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iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed,iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro,phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation,validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data,typically by relating the obtained levels of gene expression to the levels of expression of a house keeping gene"� View Publication

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells,it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system,aerobic glycolysis,and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells,while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass,increased proton leak,and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference,and highlight the importance of further research concerning energy metabolism of stem cells. View Publication

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties,Sca-1 expression (Sca-1+),lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-),and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in textgreater or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients,respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor,interleukin-6 (IL-6),and erythropoietin (with or without IL-3),a large increase in total cell number,including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly,this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo,as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture,providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype. View Publication

Mouse embryonic stem (ES) cells cultured as aggregates and exposed to retinoic acid are induced to express multiple phenotypes normally associated with neurons. A large percentage of treated aggregates produce a rich neuritic outgrowth. Dissociating the induced aggregates with trypsin and plating the cells as a monolayer results in cultures in which a sizable percentage of the cells have a neuronal appearance. These neuron-like cells express class III beta-tubulin and the neurofilament M subunit. Induced cultures express transcripts for neural-associated genes including the neurofilament L subunit,glutamate receptor subunits,the transcription factor Brn-3,and GFAP. Levels of neurofilament L and GAD67 and GAD65 transcripts rise dramatically upon induction. Physiological studies show that the neuron-like cells generate action potentials and express TTX-sensitive sodium channels,as well as voltage-gated potassium channels and calcium channels. We conclude that a complex system of neuronal gene expression can be activated in cultured ES cells. This system should be favorable for investigating some of the mechanisms that regulate neuronal differentiation. View Publication

The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways,the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK,i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose,heat shock),AICAR does not perturb the cellular contents of ATP,ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation,causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase),and almost total cessation of two of the known target pathways,i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase,previously demonstrated in cell-free assays,also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade. View Publication

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid,myeloid,and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required,and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells,this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells. View Publication

Individually,transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) alter the growth and differentiation of normal and transformed osteoblast-like (OB) cells. Although recent evidence suggests interactions between TGF beta and 1,25(OH)2D3 may occur,little is known of the individual or combined effects of these hormones on the expression of the osteoblast phenotype at the cytochemical and biochemical levels in normal human OB (hOB) cells. Primary cultures of hOBs were treated with TGF beta (0.001-10 ng/ml) and 1,25(OH)2D3 (0.1 pM-100 nM) either alone or in combination. TGF beta and 1,25(OH)2D3 stimulated spindle-shaped cells to become stellate in appearance and increased the number of cytoplasmic processes. TGF beta increased 3H-thymidine incorporation and 1,25(OH)2D3 reduced this effect. Conversely,procollagen type-I synthesis and secretion were increased in a dose-dependent manner in the presence of TGF beta but were not significantly affected in the presence of 1,25(OH)2D3. TGF beta and 1,25(OH)2D3 each marginally increased alkaline phosphatase (ALP) activity,but the combination synergistically increased ALP activity in a dose- and time-dependent manner at the cytochemical and biochemical level (three to tenfold over vehicle controls; n = 12). In contrast,TGF beta reduced 1,25(OH)2D3-stimulated osteocalcin secretion. These data suggest that TGF beta stimulates hOB cells to actively produce collagen matrix and proliferate. The combination of TGF beta and 1,25(OH)2D3,however,produces a synergistic increase in ALP activity and maintenance of collagen synthesis. 1,25(OH)2D3 stimulation may induce cells to advance to an endstage where cell proliferation is reduced and osteocalcin expression is promoted. Interactions between TGF beta and 1,25(OH)2D3 may represent important steps in the regulation of osteoblast differentiation and matrix production. View Publication

The epithelial glycoprotein 40 (EGP40,also known as GA733-2,ESA,KSA,and the 17-1A antigen),encoded by the GA-733-2 gene,is expressed on the baso-lateral cell surface in most human simple epithelia. The protein is also expressed in the vast majority of carcinomas and has attracted attention as a tumor marker. The function of the protein is unknown. We demonstrate here that EGP40 is an epithelium-specific intercellular adhesion molecule. The molecule mediates,in a Ca(2+)-independent manner,a homophilic cell-cell adhesion of murine cells transfected with the complete EGP40 cDNA. Two murine cell lines were tested for the effects of EGP40 expression: fibroblastic L cells and dedifferentiated mammary carcinoma L153S cells. The expression of the EGP40 protein causes morphological changes in cultures of transfected cells--increasing intercellular adhesion of the transfectants--and has a clear effect on cell aggregating behavior in suspension aggregation assays. EGP40 directs sorting in mixed cell populations,in particular,causes segregation of the transfectants from the corresponding parental cells. EGP40 expression suppresses invasive colony growth of L cells in EHS-matrigel providing tight adhesions between cells in growing colonies. EGP40 can thus be considered a new member of the intercellular adhesion molecules. In its biological behavior EGP40 resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs),although no immunoglobulin-like repeats are present in the EGP40 molecule. Certain structural similarities in general organization of the molecule exist between EGP40 and the lin-12/Notch proteins. A possible role of this adhesion molecule in formation of architecture of epithelial tissues is discussed. To reflect the function of the molecule the name Ep-CAM for EGP40 seems appropriate. View Publication

The bcr-abl oncogene,present in 95% of patients with chronic myelogenous leukemia (CML),has been implicated as the cause of this disease. A compound,designed to inhibit the Abl protein tyrosine kinase,was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML,there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias. View Publication

The classical definition of lymphohematopoietic stem cells (LHSC),the most primitive progenitors of all blood cells,requires that they have the capacity for self-renewal and for the long-term production of all blood cell lineages. However,other characteristics of LHSC have been debated. Our previous data suggested that mouse LHSC are very slowly proliferating cells that generate delayed multilineage engraftment,while radioprotection" (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) results from more committed progenitors. Alternatively� View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

We examined the stem cell compartment of patients with acquired aplastic anemia (AA) using the long-term culture-initiating cell assay (LTC-IC),in parallel with measurements of CD34+ cells and mature hematopoietic progenitors. Secondary colonies from cells surviving 5 weeks of long-term bone marrow culture (LTBMC) were determined for the peripheral blood (PB) of 68 AA patients and 13 normal controls and for BM of 49 AA patients and 14 controls; because of low cell numbers,formal limiting dilution analysis could only be performed in 10 patients. The relationship of cell input in LTBMC and the output of secondary colonies was linear,allowing quantification of LTC-IC number from bulk cultures. Secondary colony formation was markedly abnormal in severe AA. In contrast to 7.8 colony-forming cells (CFC)/10(5) mononuclear cells in normal BM and 0.14 CFC/10(5) normal PB mononuclear cells,patients with severe disease showed 0.024 CFC/10(5) in BM and 0.0068 CFC/10(5) in PB. Under limiting dilution conditions,patients' cells also showed markedly lower colony-forming ability. In contrast to 4.3 +/- 1 colonies/normal LTC-IC,we obtained only 1.27 +/- 0.09 and 2.0 +/- 0.35 colonies from BM of acute and recovered cases,respectively. These values were used to extrapolate LTC-IC numbers from secondary colony formation in suspension cultures. In PB,calculated LTC-IC were decreased 7.4-fold in new and relapsed severe AA and 2.8-fold in recovered AA. In BM,LTC-IC were decreased 10-fold in new and relapsed AA and sixfold in recovered cases. Compared with measurements obtained on presentation,LTC-IC were lower in post-treatment samples from patients who had failed to recover after intensive immunosuppression and relatively higher in cases at relapse. In recovered patients,LTC-IC number increased but remained below the normal range in 20 of 25. In patients studied serially for 3 to 12 months after treatment,LTC-IC numbers remained stable but low. LTC-IC number correlated with concurrently determined CD34+ cell number and primary hematopoietic colony formation. These results indicate that stem cell numbers,as quantitated by the LTC-IC assay,are markedly diminished in number in all severe AA. Additionally,the function of the stem cell or the stem cell compartment in AA is also abnormal,as inferred from the low clonogenic potential in secondary colony assays. Early hematologic improvement in some patients occurs without increasing numbers of LTC-IC,and a minority of recovered cases show apparent repopulation of the LTC-IC compartment years after treatment. View Publication