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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. Electronic supplementary material The online version of this article (10.1007/s00401-018-1941-9) contains supplementary material, which is available to authorized users.

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.

Stem cell growth and differentiation is controlled by intrinsic and extrinsic factors. The latter includes growth factors, which are conventionally supplied in vitro in media exchanged daily. Here, we illustrate the use of affinity targeted biodegradable nanoparticles to mediate paracrine stimulation as an alternative approach to sustain the growth and pluripotency of mouse embryonic stem cells. Leukaemia Inhibitory Factor (LIF) was encapsulated in biodegradable nanoparticles and targeted to the cell surface using an antibody to the oligosaccharide antigen SSEA-1. Sustained release of LIF from nanoparticles composed of a solid Poly(lactide-co-glycolic acid) polyester or a hydrogel-based liposomal system, we term Nanolipogel, replenished once after each cell passage, proved as effective as daily replenishment with soluble LIF for maintenance of pluripotency after 5 passages using 104-fold less LIF. Our study constitutes an alternative paradigm for stem cell culture, providing dynamic microenvironmental control of extrinsic bioactive factors benefiting stem cell manufacturing.

Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific.

Susceptibility to prion infection involves interplay between the prion strain and host genetics, but expression of the host-encoded cellular prion protein is a known prerequisite. Here we consider human embryonic stem cell (hESC) susceptibility by characterizing the genetics and expression of the normal cellular prion protein and by examining their response to acute prion exposure. Seven hESC lines were tested for their prion protein gene codon 129 genotype and this was found to broadly reflect that of the normal population. hESCs expressed prion protein mRNA, but only low levels of prion protein accumulated in self-renewing populations. Following undirected differentiation, up-regulation of prion protein expression occurred in each of the major embryonic lineages. Self-renewing populations of hESCs were challenged with infectious human and animal prions. The exposed cells rapidly and extensively took up this material, but when the infectious source was removed the level and extent of intracellular disease-associated prion protein fell rapidly. In the absence of a sufficiently sensitive test for prions to screen therapeutic cells, and given the continued use of poorly characterized human and animal bioproducts during hESC derivation and cultivation, the finding that hESCs rapidly take up and process abnormal prion protein is provocative and merits further investigation.

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

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 (

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.

The expansive growth and differentiation potential of human embryonic stem cells (hESCs) make them a promising source of cells for regenerative medicine. However, this promise is off set by the propensity for spontaneous or uncontrolled differentiation to result in heterogeneous cell populations. Cell elasticity has recently been shown to characterize particular cell phenotypes, with undifferentiated and differentiated cells sometimes showing significant differences in their elasticities. In this study, we determined the Young’s modulus of hESCs by atomic force microscopy using a pyramidal tip. Using this method we are able to take point measurements of elasticity at multiple locations on a single cell, allowing local variations due to cell structure to be identified. We found considerable differences in the elasticity of the analyzed hESCs, reflected by a broad range of Young’s modulus (0.05-10 kPa). This surprisingly high variation suggests that elasticity could serve as the basis of a simple and efficient large scale purification/separation technique to discriminate subpopulations of hESCs.

Dielectrophoresis can discriminate distinct cellular identities in heterogeneous populations, and monitor cell state changes associated with activation and clonal expansion, apoptosis, and necrosis, without the need for biochemical labels. Demonstrated capabilities include the enrichment of haematopoetic stem cells from bone marrow and peripheral blood, and adult stem cells from adipose tissue. Recent research suggests that this technique can predict the ultimate fate of neural stem cells after differentiationbeforethe appearance of specific cell-surface proteins. This review summarises the properties of cells that contribute to their dielectrophoretic behaviour, and their relevance to stem cell research and translational applications.

Revolutionary new opportunities to study human disease were heralded by the development of two new cell-based techniques. These are the methods able to obtain normal development after somatic cell nuclear transfer (SCNT) [45] and to derive stem cells from human embryos [38]. Use of these two new techniques together will provide opportunities to study inherited human disease that are not available in any other way. Such cells would provide new opportunities to study the pathophysiology of inherited human diseases and in turn make it possible to use high-throughput screens to identify drugs able to prevent symptoms of the disease.

The work on human embryonic stem cells is being performed with a variety of cell lines using a variety of culture conditions; a situation that makes standardisation between projects and publications very difficult. Clearly the consequence of using such cells would be wasted time and resources but, more seriously, the generation of erroneous data in the literature which could both confuse and delay scientific progress in this area. This guidance document represents the outcome of the first meeting of the group named The International Stem Cell Banking Initiative held in October 2007. The document has been prepared from the perspective of hESC culture but, in many respects, is broadly applicable to all human stem cell lines including induced pluripotent stem cell lines.

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel™. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.

Harnessing the unparalleled properties of human embryo stem cells (hESCs) for the therapeutic treatment of disease and injury will require a convergence of scientific developments with regulatory standards. In the case of the latter, it is especially critical that standards for clinically assisted reproduction be harmonized with those governing human cell and tissue transplantation, most notably with respect to procurement, donation, testing, processing, preservation, storage and distribution of cells. In the UK, existing infrastructure to address these considerations is undergoing extensive reorganization to keep pace with evolving European Union standards. The present best paradigm for defining standards for the therapeutic use of embryo-derived stem cells is experience with adult haematopoietic stem cells (HSC). However, compared with adult-derived stem cell, the origin of embryo-derived stem cells from limiting quantities of tissue and their absolute dependence on in vitro culture to realise their therapeutic potential, makes optimization of their isolation and cultivation of even greater importance. Most notable is the requirement to create animal cell product-free culture environments to reduce the risk of cross-specific disease transmission. In the present paper, we review present and emerging standards in the isolation and banking of human embryo-derived stem cells for therapeutic use in the UK and international progress in the development of defined culture systems for this purpose.

Realising the therapeutic promise of embryo-derived stem cells is absolutely dependent on the evolution of defined and quality assured in vitro culture systems to mediate their isolation, expansion and delivery. Critical to the safety of these systems, both for the transplant recipient and the general population, is the necessity to eliminate dependence on animal cell-derived reagents to reduce the risk of cross-specific disease transmission. This paper discusses progress in the development of defined culture systems for human embryonic stem cell isolation and the challenges that still must be overcome.

In this issue of Cell Stem Cell, Kim et al. (2007b) show, by genome-wide single-nucleotide polymorphism analysis, that human ES cells claimed to originate from a somatic cell nuclear transfer (SCNT) embryo are actually parthenogenetic. This now permits an appreciation of the future therapeutic utility of this type of cell.