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

Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. In the absence of Ikaros,there is a lack of markers defining fate restriction along lympho-myeloid pathways,but it is unclear whether formation of specific progenitors or expression of their markers is affected. Here we use a reporter based on Ikaros regulatory elements to separate early progenitors in wild-type and Ikaros-null mice. We found previously undetected Ikaros-null lympho-myeloid progenitors lacking the receptor tyrosine kinase Flt3 that were capable of myeloid but not lymphoid differentiation. In contrast,lack of Ikaros in the common myeloid progenitor resulted in increased formation of erythro-megakaryocytes at the expense of myeloid progenitors. Using this approach,we identify previously unknown pivotal functions for Ikaros in distinct fate 'decisions' in the early hematopoietic hierarchy. View Publication

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here,we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably,1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression,HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters,a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions. View Publication

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T),cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18),and they displayed the ability to generate NP-like tissue in vitro,which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs,and eventually towards regenerative therapies for disc degeneration. View Publication

Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However,treatment is restricted to corneal transplantation,which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study,hiPSCs were successfully differentiated into neural crest cells (NCCs),the embryonic precursor to keratocytes,and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies. View Publication

We report an injectable hydrogel scaffold system with tunable stiffness for controlling the proliferation rate and differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) context in normal growth media. The hydrogels composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate were formed using the oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H(2)O(2)) and horseradish peroxidase (HRP). The stiffness of the hydrogels was readily tuned by varying the H(2)O(2) concentration without changing the concentration of polymer precursor. We found that the hydrogel stiffness strongly affected the cell proliferation rates. The rate of hMSC proliferation increased with the decrease in the stiffness of the hydrogel. Also,the neurogenesis of hMSCs was controlled by the hydrogel stiffness in a 3D context without the use of any additional biochemical signal. These cells which were cultured in hydrogels with lower stiffness for 3 weeks expressed much more neuronal protein markers compared to those cultured within stiffer hydrogels for the same period of time. View Publication

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature,with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries,including planar 2-D films,macroporous 3-D sponges,and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However,synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion,viability,proliferation,self-renewal,and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover,the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment,where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus,synthetic substrates with specific 3-D microgeometries can support hESC colony formation,self-renewal,and directed differentiation to multiple lineages while obviating the stringent needs for complex,exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions. View Publication

The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover,because of its barrier properties,this endothelial interface restricts uptake of neurotherapeutics. Thus,a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues,including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes,including well-organized tight junctions,appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably,they respond to astrocytes,acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2),and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients. View Publication

Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible,cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. The reproducibility of the assay was first demonstrated by seeding hESC clumps at specific cell densities ranging 0.05[x02013]2x105 cells/well and then secondly by using the assay to count cell numbers after different growth conditions. Validation tests showed that consistent seeding densities are important in maintaining undifferentiated hESC culture and that the assay can be used to estimate relative cell numbers and growth curves with high accuracy. View Publication

Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo,but after isolation can be expanded considerably,generating clinically useful numbers of cells. In this study,we demonstrate the use of a defined embryonic stem cell expansion medium,mTeSR (Stem Cell Technologies),for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates,demonstrate tri-lineage differentiation potential,and show similar surface marker profiles (CD29(+),CD44(+),CD49a(+),CD73(+),CD90(+),CD105(+),CD146(+),CD166(+),CD34(-),and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However,expression of early differentiation transcription factors runt-related transcription factor 2,sex-determining region Y box 9,and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated,whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS,adipogenic differentiation was significantly decreased in mTeSR-expanded cells,both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further,this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs,such as bone repair and cartilage generation. View Publication

DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway,which promotes error-free repair of DNA double-strand breaks,is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus,cells from Fanconi anemia patients,which lack this critical pathway,fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study,we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6,but not E7,recovers FA iPSC colony formation and,furthermore,that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase,NANOG,and Tra-1-60,indicating that they were fully reprogrammed into pluripotent cells. However,FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression,whereas their FA-complemented counterparts grew efficiently. Thus,we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53. View Publication

Despite its wide use as a marker for hematopoietic stem cells (HSCs),the function of stem cell antigen-1 (Sca-1) (also known as lymphocyte activation protein-6A [Ly-6A]) in hematopoiesis remains poorly defined. We have previously established that Sca-1(-/-) T cells develop normally,although they are hyperresponsive to antigen. Here,we report detailed analysis of hematopoiesis in Sca-1-deficient animals. The differentiation potential of Sca-1-null bone marrow was determined from examination of the most mature precursors (culture colony-forming units [CFU-Cs]) to less committed progenitors (spleen CFUs [CFU-Ss]) to long-term repopulating HSCs. Sca-1-null mice are mildly thrombocytopenic with a concomitant decrease in megakaryocytes and their precursors. Bone marrow cells derived from Sca-1(-/-) mice also have decreased multipotential granulocyte,erythroid,macrophage,and megakaryocyte CFU (GEMM-CFU) and CFU-S progenitor activity. Competitive repopulation assays demonstrated that Sca-1(-/-) HSCs are at a competitive disadvantage compared with wild-type HSCs. To further analyze the potential of Sca-1(-/-) HSCs,serial transplantations were performed. While secondary repopulations using wild-type bone marrow completely repopulated Sca-1(-/-) mice,Sca-1(-/-) bone marrow failed to rescue one third of lethally irradiated wild-type mice receiving secondary bone marrow transplants from irradiation-induced anemia and contributed poorly to the surviving transplant recipients. These data strongly suggest that Sca-1 is required for regulating HSC self-renewal and the development of committed progenitor cells,megakaryocytes,and platelets. Thus,our studies conclusively demonstrate that Sca-1,in addition to being a marker of HSCs,regulates the developmental program of HSCs and specific progenitor populations. View Publication

The hematopoietic stem cell (HSC) gives rise to all mature,terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure,characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn,these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2. View Publication