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

Most primitive hematopoietic cells appear to be normally quiescent in vivo,whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system,where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of primitive" (high proliferative potential)� View Publication

In recent years,studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this,cancer is sustained by highly positioned,chemoresistant cells with extensive capacity of self renewal,which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling,we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1),the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study,we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker,most of them positive also for the stemness marker ALDH1A1,thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore,SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells. View Publication

Despite many years of intensive effort,there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison,the study of cancer stem cells is still in its infancy; so,unsurprisingly,there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal,clonogenicity,multipotentiality,adherence to the niche,and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs),probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules,and contributing to drug resistance. Antibodies are available against the ALDH enzyme family,but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours,but notably breast cancer,cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodeficient mice,and indeed the frequency of so-called ALDH(bri) cells in many tumours can be an independent prognostic indicator. View Publication

The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts. In situ hybridization revealed that Pax7 was also expressed in satellite cells residing in adult muscle. Cell culture and electron microscopic analysis revealed a complete absence of satellite cells in Pax7(-/-) skeletal muscle. Surprisingly,fluorescence-activated cell sorting analysis indicated that the proportion of muscle-derived stem cells was unaffected. Importantly,stem cells from Pax7(-/-) muscle displayed almost a 10-fold increase in their ability to form hematopoietic colonies. These results demonstrate that satellite cells and muscle-derived stem cells represent distinct cell populations. Together these studies suggest that induction of Pax7 in muscle-derived stem cells induces satellite cell specification by restricting alternate developmental programs. View Publication

To test the hypothesis that aging has negative effects on stem-cell homing and engraftment,young or old C57BL/6 bone marrow (BM) cells were injected,using a limiting-dilution,competitive transplantation method,into old or young Ly5 congenic mice. Numbers of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) recovered from BM or spleen were measured and compared with the numbers initially transplanted. Although the frequency of marrow competitive repopulation units (CRUs) increased approximately 2-fold from 2 months to 2 years of age,the BM homing efficiency of old CRUs was approximately 3-fold lower than that of young CRUs. Surprisingly,the overall size of individual stem-cell clones generated in recipients receiving a single CRU was not affected by donor age. However,the increased ages of HSC donors and HSC transplant recipients caused marked skewing of the pattern of engraftment toward the myeloid lineage,indicating that HSC-intrinsic and HSC-extrinsic (microenvironmental) age-related changes favor myelopoiesis. This correlated with changes after transplantation in the rate of recovery of circulating leukocytes,erythrocytes,and platelets. Recovery of the latter was especially blunted in aged recipients. Collectively,these findings may have implications for clinical HSC transplantation in which older persons increasingly serve as donors for elderly patients. View Publication

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells,we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells,whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover,long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage,strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether,these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors,and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis. View Publication

Human pluripotent stem cells (PSCs) are critical in vitro tools forbackslashnunderstanding mechanisms that regulate lineage differentiation inbackslashnthe human embryo as well as a potentially unlimited supply of stembackslashncells for regenerative medicine. Pluripotent human and mouse embryonicbackslashnstem cells (ESCs) derived from the inner cell mass of blastocystsbackslashnshare a similar transcription factor network to maintain pluripotencybackslashnand self-renewal,yet there are considerable molecular differencesbackslashnreflecting the diverse environments in which mouse and human ESCsbackslashnare derived. In the current study we evaluated the role of Proteinbackslashnarginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewalbackslashnand pluripotency given its critical role in safeguarding mouse ESCbackslashnpluripotency. Unlike the mouse,we discovered that PRMT5 has no rolebackslashnin hESC pluripotency. Using microarray analysis we discovered thatbackslashna significant depletion in PRMT5 RNA and protein from hESCs changedbackslashnthe expression of only 78 genes,with the majority being repressed.backslashnFunctionally,we discovered that depletion of PRMT5 had no effectbackslashnon expression of OCT4,NANOG or SOX2,and did not prevent teratomabackslashnformation. Instead,we show that PRMT5 functions in hESCs to regulatebackslashnproliferation in the self-renewing state by regulating the fractionbackslashnof cells in Gap 1 (G1) of the cell cycle and increasing expressionbackslashnof the G1 cell cycle inhibitor P57. Taken together our data unveilsbackslashna distinct role for PRMT5 in hESCs and identifies P57 as new target. View Publication

Telomeres as the protective ends of linear chromosomes,are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes,telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study,we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently,the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect,highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies. The online version contains supplementary material available at 10.1007/s00018-024-05239-7. View Publication

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin,glucagon,somatostatin,pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm,gut-tube endoderm,pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells,they release C-peptide in response to multiple secretory stimuli,but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy. View Publication

UNLABELLED The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next,the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation,these cells expressed the adult liver cell markers tyrosine aminotransferase,tryptophan oxygenase 2,phosphoenolpyruvate carboxykinase (PEPCK),Cyp7A1,Cyp3A4 and Cyp2B6. Furthermore,these cells exhibited functions associated with mature hepatocytes including albumin secretion,glycogen storage,indocyanine green,and low-density lipoprotein uptake,and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice,these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition,we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development,and form the basis for hepatocyte transplantation and drug tests. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia,haploid spermatocytes,or spermatids. Here,we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages,including postmeiotic,spermatid-like cells,in vitro without genetic manipulation. Furthermore,our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-,PLZF-,and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin,transition protein 1,and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro View Publication

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed,we report that the cells survive and differentiate upon transplant into newborn rat brain. View Publication