hematopoietic defferentiation model, immunofluorescence, flow cytometry and real-time PCR; moreover the role of I-BET151 in process of hematopoietic differentiation was explored by adding I-BET151 in different differentiation stages.

RESULTSThe analysis results of immunofluorescence, flow cytometry and real-time PCR showed that I-BET 151 significantly inhibited the generation of CD43 positive hematopoietic stem and progenitor cells (HSPCs). It was found that the addition of I-BET 151 in different stages, including APLNR lateral plate mesoderm production, CD34CD31 hemogenic endothelium (HEP) generation and endothelial-to-hematopoietic transition, significantly suppressed the generation of CD43 positive hematopoietic progenitor cells.

CONCLUSIONI-BET 151 inhibites hematopoietic differentiation from hESCs at several stages, suggesting that the BET bromodomain plays important roles in multiple stages of hematopoietic differentiation from hESCs.

OBJECTIVETo investigate the effect of embryonic stem cells on the proliferation and apoptosis in human acute myeloid leukemia cell line KG-1a and to explore its potential mechanism.

METHODSThe direct co-culture system between human embryonic stem cells H9 and human acute myeloid leukemia cell line KG-1a was established, and CCK8 assay was used to detect the proliferation of KG-1a cells. The changes of cell cycle and apoptosis were detected by flow cytometry (FCM). The mRNA expressions of BCL-2, BAX, Caspase-3 were assessed by RT-PCR. Meanwhile, the protein-expressions of BCL-2, BAX, Caspase-3 were detected by Western blot.

RESULTSThe proliferation level of KG-1a cells was significantly inhibited by H9, and the apoptotic rate increased, and the cell cycle was blocked at G/M phase. The mRNA-expression and the protein-expression of BAX and Caspase-3 increased, the mRNA and protein-expression of BCL-2 decreased.

CONCLUSIONEmbryonic stem cells can inhibit the proliferation of KG-1a and induce the apoptosis that maybe relate with the down-regulation of BCL-2 expression and up-regulation of BAX and caspase-3 expression.

BACKGROUND: Human embryonic stem cells (hESCs) have the potential to treat various human disorders currently labeled as incurable and/or terminal illness. However, the fear that the patients' immune system would recognize them as non self and lead to an immune rejection has hampered their use. The main cause for immune rejection is usually the incompatibility of both donor and recipient's major histocompatibility complex (MHC). METHODS: We describe a hESC line developed through a patented technology that does not lead to immune reaction upon transplantation. We have transplanted these cells in >1,400 patients with chronic/terminal conditions and did not observe any immune reaction. No immunosuppressant were administered to these patients. We analyzed the expression levels of MHC-I and MHC-II on the surface of these hESCs using microarray technology. The gene targets for miRNA were analyzed using Gene ontology and DAVID database and pathways for these genes were determined using Reactome and Panther databases. RESULTS: Our results showed that the levels of expression of MHC-I and MHC-II on hESCs is almost negligible and thus the hESCs are less susceptible to an immune rejection. CONCLUSIONS: The hESCs cultured at our facility expresses low levels of MHC-I and do not produce an immune reaction. These can be administered universally and need no cross matching before transplantation.
Cell Line ; Gene Ontology ; Human Embryonic Stem Cells ; Humans ; Immune System ; Major Histocompatibility Complex ; MicroRNAs ; Neurons ; Tissue Donors ; Zygote

PURPOSE: To evaluate the therapeutic effect of human embryonic stem cell (hESC)-derived multipotent mesenchymal stem cells (M-MSCs) on ketamine-induced cystitis (KC) in rats. METHODS: To induce KC, 10-week-old female rats were injected with 25-mg/kg ketamine hydrochloride twice weekly for 12 weeks. In the sham group, phosphate buffered saline (PBS) was injected instead of ketamine. One week after the final injection of ketamine, the indicated doses (0.25, 0.5, and 1×106 cells) of M-MSCs (KC+M-MSC group) or PBS vehicle (KC group) were directly injected into the bladder wall. One week after M-MSC injection, the therapeutic outcomes were evaluated via cystometry, histological analyses, and measurement of gene expression. Next, we compared the efficacy of M-MSCs at a low dose (1×105 cells) to that of an identical dose of adult bone marrow (BM)-derived MSCs. RESULTS: Rats in the KC group exhibited increased voiding frequency and reduced bladder capacity compared to rats of the sham group. However, these parameters recovered after transplantation of M-MSCs at all doses tested. KC bladders exhibited markedly increased mast cell infiltration, apoptosis, and tissue fibrosis. Administration of M-MSCs significantly reversed these characteristic histological alterations. Gene expression analyses indicated that several genes associated with tissue fibrosis were markedly upregulated in KC bladders. However the expression of these genes was significantly suppressed by the administration of M-MSCs. Importantly, M-MSCs ameliorated bladder deterioration in KC rats after injection of a low dose (1×105) of cells, at which point BM-derived MSCs did not substantially improve bladder function. CONCLUSIONS: This study demonstrates for the first time the therapeutic efficacy of hESC-derived M-MSCs on KC in rats. M-MSCs restored bladder function more effectively than did BM-derived MSCs, protecting against abnormal changes including mast cell infiltration, apoptosis and fibrotic damage.
Adult ; Animals ; Apoptosis ; Bone Marrow ; Cystitis ; Female ; Fibrosis ; Gene Expression ; Human Embryonic Stem Cells ; Humans ; Ketamine ; Mast Cells ; Mesenchymal Stromal Cells ; Multipotent Stem Cells ; Pelvic Pain ; Rats ; Urinary Bladder

Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) are two of the best characterized human progeroid syndromes. HGPS is caused by a point mutation in lamin A (LMNA) gene, resulting in the production of a truncated protein product-progerin. WS is caused by mutations in WRN gene, encoding a loss-of-function RecQ DNA helicase. Here, by gene editing we created isogenic human embryonic stem cells (ESCs) with heterozygous (G608G/+) or homozygous (G608G/G608G) LMNA mutation and biallelic WRN knockout, for modeling HGPS and WS pathogenesis, respectively. While ESCs and endothelial cells (ECs) did not present any features of premature senescence, HGPS- and WS-mesenchymal stem cells (MSCs) showed aging-associated phenotypes with different kinetics. WS-MSCs had early-onset mild premature aging phenotypes while HGPS-MSCs exhibited late-onset acute premature aging characterisitcs. Taken together, our study compares and contrasts the distinct pathologies underpinning the two premature aging disorders, and provides reliable stem-cell based models to identify new therapeutic strategies for pathological and physiological aging.
Aging ; genetics ; physiology ; DNA Helicases ; genetics ; Human Embryonic Stem Cells ; metabolism ; physiology ; Humans ; Kinetics ; Lamin Type A ; genetics ; Mesenchymal Stem Cells ; metabolism ; physiology ; Mutation ; Progeria ; genetics ; physiopathology ; Werner Syndrome ; genetics ; physiopathology

Although microRNAs have emerged as key regulators in diverse cellular processes, the roles of microRNAs are poorly understood in human embryonic stem cells (hESCs) during differentiation into specialized cell types. In this study, we used a microRNA array with 799 human microRNA probes to examine the expression profiles of microRNAs in hESCs during differentiation into endodermal and mesodermal lineages in vitro. Among the microRNAs analyzed, 7 and 20 microRNAs were enriched in the developmental process of hESCs into mesodermal and endodermal lineages, respectively. In particular, the expression levels of miR-200 family, which is known to regulate the epithelial to mesenchymal transition (EMT), gradually increased in hESCs during differentiation into hepatocytes while they gradually decreased during differentiation into vascular endothelial cells. Downregulation of ZEB1, a direct target of miR-200 family, and E-CADHERIN, a target protein of ZEB1, was observed in hESCs during differentiation into endodermal and mesodermal lineages, respectively. These results indicate that miR-200 family has an important role in determining the cell fate between endodermal and mesodermal lineages from the pluripotent state.
Cadherins ; Down-Regulation ; Endoderm ; Endothelial Cells ; Hepatocytes ; Human Embryonic Stem Cells* ; Humans ; Humans* ; In Vitro Techniques* ; Mesoderm ; MicroRNAs

BACKGROUND: Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. METHODS: Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. RESULTS: Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34⁺CD43⁺ hematopoietic progenitor cells (HPCs) and CD34⁺CD45⁺ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro. CONCLUSION: In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.
Animals ; Cytokines ; Embryonic Stem Cells ; Hematopoietic Stem Cells* ; Human Embryonic Stem Cells* ; Humans* ; In Vitro Techniques ; Methods* ; Pluripotent Stem Cells ; Stem Cells