Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture.

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.

EBiSC best practice: How to ensure optimal generation, qualification, and distribution of iPSC lines.

Disease-relevant human induced pluripotent stem cells (iPSCs) are generated worldwide for research purposes; however, without robust and practical ethical, legal, and quality standards, there is a high risk that their true potential will not be realized. Best practices for tissue procurement, iPSC reprogramming, day-to-day cultivation, quality control, and data management aligned with an ethical and legal framework must be included into daily operations to ensure their promise is maximized. Here we discuss key learning experiences from 7 years of operating the European Bank for induced Pluripotent Stem Cells (EBiSC) and recommend how to incorporate solutions into a daily management framework.

Sufficiency of hypoxia-inducible 2-oxoglutarate dioxygenases to block chemical oxidative stress-induced differentiation of human embryonic stem cells.

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.

Renewed assessment of the risk of emergent advanced cell therapies to transmit neuroproteinopathies.

The inadvertent transmission of long incubating, untreatable and fatal neurodegenerative prionopathies, notably iatrogenic Creutzfeldt-Jakob disease, following transplantation of cadaver-derived corneas, pituitary growth, hormones and dura mater, constitutes a historical precedent which has underpinned the application of precautionary principles to modern day advanced cell therapies. To date these have been reflected by geographic or medical history risk-based deferral of tissue donors. Emergent understanding of other prion-like proteinopathies, their potential independence from prions as a transmissible agent and the variable capability of scalably manufacturable stem cells and derivatives to take up and clear or to propagate prions, substantiate further commitment to qualifying neurodegenerative proteinopathy transmission risks. This is especially so for those involving direct or facilitated access to a recipient's brain or connected visual or nervous system such as for the treatment of stroke, retinal and adult onset neurodegenerative diseases, treatments for which have already commenced. In this review, we assess the prospective global dissemination of advanced cell therapies founded on transplantation or exposure to allogeneic human cells, recap lessons learned from the historical precedents of CJD transmission and review recent advances and current limits in understanding of prion and other neurodegenerative disease prion-like susceptibility and transmission. From these we propose grounds for a reassessment of the risks of emergent advanced cell therapies to transmit neuroproteinopathies and suggestions to ACT developers and regulators for risk mitigation and extension of criteria for deferrals.

Quality Assessment and Production of Human Cells for Clinical Use.

Cell transplantation therapy aspires to repair and restore lost function while minimizing the risk of harm. The potential for harm arises from cell instability, variability, inappropriate behavior, and/or transmission of adventitious pathogens. Quality assured and controlled assessment and production of human cells for clinical use ensures that the risk of harm is minimized. Application of quality standards requires thorough planning and consultation with regulatory authorities on process and product specifications, as early as possible at the research and development (R&D) stage. Here we outline considerations applicable to all human cells in relation to regulatory governance, the route to the clinic and Cell Therapy Product (CTP) characterization, with special emphasis on human pluripotent stem cells (hPSC).

Hot Start to European Pluripotent Stem Cell Banking.

Achieving consistency in standards of access to and quality of human induced pluripotent stem cells has lagged behind their use. In Europe, a network of academic and industrial partners has been established to overcome this challenge. The experience reveals the devil in the detail of worthy ambitions informing future efforts.

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC) - the Hot Start experience.

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.

Datasets of genes coexpressed with FBN1 in mouse adipose tissue and during human adipogenesis.

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.

Expression of FBN1 during adipogenesis: Relevance to the lipodystrophy phenotype in Marfan syndrome and related conditions.

Fibrillin-1 is a large glycoprotein encoded by the FBN1 gene in humans. It provides strength and elasticity to connective tissues and is involved in regulating the bioavailability of the growth factor TGFß. Mutations in FBN1 may be associated with depleted or abnormal adipose tissue, seen in some patients with Marfan syndrome and lipodystrophies. As this lack of adipose tissue does not result in high morbidity or mortality, it is generally under-appreciated, but is a cause of psychosocial problems particularly to young patients. We examined the role of fibrillin-1 in adipogenesis. In inbred mouse strains we found significant variation in the level of expression in the Fbn1 gene that correlated with variation in several measures of body fat, suggesting that mouse fibrillin-1 is associated with the level of fat tissue. Furthermore, we found that FBN1 mRNA was up-regulated in the adipose tissue of obese women compared to non-obese, and associated with an increase in adipocyte size. We used human mesenchymal stem cells differentiated in culture to adipocytes to show that fibrillin-1 declines after the initiation of differentiation. Gene expression results from a similar experiment (available through the FANTOM5 project) revealed that the decline in fibrillin-1 protein was paralleled by a decline in FBN1 mRNA. Examination of the FBN1 gene showed that the region commonly affected in FBN1-associated lipodystrophy is highly conserved both across the three human fibrillin genes and across genes encoding fibrillin-1 in vertebrates. These results suggest that fibrillin-1 is involved as the undifferentiated mesenchymal stem cells transition to adipogenesis but then declines as the developing adipocytes take on their final phenotype. Since the C-terminal peptide of fibrillin-1 is a glucogenic hormone, individuals with low fibrillin-1 (for example with FBN1 mutations associated with lipodystrophy) may fail to differentiate adipocytes and/or to accumulate adipocyte lipids, although this still needs to be shown experimentally.

The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines.

The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson's, Huntington's, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised.

Thermoresponsive hydrogel maintains the mouse embryonic stem cell "naïve" pluripotency phenotype.

A chemically defined thermoresponsive hydrogel, poly(AEtMA-Cl-co-DEAEA) cross-linked with N,N'-methylenebisacrylamide, which allows enzyme-free passaging, was used as a substrate to culture murine embryonic stem cells (mESCs) under defined and undefined conditions. Analysis of 14 stem cell markers showed that the mESCs remained in a "naïve" state of pluripotency with differentiation potential to form endoderm, mesoderm, and ectoderm derived lineages. These results validate the use of a chemically defined hydrogel for standardised and inexpensive mESC culture.

Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

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.

White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion.

White matter abnormalities due to age-related cerebrovascular alterations is a common pathological hallmark associated with functional impairment in the elderly which has been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modifications that have been recently linked with age-related neurodegeneration and cerebrovascular pathology. Here we conducted a pilot investigation of whether chronic cerebral hypoperfusion might affect genomic distribution of these modifications and/ or a Ten-Eleven Translocation protein 2 (TET2) which catalyses hydroxymethylation in white and grey matter regions of this animal model. Immunohistochemical evaluation of sham and chronically hypoperfused mice a month after surgery revealed significant (p<0.05) increases in the proportion of 5hmC positive cells, Iba1 positive inflammatory microglia, and NG2 positive oligodendroglial progenitors in the hypoperfused corpus callosum. In the same white matter tract there was an absence of hypoperfusion-induced alterations in the proportion of 5mC, TET2 positive cells and CC1 positive mature oligodrendrocytes. Correlation analysis across animals within both treatment groups demonstrated a significant association of the elevated 5hmC levels with increases in the proportion of inflammatory microglia only (p=0.01) in the corpus callosum. In vitro studies revealed that 5hmC is lost during oligodendroglial maturation but not microglial activation. Additionally, TET1, TET2, and TET3 protein levels showed dynamic alterations during oligodendroglial development and following oxidative stress in vitro. Our study suggests that 5hmC exhibits white matter tract and cell type specific dynamics following chronic cerebral hypoperfusion in mice.

Long term mesenchymal stem cell culture on a defined synthetic substrate with enzyme free passaging.

Mesenchymal stems cells (MSCs) are currently the focus of numerous therapeutic approaches in tissue engineering/repair because of their wide multi-lineage potential and their ability to modulate the immune system response following transplantation. Culturing these cells, while maintaining their multipotency in vitro, currently relies on biological substrates such as gelatin, collagen and fibronectin. In addition, harvesting cells from these substrates requires enzymatic or chemical treatment, a process that will remove a multitude of cellular surface proteins, clearly an undesirable process if cells are to be used therapeutically. Herein, we applied a high-throughput 'hydrogel microarray' screening approach to identify thermo-modulatable substrates which can support hES-MP and ADMSC growth, permit gentle reagent free passaging, whilst maintaining multi-lineage potential. In summary, the hydrogel substrate identified, poly(AEtMA-Cl-co-DEAA) cross-linked with MBA, permitted MSCs to be maintained over 10 passages (each time via thermo-modulation), with the cells retaining expression of MSC associated markers and lineage potency. This chemically defined system allowed the passaging and maintenance of cellular phenotype of this clinically important cell type, in the absence of harsh passaging and the need for biological substrates.

5-azacytidine improves the osteogenic differentiation potential of aged human adipose-derived mesenchymal stem cells by DNA demethylation.

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 (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors.

High-density polymer microarrays: identifying synthetic polymers that control human embryonic stem cell growth.

The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance.

A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells.

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth, while mechanical, enzymatic or chemical cell dissociation methods are used for cellular passaging. However, these methods are ill defined, thus introducing variability into the system, and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate, which support long-term human embryonic stem cell growth and pluripotency over a period of 2-6 months. The hydrogels permitted gentle, reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used, undefined biological substrates represent a flexible and scalable approach for improving the definition, efficacy and safety of human embryonic stem cell culture systems for research, industrial and clinical applications.

A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture.

Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-, G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCß, intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly, treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs, lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (p<0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture.