Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells.

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and ßIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.

Self-renewal and differentiation capabilities are variable between human embryonic stem cell lines I3, I6 and BG01V.

BACKGROUND: A unique and essential property of embryonic stem cells is the ability to self-renew and differentiate into multiple cell lineages. However, the possible differences in proliferation and differentiation capabilities among independently-derived human embryonic stem cells (hESCs) are not well known because of insufficient characterization. To address this question, a side-by-side comparison of 1) the ability to maintain an undifferentiated state and to self-renew under standard conditions; 2) the ability to spontaneously differentiate into three primary embryonic germ lineages in differentiating embryoid bodies; and 3) the responses to directed neural differentiation was made between three NIH registered hES cell lines I3 (TE03), I6 (TE06) and BG01V. Lines I3 and I6 possess normal XX and a normal XY karyotype while BG01V is a variant cell line with an abnormal karyotype derived from the karyotypically normal cell line BG01. RESULTS: Using immunocytochemistry, flow cytometry, qRT-PCR and MPSS, we found that all three cell lines actively proliferated and expressed similar "stemness" markers including transcription factors POU5F1/Oct3/4 and NANOG, glycolipids SSEA4 and TRA-1-81, and alkaline phosphatase activity. All cell lines differentiated into three embryonic germ lineages in embryoid bodies and into neural cell lineages when cultured in neural differentiation medium. However, a profound variation in colony morphology, growth rate, BrdU incorporation, and relative abundance of gene expression in undifferentiated and differentiated states of the cell lines was observed. Undifferentiated I3 cells grew significantly slower but their differentiation potential was greater than I6 and BG01V. Under the same neural differentiation-promoting conditions, the ability of each cell line to differentiate into neural progenitors varied. CONCLUSION: Our comparative analysis provides further evidence for similarities and differences between three hESC lines in self-renewal, and spontaneous and directed differentiation. These differences may be associated with inherited variation in the sex, stage, quality and genetic background of embryos used for hESC line derivation, and/or changes acquired during passaging in culture.

Comparison of neural differentiation potential of human pluripotent stem cell lines using a quantitative neural differentiation protocol.

Neural differentiation of human embryonic (ES) and induced pluripotent (iPS) stem cell lines has been used for research in early human development, drug discovery, and cell replacement therapies. It is critical to establish generic differentiation protocols to compare the neural specification potential of each individually derived pluripotent stem cell line and identify the efficacious lines for research and therapeutic use. Here, we describe a reproducible and quantitative protocol to assess the neural progenitor (NP) generation of human pluripotent stem cell lines. This method includes a robust and well-defined neural inducing platform for Pax6(+) neural rosette (neuroectodermal cells) generation, propagation, and subsequent differentiation into nestin(+) NPs. A side-by-side comparison under common culture conditions among three human ES cell lines, TE03, TE06, and BG01V, and one iPS cell line, HD02, showed highly variable efficiency in their differentiation into NPs.

Reconstruction of functional cortical-like tissues from neural stem and progenitor cells.

Neural stem and progenitor cells isolated from embryonic day 13 rat cerebral cortex were immobilized in three-dimensional type I collagen gels, and then the cell-collagen constructs were transferred to rotary wall vessel bioreactors and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) combined with brain-derived neurotrophic factor for up to 10 weeks. Remarkably, the collagen-entrapped cells formed a complex two-layered structure that emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The surface layer (layer I) composed primarily of proliferating neural progenitor cells (nestin(+), vimentin(+), and PCNA(+)) predominantly expressed functional neurotransmitter receptors for cholinergic and purinergic agonists while differentiating cells (TuJ1(+) and GFAP(+)) in the deeper layer (layer II) contained differentiated neurons and astrocytes and mainly responded to GABAergic and glutamatergic agonists and to veratridine, which activates voltage-dependent Na(+) channels. An active synaptic vesicle recycling was demonstrated by neuronal networks in the deeper layer using the endocytotic marker FM1-43. Cell polarization forming the characteristic two-layered structure was found to associate with the bFGF and FGF receptor signaling. These engineered functional tissue constructs have a potential use as tissue surrogates for drug screening and detection of environmental toxins, and in neural cell replacement therapy.

Secular trends in mortality associated with bloodstream infections in 4268 patients hospitalized in a cancer referral center between 1975 and 1989.

OBJECTIVE: To study the trends in mortality over 15 years in hospitalized cancer patients with bloodstream infection. METHODS: The yearly incidence rates and risk of death, by type of microorganism, were calculated for 4268 cancer patients hospitalized between 1975 and 1989 in a French cancer referral center. The relative risk of death (RR) associated with each type of microorganism was estimated using the proportional hazards model, taking into account age, hospital ward, underlying disease, geographical origin and year of the first positive blood culture. RESULTS: The incidence of these infections was five-fold higher in 1989 than in 1975. The largest increases were for coagulase-negative staphylococci (CNS), yeasts and Staphylococcus aureus. For the 3756 patients who had a single-microorganism bloodstream infection, the risk of death compared with that of patients with CNS infection was significantly increased in those with Pseudomonadaceae (RR=5.0), yeasts (RR=3.4), Enterobacteriaceae (RR=3.2), S. aureus (RR=2.8) and streptococci (RR=2.1). The risk of death was not significantly different between patients with a single or several positive blood cultures nor between those with nosocomial or non-nosocomial infections. When the study period was divided in two time periods (1975 to 1982 vs 1983 to 1989), a significant variation (p=0.001) in risk of death associated with the different microorganisms was observed. Most risks were lower from 1983 to 1986 than before 1982. This decrease reached 60% for both S. aureus and Pseudomonadaceae. CONCLUSIONS: These data support of continuing use of aggressive empirical antimicrobial therapy for cancer patients with fever.