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BACKGROUND AIMS: We evaluated hematopoietic stem cells according to CD34 expression and aldehyde dehydrogenase (ALDH) activity in peripheral blood and apheresis product samples from patients after mobilization with granulocyte-colony-stimulating factor (G-CSF) alone or G-CSF after high-dose cyclophosphamide (4 g/m²) once daily, intravenously on day 1). We also investigated the relationship between the number of SSC(lo) CD45(dim) CD34(hi) cells, SSC(lo) ALDH(br) cells and engraftment. METHODS: Thirty patients (20 males and 10 females), who were candidates for autologous peripheral blood stem cell transplantation, were included in the study. Cyclophosphamide + G-CSF was used for 17 and G-CSF alone for 24 mobilizations. Primary diagnoses were multiple myeloma (n = 14), Hodgkin's lymphoma (n = 7), non-Hodgkin's lymphoma (n = 2), acute myloid leukemia (n = 2), chronic lymphocytic leukemia (n = 1) and germ cell testis tumor (n = 1). RESULTS: Numbers of SSC(lo) CD45(dim) CD34(hi) cells and SSC(lo) ALDH(br) cells were highly correlated in both peripheral blood and apheresis products (P textless 0.001). We could not find a relationship between the transplanted SSC(lo) CD45(dim) CD34(hi) cell dose or SSC(lo) ALDH(br) cell dose and platelet or neutrophil recovery. The optimal thresholds for SSC(lo) CD45(dim) CD34(hi) cells were 5.40 × 10�?�/kg for neutrophil recovery and 7.22 x 10�?�/kg for platelet recovery. The optimal thresholds for SSC(lo) ALDH(br) cells were 6.53 x 10�?�/kg for neutrophil recovery and 8.72 x 10�?�/kg platelet recovery. CONCLUSIONS: According to our data, numbers of SSC(lo) ALDH(br) cells are in very good agreement with numbers of SSC(lo) CD45(dim) CD34(hi) cells and can be a predictor of stem cell mobilization.

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair, eye, and the mammary gland. In this study, we validate a protocol that utilizes BMP4 and the $$-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGF$$ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGF$$-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development, studying disease pathogenesis, and development of regenerative medicine approaches.

In culture conditions, human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks, which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons, the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study, we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes.

BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential, a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation, the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs, which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating, EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore, this 3D system can also be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications, with the potential of developing to a GMP-compatible scalable system. View Publication

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous, de novo, premature termination codon (PTC) mutation (NM024757.4(EHMT1):c.3413GtextgreaterA; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome, also for drug testing, early biomarker discovery and gene therapy studies.

Long non-coding RNAs (lncRNAs) are a numerous class of newly discovered genes in the human genome, which have been proposed to be key regulators of biological processes, including stem cell pluripotency and neurogenesis. However, at present very little functional characterization of lncRNAs in human differentiation has been carried out. In the present study, we address this using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. With a newly developed method, hESCs were robustly and efficiently differentiated into neurons, and we profiled the expression of thousands of lncRNAs using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified, and shown to physically interact with SOX2, and PRC2 complex component, SUZ12. Using a similar approach, we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis, and immunoprecipitation studies revealed physical association with REST and SUZ12. This study indicates that lncRNAs are important regulators of pluripotency and neurogenesis, and represents important evidence for an indispensable role of lncRNAs in human brain development.

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.

Intercellular interactions in the cell microenvironment play a critical role in determining cell fate, but the effects of these interactions on pathways governing human embryonic stem cell (hESC) behavior have not been fully elucidated. We and others have previously reported that 3-D culture of hESCs affects cell fates, including self-renewal and differentiation to a variety of lineages. Here we have used a microwell culture system that produces 3-D colonies of uniform size and shape to provide insight into the effect of modulating cell-cell contact on canonical Wnt/??-catenin signaling in hESCs. Canonical Wnt signaling has been implicated in both self-renewal and differentiation of hESCs, and competition for ??-catenin between the Wnt pathway and cadherin-mediated cell-cell interactions impacts various developmental processes, including the epithelial-mesenchymal transition. Our results showed that hESCs cultured in 3-D microwells exhibited higher E-cadherin expression than cells on 2-D substrates. The increase in E-cadherin expression in microwells was accompanied by a downregulation of Wnt signaling, as evidenced by the lack of nuclear ??-catenin and downregulation of Wnt target genes. Despite this reduction in Wnt signaling in microwell cultures, embryoid bodies (EBs) formed from hESCs cultured in microwells exhibited higher levels of Wnt signaling than EBs from hESCs cultured on 2-D substrates. Furthermore, the Wnt-positive cells within EBs showed upregulation of genes associated with cardiogenesis. These results demonstrate that modulation of intercellular interactions impacts Wnt/??-catenin signaling in hESCs. ?? 2011 Elsevier Ltd.

The elderly often suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging owing in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of MuSCs from aged mice are intrinsically defective relative to MuSCs from young mice, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation. This deficiency is correlated with a higher incidence of cells that express senescence markers and is due to elevated activity of the p38α and p38β mitogen-activated kinase pathway. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the MuSC population from aged mice to transient inhibition of p38α and p38β in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional MuSC population from aged mice, rejuvenating its potential for regeneration and serial transplantation as well as strengthening of damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy for the elderly.

Success in the differentiating human embryonic stem cells (hESCs) into insulin-secreting β cells raises new hopes for diabetes treatment. In this work, we demonstrated the feasibility of developing islet organoids from hESCs within biomimetic 3D scaffolds. We showed that such a 3D microenvironment is critical to the generation of pancreatic endoderm and endocrine from hESCs. The organoids formed consisted of pancreatic α, β, δ, and pancreatic polypeptide (PP) cells. A high-level co-expression of PDX1, NKX6.1, and NGN3 in these cells suggests the characteristics of pancreatic β cells. More importantly, most insulin-secreting cells generated did not express glucagon, somatostatin, or PP. The expression of mature β cell marker genes such as Pdx1, Ngn3, Insulin, MafA, and Glut2 was detected in these 3D-induced cell clusters. A high-level expression of C-peptide confirmed the de novo endogenous insulin production in these 3D induced cells. Insulin-secretory granules, an indication of β cell maturity, were detected in these cells as well. Glucose challenging experiments suggested that these cells are sensitive to glucose levels due to their elevated maturity. Exposing the cells to a high concentration of glucose induced a sharp increase in insulin secretion.

Bone morphogenetic proteins (BMPs) initiate differentiation in human embryonic stem cells (hESCs) but the exact mechanisms have not been fully elucidated. We demonstrate here that SLUG and MSX2, transcription factors involved in epithelial-mesenchymal transitions, essential features of gastrulation in development and tumor progression, are important mediators of BMP4-induced differentiation in hESCs. Phosphorylated Smad1/5/8 colocalized with the SLUG protein at the edges of hESC colonies where differentiation takes place. The upregulation of the BMP target SLUG was direct as shown by the binding of phosphorylated Smad1/5/8 to its promoter, which interrupted the formation of adhesion proteins, resulting in migration. Knockdown of SLUG by short hairpin RNA blocked these changes, confirming an important role for SLUG in BMP-mediated mesodermal differentiation. Furthermore, BMP4-induced MSX2 expression leads to mesoderm formation and then preferential differentiation toward the cardiovascular lineage.