搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Neural crest (NC) cells contribute to the development of many complex tissues of all three germ layers during embryogenesis,and its abnormal development accounts for several congenital birth defects. Generating NC cells-including specific subpopulations such as cranial,cardiac,and trunk NC cells-from human pluripotent stem cells will provide a valuable model system to study human development and disease. Here,we describe a rapid and robust NC differentiation method called LSB-short" that is based on dual SMAD pathway inhibition. This protocol yields high percentages of NC cell populations from multiple human induced pluripotent stem and human embryonic stem cell lines in 8 days. The resulting cells can be propagated easily� View Publication

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus,they are increasingly employed as models for cancer and drug research,as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures,and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore,SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells,providing a pathway to enable our understanding of morphogenesis in 3D cultures. View Publication

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair,underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here,we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines,including a WT1-2A-eGFP knock-in reporter line,under chemically-defined,xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells,resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo,as determined by morphological and functional assays,including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies. View Publication

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells,and overexpression of Hoxa-9 markedly expands hematopoietic stem cells,suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However,sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia,indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays,Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability,a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures,Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation,with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo. View Publication

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha,we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally,enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus,the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling,and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution. View Publication

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However,attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions,in up to 63% of 59 low-volume UCB units,cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition,osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However,in contrast to MSCs,MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours,a net volume of more than 33 ml,and an MNC count of more than 1 x 10(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations,we regard UCB as an additional stem cell source for experimental and potentially clinical purposes. View Publication

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs),the molecular mechanism involved remains to be fully elucidated. Here,we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover,BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together,our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy,however,is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs,implying that activation of JNKs is essential for BMP9 osteoinductive activity. View Publication

OBJECTIVE: The aim of this narrative review was to evaluate the role of cancer stem cells (CSCs) and sex steroids in the pathophysiology of human breast cancer. METHODS: A key-word search was performed using the Scopus database. Preference was given to studies using human cells and tissues. RESULTS: Long-term estrogen-progestin hormone therapy is known to increase breast cancer risk,although the mechanisms are poorly understood. In the last few years,it has become clear that many human breast cancers contain CSCs,which may be responsible for much of the tumor's malignant behavior. Very recently,the impact of estrogen,progesterone,and progestins on breast CSCs and their progeny has been studied and clarified. Most breast CSCs are estrogen receptor negative and progesterone receptor negative,although some intermediary progenitor forms have hormone receptors,especially progesterone receptor. Most mature human breast cancer cellsare estrogen receptor positive and can thus be stimulated by estrogen. Breast CSCs usually elaborate CD44+,CD24j/low and/or ALDEFLUOR+ cell markers and are lineage markers negative. One of the main roles of progesterone and progestin seems to be on certain breast cancer stem intermediate forms,inducing them to revert back to a more primitive breast CSC form. CONCLUSIONS: As the pathophysiology of human breast CSC is clarified,it is probable that this will lead to novel,effective breast cancer treatments and,perhaps,new breast cancer preventive agents. This research may also lead to safer hormone therapy regimens. View Publication

Thyroid carcinoma is the most common endocrine malignancy and the first cause of death among endocrine cancers. We show that the tumorigenic capacity in thyroid cancer is confined in a small subpopulation of stem-like cells with high aldehyde dehydrogenase (ALDH(high)) activity and unlimited replication potential. ALDH(high) cells can be expanded indefinitely in vitro as tumor spheres,which retain the tumorigenic potential upon delivery in immunocompromised mice. Orthotopic injection of minute numbers of thyroid cancer stem cells recapitulates the behavior of the parental tumor,including the aggressive metastatic features of undifferentiated thyroid carcinomas,which are sustained by constitutive activation of cMet and Akt in thyroid cancer stem cells. The identification of tumorigenic and metastagenic thyroid cancer cells may provide unprecedented preclinical tools for development and preclinical validation of novel targeted therapies. View Publication

The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells,including fibroblasts,keratinocytes and peripheral blood cells,with variable reprogramming efficiencies and kinetics. Here,we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells,with similar efficiencies to keratinocytes,which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure. View Publication

Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones,three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation,but it does not require culture preadaptation,use of microcarriers or any other matrices. Over a time course of 4-7 d,hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly,hPSCs maintain pluripotency and karyotype stability for more than ten passages. View Publication

Histone deacetylases (HDAC) are key enzymes in the epigenetic control of gene expression. Recently,inhibitors of class I and class II HDAC have been successfully employed for the treatment of different inflammatory diseases such as rheumatoid arthritis,colitis,airway inflammation and asthma. So far,little is known so far about a similar therapeutic effect of inhibitors specifically directed against sirtuins,the class III HDAC. In this study,we investigated the expression and localization of endogenous sirtuins in primary human dermal microvascular endothelial cells (HDMEC),a cell type playing a key role in the development and maintenance of skin inflammation. We then examined the biological activity of sirtinol,a specific sirtuin inhibitor,in HDMEC response to pro-inflammatory cytokines. We found that,even though sirtinol treatment alone affected only long-term cell proliferation,it diminishes HDMEC inflammatory responses to tumor necrosis factor (TNF)α and interleukin (IL)-1β. In fact,sirtinol significantly reduced membrane expression of adhesion molecules in TNFã- or IL-1β-stimulated cells,as well as the amount of CXCL10 and CCL2 released by HDMEC following TNFα treatment. Notably,sirtinol drastically decreased monocyte adhesion on activated HDMEC. Using selective inhibitors for Sirt1 and Sirt2,we showed a predominant involvement of Sirt1 inhibition in the modulation of adhesion molecule expression and monocyte adhesion on activated HDMEC. Finally,we demonstrated the in vivo expression of Sirt1 in the dermal vessels of normal and psoriatic skin. Altogether,these findings indicated that sirtuins may represent a promising therapeutic target for the treatment of inflammatory skin diseases characterized by a prominent microvessel involvement. View Publication