Top PDF Stepwise development of hematopoietic stem cells from embryonic stem cells.

Stepwise development of hematopoietic stem cells from embryonic stem cells.

Stepwise development of hematopoietic stem cells from embryonic stem cells.

Humphries (Terry Fox Laboratory, Vancouver, Canada). The Tet- off regulated gene expression plasmid comprised a splice-acceptor (SA) sequence; a loxP-flanked neomycin phosphotransferase gene (neor) gene, including a polyA signal; the Tet-controlled transcrip- tional activator (tTA) gene, including a polyA signal; an insulator sequence; the tTA-responsive element (TRE), followed by the minimal immediate-early promoter from Cytomegalovirus (CMV); the rabbit beta-globin 2nd intron; human HOXB4 cDNA; an internal ribosome entry site (ires); EGFP cDNA; and a polyA signal. The constructed vector was amplified in E. coli Stabl2 cells (Invitrogen), purified using a GENOPURE plasmid maxi kit (Roche), linearized by SwaI digestion, and used to transfect ESCs. The Tet-regulated HOXB4/EGFP expression cassette was inte- grated into the constitutively active ROSA26 locus in EB3 cells by homologous recombination. In brief, EB3 ESCs were electroporated with the linearized vector and were selected with G418 (150– 200 mg/ml). G418-resistant colonies were picked and ES clones carrying a targeted integration of the vector in the ROSA26 locus were identified by long distance-PCR analysis using the following primers: forward (ROSA26 locus 1st exon), 59-CCTCGGCTAGG- TAGGGGATCGGGACTCT-39; reverse (neor gene), 59-CGGA- GAACCTGCGTGCAATCCATCTTGTTC-39; forward (EGFP), 59-GGATCACTCTCGGCATGGACGAGCTGTAC-39; and re- verse (ROSA26 locus 2nd exon), 59-AGCCTTAAACAAG- CACTGTCCTGTCCTCAAG-3 9. The PCR cycles consisted of one cycle at 94 uC for 1 min, 32 cycles at 98uC for 20 s, 66uC for 30 s, 68 uC for 4 min, and one cycle at 72uC for 10 min. To remove the loxP-flanked neor gene, Cre recombinase was transiently expressed in the selected clones by transfection with the pCAG- cre-IRES-puro plasmid. The resultant ES cell line was named ‘‘inducible HOXB4-EGFP ESCs’’ (iHOXB4 ESCs). (B) In the absence of doxycycline (Dox), tTA binds to the TRE, resulting in activation of HOXB4/EGFP transcription. In the presence of Dox, Dox binds tTA, preventing tTA binding to the TRE. We tested if this inducible expression system works in HOXB4 ES clones. Representative results from Western blot analysis for 4 clones are shown. HOXB4 was not detected when ESCs were cultured in the presence of Dox. HOXB4 was detected when ESCs were cultured in the absence of Dox. Anti-FLAG antibody was used to detect HOXB4. EGFP expression in these ES clones was consistent with results from Western blots (data not shown).
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From embryonic stem cells to sensory hair cells : a programming approach

From embryonic stem cells to sensory hair cells : a programming approach

, impairments in the establishment of apico-basal polarity and development of proper stereocilliary bundles is likely to occur in induced HCs. In fact, a recent study has discovered a novel mechanism by which supporting cells can transmit polarity information to HCs. Supporting cells express Ptk7, a novel regulator of the PCP (planar cell polarity), which regulates a apical and contractile myosin II network. This apical myosin II network exerts polarized contractile tension on neighboring HCs to align their PCP (Lee et al., 2012). Another recent study also has shown abnormal hair bundle morphology and orientation when the normal checkerboard-like cell pattern is disrupted and HCs are contacting each other inappropriately instead of supporting cells (Fukuda et al., 2014). This study clearly demonstrates the importance of proper HC and supporting cell-cell contacts in the correct formation of the hair bundle structure.
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Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

The mammary microenvironment plays important function in the regeneration of mammary gland. Mammary stem cells are maintained within specific microenvironments (niches) in the mammary gland and these mammary stem cell niches can be reconstituted de novo by mammary epithelial cells as shown in several studies [7,8,9,10,11,12,13]. Transplanted epithelial cells from the glands can induce mammary regeneration by providing a niche with local signaling cells and extracellular matrix that sustain stem cells [14]. Additionally, parity-identified mammary epithelial cells (PI-MEC), originating from differentiating cells during pregnancy, were shown to possess pluripotency and self-renewal upon transplantation and contributed progeny directly to the formation of secretory acini upon subsequent pregnancies [11,13]. Interestingly, a recent study [15] showed that testicular cells can interact with the mammary epithelial cells within the mammary stroma, proliferate and contribute to epithelial cell progeny, resulting in mammary outgrowth. These testicular cells, when mixed with mammary epithelial cells and when transplanted into denuded mammary fat pads, were able to adopt a mammary epithelial cell phenotype, indicating that the mammary niche can redirect spermatogenic cell fate into mammary epithelial cell fate in vivo . Further, murine fetal and adult neural stem cells (NSCs) when mixed with equal numbers of mammary epithelial cells (MECs) and inoculated into epithelium-divested mammary fat pads of prepubertal female mice, the NSCs contributed to mammary epithelial specific progeny and mammary outgrowths (16). Thus, non-mammary tissues could be altered from their initial cell fate lineage to adopt mammary epithelial characteristics upon interaction with mammary epithelial cells during recon- struction of mammary epithelium in regenerating mammary tissue in vivo (17). However, it is unknown whether other sources of stem cells can generate epithelial progenitors that can reconstitute mammary tissues without the contribution of MECs. Here, in this study, we examined whether murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, can support the recon- stitution of mammary outgrowth when transplanted into epithe- lium-divested mammary fat pads.
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The gene expression profiles of induced pluripotent stem cells (iPSCs) generated by a non-integrating method are more similar to embryonic stem cells than those of iPSCs generated by an integrating method

The gene expression profiles of induced pluripotent stem cells (iPSCs) generated by a non-integrating method are more similar to embryonic stem cells than those of iPSCs generated by an integrating method

Induced pluripotent stem cells (iPSCs) obtained by the ectopic expression of defined transcription factors have tre- mendous promise and therapeutic potential for regenerative medicine. Many studies have highlighted important dif- ferences between iPSCs and embryonic stem cells (ESCs). In this work, we used meta-analysis to compare the global transcriptional profiles of human iPSCs from various cellular origins and induced by different methods. The in- duction strategy affected the quality of iPSCs in terms of transcriptional signatures. The iPSCs generated by non-integrating methods were closer to ESCs in terms of transcriptional distance than iPSCs generated by integrat- ing methods. Several pathways that could be potentially useful for studying the molecular mechanisms underlying transcription factor-mediated reprogramming leading to pluripotency were also identified. These pathways were mostly associated with the maintenance of ESC pluripotency and cancer regulation. Numerous genes that are up-regulated during the induction of reprogramming also have an important role in the success of human preimplan- tation embryonic development. Our results indicate that hiPSCs maintain their pluripotency through mechanisms similar to those of hESCs.
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Differentiation Potential of O Bombay Human-Induced Pluripotent Stem Cells and Human Embryonic Stem Cells into Fetal Erythroid-Like Cells

Differentiation Potential of O Bombay Human-Induced Pluripotent Stem Cells and Human Embryonic Stem Cells into Fetal Erythroid-Like Cells

Identiication of hemangioblast functionality As previously mentioned, cells from early- stage aggregates (14-day) were cultured in con- ditions known to support the growth of blast colonies. As shown in figure 4A, colonies with grape-like morphology of hemangioblast colo- nies were detected in ES and iPSC lines after seeding on a thin layer of matrigel for six days. Cells isolated from these colonies at days 3, 4, 5, and 6 were sub-cultured on methylcellulose to form hematopoietic progenitor cells. As shown in figure 4B, six-day-old colonies formed two types of cells on methylcellulose, adhesive and non-adhesive (or loosely adhesive). Interest- ingly, non-adhesive cells formed small color- ed colonies, their color changed to red pale and more than 80% of them expressed fetal hemoglobin (Fig 4B, C). It seems the culture includes mixed cells. For further evaluation of the erythroid cells, we chose colonies cultured on methylcellulose to be pooled and analyzed for CD71 and GPA expressions by flow cytom- etry. According to our findings, about 5-8% of cells from all lines expressed CD71 + GPA +
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A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.

A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the ability to proliferate indefinitely in an undifferentiated state, and to differentiate to virtually all mature cell types found in the human body when induced with the appropriate combination of growth factors and cytokines. Pluripotent cells offer a powerful system to create in vitro models of human development and disease, provide a valuable source of large quantities of mature cell types of consistent quality and purity for drug discovery and testing, and have strong potential for clinical cell replacement therapies. The hematopoi- etic system is of particular interest for these applications due to the wide range of progenitor and mature blood cell types, which could be generated from pluripotent cells, and for the already available large amount of information on the development and character- ization of these cells. Moreover, establishing a protocol to induce differentiation of hESCs into hematopoietic progenitors provides an easy approach to access to initial steps of hematopoiesis during human ontogeny, which occur in the first weeks of the developing embryo and are therefore impractical to study in vivo. Finally, a robust differentiation method together with the accessibility of patient-specific pluripotent cell lines provide a novel approach to study blood disorders [1], and generation of patient-specific multipotent hematopoietic progenitors could eventually be used in cellular therapy.
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Xinghui Song1 , Yanwei Li1 , Xiao Chen2

Xinghui Song1 , Yanwei Li1 , Xiao Chen2

The difficulties associated with the surgical treatment of trauma, burns, tumor resection, congenital malforma- tions and other causes of soft tissue defects are well-known. With increased development of adipose tissue engineering, new ideas have been developed in relation to the treatment of soft tissue defects and expansion. Adipose-derived stem cells are ideal seed cells for adipose tissue engineering (Rodeheffer et al., 2008). Adipocytes differentiate primar- ily from MSCs through the embryonic body (EB) with the help of induction factors (Dani et al., 1997). But long in- duction times and a low differentiation efficiency are still major problems. Basic fibroblast growth factor (bFGF) is one of the most effective induction factors in cellular differ- entiation and promotes adipocyte differentiation through activation of Ras-MAP in vivo (Hutley et al., 2004; Newell
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Stromal cells in the hematopoietic-stem-cell niche

Stromal cells in the hematopoietic-stem-cell niche

Multiple myeloma (MM) is a B-cell neoplasia character- ized by infiltration and accumulation of clonal plasma cells in bone marrow. This causes a build-up of monoclonal im- munoglobulin in blood and/or urine, and the presence of a peak in the gamma zone of serum and/or urine protein electrophoresis. MSC may play a role in the development of bone disease in MM patients. Based on this hypothesis, our group performed an array-based comparative genomic hy- bridization analysis showing that, while healthy donor MSC were devoid of genomic imbalances, several non-recurrent chromosomal gains and losses (>1 Mb in size) were de- tected in MM-MSC (54). These abnormalities were not found in the myelomatous cells from the same patient.
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Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells.

Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells.

cells with noggin, which is a pSMAD suppressor (14). We cultured CHA13/15 hESCs on nog- gin-expressing cells during the early stage of co-culture differentiation (Fig 4A). Undifferenti- ated CHA13 and CHA15 cells were transferred onto γ-irradiated MEF-noggin feeder cells for 7–10 days before placing them on a MS5-noggin feeder layer. At the end of the stage 1 process, a change in the morphology of the hESCs was evident (Fig 4B-1 and 4C-1, stage 1). Moreover, pSMAD1/5/8 protein and Smad 1, 5, 8 mRNA levels decreased during stage 1 (Fig 4B-2, 3 and 4C-2, 3) compared to levels of these markers in hESCs grown on MEF feeder cells. After 7–10 days, stage 1 cells were split into small clusters, re-seeded on γ-irradiated MS5-noggin cells for 7–10 days (stage 2–1), and then transferred to γ-irradiated MS5-shh cells for another 7–10 days (stage 2–2). Previous studies have established that shh is a crucial factor in the specifica- tion of midbrain DA neurons for mouse ES cell differentiation in culture [22]. Under our cul- ture conditions, rosette structures were clearly observed as shown in Fig 4B-4 and 4C-4 (Insets are high magnification views). Next, we isolated the rosette–like cells mechanically and seeded them on a PLO/FN coated culture dish under ITS + AA + bFGF culture conditions [Fig 4B-5 and 4C-5, stage 3, hESC-derived neural precursor cells (hES-NPCs)]. hES-NPCs were continu- ously expanded following passages. After the final differentiation step, cells expressed the neu- ronal marker TuJ1 and DA marker TH by immunofluorescence (Fig 4B-6 and 4C-6, stage 4). CHA13-derived NPCs expressed the NSC-specific markers nestin and SOX2 (Fig 5A and 5B). These cells were stably expandable without loss of self-renewing potential (Fig 5A and 5B, P4; SOX2, 73.2 ± 1.24%, nestin, 80.8 ± 0.58%, P6; SOX2, 76.6 ± 0.72%, nestin, 84.7 ±1.03%). Dur- ing the final differentiation step, the proportion of TuJ1+ cells (TuJ1/DAPI) and TH+ cells (TH/DAPI) increased as well as the proportion of TH+ cells out of TuJ1+ cells (TH,TuJ1/TH) [Fig 5C and 5D, D6; 27.3 ± 2.22% (TH/DAPI), 43.0 ± 1.94% (TuJ1/DAPI), 68.3 ± 1.71% (TH, TuJ1/TH), D12; 38.2 ± 2.15% (TH/DAPI), 52.5 ± 2.56% (TuJ1/DAPI), 75.0 ± 3.02% (TH,TuJ1/ TH)] based on immunostaining. Semi-quantitative RT-PCR analyses revealed that expression of markers of midbrain DA development, including En-1, Nurr1 and Lmx1b, was induced dur- ing in vitro differentiation (Fig 5E). Expression of the A9-type (nigral) mDA neuron marker Girk2 was also detected (Fig 5E). Similarly, CHA15-derived NPCs also effectively generated NPC and DA neurons (Fig 5F–5J). However, Lmx1b and Girk2 were expressed at low levels in CHA15-derived DA neurons. These findings suggested that highly expressed pSMAD signaling was mitigated by early inhibition of pSMAD signaling by noggin, resulting in the generation of NPCs/DA neurons.
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Rev. Bras. Hematol. Hemoter.  vol.22 número3

Rev. Bras. Hematol. Hemoter. vol.22 número3

Techniques of separation, purification as well as cryopreservation of marrow, blood, cord blood or tissue cells have been developed in blood banks. Transfusion medicine, over the last 20 years, has advanced radically from total blood transfusion to cell and ex vi vo gene th erap y, go i n g th ro u gh d i f f eren t stages, including the production of w ell-defined cell products (red blood cells, platelets, granulocytes), T-cell depletion, as well as cell purification and expansion (hematopoietic stem cells [HSC], immunocompetent cells). Given the experience acquired by blood transfusion centers, one of the main objectives is to adapt modern transfusion medicine for these new types of blood or marrow or tissue products. Transfusion medicine is facing real challenges in various fields of biotechnology, th ese n o v el ap p r o ach es r ep r esen t an ex tr ao r d i n ar y th er ap eu ti c p o ten ti al an d development tool (1, 2).
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Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation.

Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation.

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naı¨ve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.
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Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting ß-cell regeneration

Secondary prevention of type 1 diabetes mellitus: stopping immune destruction and promoting ß-cell regeneration

Type 1 diabetes mellitus results from a cell-mediated autoimmune attack against pancreatic ß-cells. Traditional treatments involve nu- merous daily insulin dosages/injections and rigorous glucose control. Many efforts toward the identification of ß-cell precursors have been made not only with the aim of understanding the physiology of islet regeneration, but also as an alternative way to produce ß-cells to be used in protocols of islet transplantation. In this review, we summarize the most recent studies related to precursor cells implicated in the regeneration process. These include embryonic stem cells, pancreas- derived multipotent precursors, pancreatic ductal cells, hematopoietic stem cells, mesenchymal stem cells, hepatic oval cells, and mature ß- cells. There is controversial evidence of the potential of these cell sources to regenerate ß-cell mass in diabetic patients. However, clinical trials using embryonic stem cells, umbilical cord blood or adult bone marrow stem cells are under way. The results of various immunosuppressive regimens aiming at blocking autoimmunity against pancreatic ß-cells and promoting ß-cell preservation are also ana- lyzed. Most of these regimens provide transient and partial effect on insulin requirements, but new regimens are beginning to be tested. Our own clinical trial combines a high dose immunosuppression with mobilized peripheral blood hematopoietic stem cell transplantation in early-onset type 1 diabetes mellitus.
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Nucleosome Organization in Human Embryonic Stem Cells.

Nucleosome Organization in Human Embryonic Stem Cells.

In eukaryotes, DNA is packaged into chromatin whose fundamental repeating unit is the nucleosome. The nucleosome is comprised of two copies of each of the core histones (H2A, H2B, H3, and H4) wrapped by 147 base pairs (bp) of DNA, with the symmetrical center being called the dyad[12]. Besides being involved in packaging DNA, nucleosome positioning (the genomic location of nucleosomes), nucleosome occupancy (how enriched a genomic location is for nucleosomes), and epigenetic modifications (post-translational modifications of histone proteins and DNA methylation) are thought to play a role in development, transcriptional reg- ulation, cellular identity, evolution, and human disease[13 – 21]. Analyses in model organisms and humans have revealed that the nucleosome organization of a genome is affected by such diverse factors as underlying DNA sequences, nucleosome remodelers, protein binding, and the transcriptional machinery[13–18, 22–31]. Currently there is considerable debate about the roles and extent these factors play, especially in humans compared to yeast[32 – 36]. Further- more, to the best of our knowledge, no one has generated genome-wide maps of nucleosomes in hESC and analyzed its potential role in pluripotency. To begin addressing these questions, we paired-end sequenced Micrococcal Nuclease (MNase) digested DNA from H1 and H9 human embryonic stem cells (hESC), yielding 180x and 70x depth of coverage of the human genome, respectively. A nucleosome occupancy score (NOS) map at single bp resolution with- out smoothing was calculated and used to call nucleosomes (Methods)[37]. The same process- ing was performed on ten other non-hESC datasets including one in vitro (IV) dataset, derived by reconstituting recombinant histones with genomic DNA from human granulocytes as a measure of the purely sequence driven component of nucleosome organization[17]. Addition- ally, nucleosome data was analyzed against a diverse set of epigenomic and genomic features [20, 38–40]. Finally, nucleosome architecture alone was used to predict transcription factor binding sites.
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Interface (Botucatu)  vol.11 número23 en a13v1123

Interface (Botucatu) vol.11 número23 en a13v1123

The vast majority of the informants accepted the legal use of human embryos in research, but this did not mean that most of their projects used this material. To the contrary: there was a clear division between those laboratories (on this point, individual opinions made no impact) which thought adult stem cells more productive and less ethically problematic and which thus preferred to direct their efforts towards this field, and those laboratories which were betting on the potential of embryonic stem cells. One researcher claimed that she preferred to not ally herself with either group, but used in her research both adult and embryonic stem cells on her animal subjects’ injuries in order to better evaluate their results. Some laboratories developed distinct lines of research, the older lines using adult stem cells and the more recent lines investigating the development of embryonic stem cell lineages. One professor, a pioneer in stem cell research, began his interview with the following declaration: “I don’t research embryonic stem cells”. Later in the interview, he remarked on the possibility that embryonic stem cells could cause tumors, but he concluded his deposition by affirming that “for me, this question of whether life begins at fertilization is quite clear. I think we should be pragmatic: if we use a donor’s organs, we also use cells from a donor embryo.” This researcher’s position exemplifies the fact that there is no direct concordance between believing that life begins at fertilization and being ethically against embryonic stem cell research. Many of the interviewees articulated similar beliefs.
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Regulation of PKM2 and Nrf2-ARE pathway during benzoquinone induced oxidative stress in yolk sac hematopoietic stem cells.

Regulation of PKM2 and Nrf2-ARE pathway during benzoquinone induced oxidative stress in yolk sac hematopoietic stem cells.

We had previously established an experimental system for assessment of the hematopoietic toxicity and leukemogenicity of benzene and its metabolites during mouse embryonic development. We found that the cytotoxic and apoptotic effects of benzene metabolites were much pronounced in embryonic YS-HSCs than in adult BM-HSCs [24]. The present study was focused on the possible involvement of ROS generation and the activation of the anti-oxidant defense system in embryonic YS-HSCs treated with BQ. We provided evidence showing that BQ is highly capable of inducing ROS, along with an increased elevation of NOX1 protein, nuclear accumulation of Nrf2 and the activation of Nrf2-ARE pathway in YS-HSCs. The expression of PKM2 protein was decreased by treatment of BQ. Inhibition of PKM2 may play an important role in determining the biological effects of benzene-induced ROS in yolk sac hematopoietic cells. Pretreatment of YS-HSCs with antioxidant NAC reversed PKM2 reduction, suggesting BQ- induced ROS generation contributed to PKM2 degradation.
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Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne dis- eases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis) ocular fluid (BuOF) to replace FBS in cryomedia. Frozen–thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4), mouse embryonic stem (mES) cells, and primary cells, such as mouse embryonic fibroblast (MEF) cells, human peripheral blood mononuclear cells (hPBMCs), and mouse bone marrow cells (mBMCs), were cryopreserved in cryomedia containing 10% DMSO (D10) with 20% FBS (D10S20) or D10 with 20% BuOF (D10O20). For all three cell lines and mES cells cryopre- served in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen–thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA) proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryo- preserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analy- sis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types using BuOF.
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Stem cells – Current concepts and Future applications

Stem cells – Current concepts and Future applications

spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis. It may become possible to generate healthy heart muscle cells in the laboratory and then transplant those cells into patients with chronic heart disease [2]. Bone-marrow have been used to treat cancer patients with conditions such as leukaemia and lymphoma; this is the only form of stem cell therapy that is widely practiced.

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Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them.

Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them.

To derive NSCs, hESC colonies were harvested using a scraper with care taken to avoid high colony fragmentation. Colonies were cultured in suspension as EBs on Petri dishes with agitation for 8 days in StemPro defined media minus FGF2. EBs were then cultured for additional 2–3 days in suspension in neural induction media containing DMEM/F12 with Glutamax, 16NEAA, 16N2 and FGF2 (20 ng/ml) prior to attachment on cell culture plates coated with CellStart. Numerous neural rosettes were formed 2–3 days after adherent culture. To obtain a pure population of NSCs, rosettes were manually isolated using stretched glass Pasteur pipette and placed in fresh culture dishes. The rosettes were then dissociated into single cells using accutase and replated onto culture dishes to obtain a homogeneous population of NSCs. The NSCs population was expanded in Neurobasal media containing 16NEAA, 16L-Glutamine (2 mM), 16B27, LIF, FGF2 20 ng/ml. To confirm that NSCs can differentiate into astrocytes, NSCs were cultured in DMEM/F12 medium supplemented with 16NEAA, L-Glutamine (2 mM), 16N2 and 16B27 for two weeks and processed for immunostaining. To initiate oligoden- drocyte differentiation, NSCs were cultured in medium containing DMEM/F12, 16NEAA, 16L-Glutamine (2 mM), 16N2, 16B27, Shh (200 ng/ml, R&D Systems) and retinoic acid (RA, 2 m M) for
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Rev. Bras. Hematol. Hemoter.  vol.37 número4

Rev. Bras. Hematol. Hemoter. vol.37 número4

Hematological relapse was detected between 21 and 1228 days (mean: 181 days; 95% confidence interval: 94.0–670.0) after transplantation in 23 of 96 patients treated for malignant dis- ease. Relapse was observed in 29.31% of the patients in the BM group and 15.79% in the UCB group (p-value = 0.117). The cumulative incidences of relapse were 45% in the BM group and 25% in the UCB group at two years (Figure 4). The stage of the disease was found to be significant (p-value = 0.005) how- ever cell source, GVHD, CMV and ABO incompatibility were not (Table 2).

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Genetic background drives transcriptional variation in human induced pluripotent stem cells.

Genetic background drives transcriptional variation in human induced pluripotent stem cells.

variability in IPS to a range of sources, including epigenetic memory [5–10,17], inherent differences between IPSCs and ESCs [4,28], artifacts of reprogramming [7] or lab environment [29]. Perhaps surprisingly, the effects of genetic background have been less well appreciated, although more recent work has highlighted its potential importance in differentiation [30]. It seems likely that at least some of variability previously reported to exist in IPS cells could in fact have arisen from genetic differences. This is particularly true of comparisons of IPS with ES that are typically derived from different individuals. It is also notable that studies including larger numbers of donors tend to find fewer transcriptional differences between IPS and ES [29,31–33]. Studies that have not controlled for genetic background when investigating epigenetic memory, such as by confounding tissue of origin and donor, may also have mistakenly attributed genetically driven differences in transcription to epige- netic memory. Our study explicitly incorporates multiple tissues from the same donors, allowing us to correct for the effects of changing genetic background. This is likely to explain why we do not find extensive apparent epigenetic memory. We note, however, that other studies that have also explicitly controlled for genetic background still report some variation in transcription, methylation and differentiation efficiencies that appear to arise from cell type of origin effects [5,8,10]. A possible explanation for the discrepancy between the results of these studies and our own is that we have also taken our samples from cells at between 10 and 13 passages and epigenetic memory effects may be transient and disappear following multiple passages [8].
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