Search

Abstract Background Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. Main Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. Conclusion Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

Hypoxia benefits undifferentiated pluripotent stem cell renewal, and 2-oxoglutarate (2OG) dioxygenases have been implicated in pluripotent stem cell induction and renewal. We show in human embryonic stem cells (hESC) that an ambient oxygen-induced oxidative stress response elicited by culture in a hypoxic atmosphere (0.5% O2) correlates with the expression of 2OG dioxygenases, which oxidise DNA (TET1, 2, 3) and histone H3 (KDM4C), the former reflected by elevation in genomic 5-hydroxymethylcytosine (5hmC). siRNA-mediated targeting of KDM4C and TET1–3 induces hESC differentiation. Under ambient atmospheric oxygen (21% O2), exposure to a low inhibitory concentration of sodium arsenite (NaAsO2, IC10), as a model of chemically-induced oxidative stress, suppresses antioxidant gene expression, reduces mitochondrial membrane potential and induces hESC differentiation. Co-administration of the antioxidant N-acetyl-L-cysteine promoted anti-oxidant, pluripotency and 2OG dioxygenase gene expression, elevated genomic hydroxymethylation and blocked induction of differentiation. Transient ectopic expression of KDM4C or TET1 in ambient atmospheric oxygen achieved the same. Our study substantiates a role for 2OG-dependent dioxygenases in hypoxia's promotion of undifferentiated hESC self-renewal.

This article contains data related to the research article entitled “Expression of FBN1 during adipogenesis: relevance to the lipodystrophy phenotype in Marfan syndrome and related conditions” [1]. The article concerns the expression of FBN1, the gene encoding the extracellular matrix protein fibrillin-1, during adipogenesis in vitro and in relation to adipose tissue in vivo. The encoded protein has recently been shown to produce a short glucogenic peptide hormone, (Romere et al., 2016) [2], and this gene is therefore a key gene for regulating blood glucose levels. FBN1 and coexpressed genes were examined in mouse strains and in human cells undergoing adipogenesis. The data show the genes that were coexpressed with FBN1, including genes coding for other connective tissue proteins and the proteases that modify them and for the transcription factors that control their expression. Data analysed were derived from datasets available in the public domain and the analysis highlights the utility of such datasets for ongoing analysis and hence reduction in the use of experimental animals.

Human embryonic stem cells (hESCs) undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency "signature" would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI) methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5) induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells.

The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (