Human midbrain dopaminergic progenitors.

Human midbrain dopaminergic progenitors (mDAPs) are one of the most representative cell types in both basic research and clinical applications. However, there are still many challenges for the preparation and quality control of mDAPs, such as the lack of standards. Therefore, the establishment of critical quality attributes and technical specifications for mDAPs is largely needed. "Human midbrain dopaminergic progenitor" jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human mDAPs in China. This standard specifies the technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for human mDAPs, which is applicable to the quality control for human mDAPs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that the publication of this guideline will facilitate the institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human mDAPs for clinical development and therapeutic applications.

Human neural stem cells.

'Human neural stem cells' jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research, is the first guideline for human neural stem cells (hNSCs) in China. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements, transportation requirements and waste disposal requirements for hNSCs, which is applicable to the quality control for hNSCs. It was originally released by the China Society for Cell Biology on 30 August 2022. We hope that publication of the guideline will facilitate institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of hNSCs for clinical development and therapeutic applications.

In situ U-Pb Dating of Calcite from the South China Antimony Metallogenic Belt.

Accurately determining the age of hydrothermal ore deposits is difficult, because of lack of suitable mineral chronometers and techniques. Here we present the first LA-MC-ICPMS U-Pb age of carbonates from hydrothermal Sb deposits. Three stages of hydrothermal carbonates from the giant South China Sb metallogenic belt were identified: (1) pre-ore dolomite (Dol-I), (2) syn-ore calcite (Cal-II), and (3) post-ore calcite (Cal-III). The U and Pb isotopic data show that Cal-II yielded a lower intercept age of 115.3 ± 1.5 Ma (MSWD = 2.0), suggesting a Sb mineralization that corresponds to an extension event occurred during the early Cretaceous in South China. Although Cal-III yielded an age of 60.0 ± 0.9 Ma (MSWD = 1.5), indicating a potential tectonothermal event occurred in this belt during the early Cenozoic. Hence, in situ U-Pb dating of calcite offers a new way to determine the age of hydrothermal ore deposits.

[Role of BET Bromodomain in Hematopoietic Differentiation from hESCs].

OBJECTIVE: To explore the role of bromodomain and extra terminal (BET) bromodomain in hematopoietic differentiation from human enbryonic stem cells (hESC). METHODS: The 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. RESULTS: The 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, CD34+CD31+ hemogenic endothelium (HEP) generation and endothelial-to-hematopoietic transition, significantly suppressed the generation of CD43 positive hematopoietic progenitor cells. CONCLUSION: I-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.

[DMSO Promotes Hematopoietic Differentiation of Human Embryonic Stem Cells in vitro].

OBJECTIVE: To explore the role of dimethyl sulfoxide (DMSO) in the hematopoietic differentiation of human embryonic stem cells (hESCs). METHODS: The role of DMSO in hematopoietic differentiation of hESC was investigated by using a established stepwise hematopoietic differentiation system from hESC, immunofluorescouse assay and flow cytometry. Furthermore, the window phase of DMSO action was explored by adding it to the different stage of hematopoietic differentiation. RESULTS: Immunofluorescence and flow cytometry analysis showed that DMSO significantly promoted the generation of CD43+ hematopoietic progenitor cells (HPC). The flow cytometry demonstrated that DMSO profoundly promoted the generation of APLNR+ lateral plate mesoderm cells and CD31+CD34+ hemogenic endothelium progenitors (HEP). The addition of DMSO in the window phase of lateral plate mesoderm cell generation could markedly improve the generation of hematopoietic progenitor cells. CONCLUSION: DMSO promotes hematopoietic differentiation of hESC through enhancing the generation of APLNR positive lateral plate mesoderm cells. The addition of DMSO in the window phase of lateral plate mesoderm cell generation can significantly improve the generation of hematopoietic progenitor cells.

[Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

Role of sonic hedgehog in maintaining a pool of proliferating stem cells in the human fetal epidermis.

BACKGROUND: The mammalian epidermis is maintained by the ongoing proliferation of a subpopulation of keratinocytes known as epidermal stem cells. Sonic hedgehog (Shh) can regulate morphogenesis of hair follicles and several types of skin cancer, but the effect of Shh on proliferation of human putative epidermal stem cells (HPESCs) is poorly understood. METHODS AND RESULTS: We first found that Shh, its receptors Patched1 (Ptc1) as well as Smoothened (Smo) and its downstream transcription factor Gli-1 were expressed in the basal layer of human fetal epidermis and freshly sorted HPESCs. Next, treatment of HPESCs with media conditioned by Shh-N-expressing cells promoted cell proliferation, whereas inhibition of Shh by cyclopamine, a specific inhibitor of Shh signalling, had an opposite effect. Interestingly, the mitogenic effect of epidermal growth factor (EGF) on HPESCs was efficiently abolished by cyclopamine. Finally, bone morphogenetic protein 4 (BMP-4), a potential downstream effector of Shh signalling, increased HPESC proliferation in a concentration-dependent manner. CONCLUSIONS: Shh is an important regulator of HPESC proliferation in the basal layer of human fetal epidermis and modulates the cell responsiveness to EGF, which will assist to unravel the mechanisms that regulate stem cell proliferation and neoplasia in the human epidermis.

Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy.

Calcyclin-binding protein (Siah-1-Interacting Protein, CacyBP/SIP), is a calcium signaling protein involved in the degradation of beta-catenin, however, little is known about its role in reproductive biology. The present study was to character its temporospatial expression pattern and regulation in mouse uterus and to investigate whether it plays a role in the regulation of normal endometrial events. While prominently expressed in both luminal and glandular epithelia, CacyBP underwent dynamic changes during early pregnancy. CacyBP expression was observed weakly from days 1-4. An intense accumulation in luminal and glandular epithelia as well as decidua surrounding the embryo at later stages (days 5-7) was observed. Most notably, CacyBP accumulation in trophoblast was pronounced at day 7. Using ovariectomized and pseudopregnant mice, we found that progesterone (P(4)) and 17beta-estradiol (E(2)) led to increased expression of CacyBP gene and this could be abolished by Ru486 and tamoxifen, respectively. Antisense oligonucleotides (ODNs) against CacyBP significantly inhibited cultured endometrial stromal cells' (ESCs) apoptosis induced by UV irradiation. Injection of antisense ODNs into mouse uterine horn severely impaired the number of implanted blastocysts. Taken together, our results suggested that CacyBP expression was positively regulated by P(4) and E(2). CacyBP may be involved in the regulation of endometrial cell apoptosis during early pregnancy and play an important role in mouse endometrial events such as pregrancy establishment.

Inhibition of the beta-catenin signaling pathway in blastocyst and uterus during the window of implantation in mice.

Beta-catenin, the mammalian homolog of Drosophila armadillo protein, was first identified as a cadherin-associated protein at cell-cell junctions. Another function of beta-catenin is the transduction of cytosolic signals to the nucleus in a variety of cellular contexts, which usually are elicited by the active form of beta-catenin. The aim of the present study was to examine the potential role of active beta-catenin in the mouse embryo and uterus during embryo implantation. Active beta-catenin was detected differentially in mouse embryos and uteri during the peri-implantation period. Aberrant activation of beta-catenin by LiCl, a well-known glycogen synthase kinase-3 inhibitor, significantly inhibited blastocyst hatching and subsequent adhesion and outgrowth on fibronectin. Results obtained from pseudopregnant and implantation-delayed mice imply an important role for implanting blastocysts in the temporal and spatial changes of active beta-catenin in the uterus during the window of implantation. Collectively, these results suggest that the beta-catenin signaling pathway is inhibited in both blastocyst and uterus during the window of implantation, which may represent a new mechanism to synchronize the development of preimplantation embryos and differentiation of the uterus during this process.

Enrichment and characterization of mouse putative epidermal stem cells.

Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.

Enrichment and identification of human 'fetal' epidermal stem cells.

BACKGROUND: Human epidermis, a continuously renewing tissue, is maintained throughout life by stem cells that proliferate and replenish worn-out or damaged cells in the tissue. Cultured human epidermal stem cells have great potential in clinical application. However, isolating and culturing a pure population of epidermal stem cells has proven to be challenging. METHODS AND RESULTS: We show that p63, a new marker for epidermal stem cells, is expressed in the basal layer of human fetal epidermis using immunohistochemistry, and that keratinocytes with the characteristics of stem cells can be isolated from the epidermis of aborted human fetuses aged >/=20 weeks based on high expression of beta(1) integrins by fluorescence-activated cell sorting. Furthermore, the enriched population showed the expression of molecular markers of putative human epidermal stem cells under a confocal microscope and a high colony formation efficiency when it was cultured at a clonal density. Under an electron microscope the sorted stem cells exhibited a high nuclear:cytoplasmic ratio and fewer organelles than the transit amplifying cells. The cultured epidermal stem cells can also be amplified and induced to terminal differentiation by suspension in vitro. CONCLUSIONS: Human 'fetal' epidermal stem cells have been successfully isolated and cultured in vitro. The cultured human epidermal stem cells could be used as a tool for studying stem cell biology and testing stem cell therapy.