There has been great progress in Huntington’s disease (HD) research. Yet, effective treatments to... more There has been great progress in Huntington’s disease (HD) research. Yet, effective treatments to halt disease before the onset of disabling symptoms are still unavailable. Scientific breakthroughs require an active and lasting commitment from families. However, they are traditionally less involved and heard in studies. Accordingly, the European Huntington Association (EHA) surveyed individuals at risk (HDRisk) and with premanifest HD (PreHD) to determine which factors affect their willingness to participate in research. Questions assessed research experience and knowledge, information sources, reasons for involvement and noninvolvement, and factors preventing and facilitating participation. The survey included 525 individuals, of which 68.8% never participated in studies and 38.6% reported limited research knowledge. Furthermore, 52% trusted patient organizations to get research information. Reasons for involvement were altruistic and more important than reasons for noninvolvement,...
<p>(A) Table showing the number of methylated CGIs (Me-CGI) in each hESC line, and for adul... more <p>(A) Table showing the number of methylated CGIs (Me-CGI) in each hESC line, and for adult somatic tissues (Illingworth et al., 2008), the percentage of methylated CGIs (% Me-CGI), number of methylated gene-associated CGIs (Me-GA-CGI) and the percentage of methylated CGIs that are gene-associated (% Me-GA-CGI). Me-CGIs are given in the supplementary file <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.s018" target="_blank">S1 Table</a>. (B) Venn Diagram shows heterogeneity of hESC CGI methylation. 1111 CGIs are methylated in all 4 lines. (C-G) Genome maps depicting locations of various CGI groups: (C) Me-CGIs in hESC lines (red, gene-associated; green, not gene-associated). (D) 201 GA-CGIs methylated in hESCs but unmethylated in somatic tissues. (E) 98 GA-CGIs unmethylated in hESCs but methylated in somatic tissues. (F) hESC-expressed genes whose associated CGIs are hESC-methylated and unmethylated in somatic tissues. (G) hESC-expressed genes whose associated CGIs are hESC-unmethylated and methylated in somatic tissues. Autosomes ordered 1–22, 23 = X, 24 = Y.</p
Effects of superovulated heifer diet type and quantity on relative mRNA abundance and pyruvate me... more Effects of superovulated heifer diet type and quantity on relative mRNA abundance and pyruvate metabolism in recovered embryos
For a long time it has been assumed that the only role of sperm at fertilization is to introduce ... more For a long time it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo1,2. However conflicting reports have challenged the existence of epigenetic marking of sperm genes3,4, and there are no functional test supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of spermand spermatid-derived frog embryos we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marking. Experimental removal of these epigenetic marks, at fertilization, deregulate gene expression in the resulting embryo...
hypoxia-inducible 2-oxoglutarate dioxygenases to block chemical oxidative stress-induced differen... more hypoxia-inducible 2-oxoglutarate dioxygenases to block chemical oxidative stress-induced differentiation of human embryonic stem cells', Stem Cell Research.
The inadvertent transmission of long incubating, untreatable and fatal neurodegenerative prionopa... more 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.
Methods in molecular biology (Clifton, N.J.), 2018
Cell transplantation therapy aspires to repair and restore lost function while minimizing the ris... more 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).
Methods in molecular biology (Clifton, N.J.), 2017
The promise of human pluripotent stem cells to serve as a scalable and renewable starting materia... more The promise of human pluripotent stem cells to serve as a scalable and renewable starting material for "off the shelf" therapeutic cell products to repair or replace cells and tissues damaged by disease or injury is unparalleled. Whether originating from embryos or the genetic manipulation of adult tissue-derived cells, this prospective impact dictates a comprehensive yet practicable standard of quality assured characterization, blending existing and bespoke standards and considerations. Here, we provide a guide to qualifying the suitability of this resource for human clinical application.
A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripot... more 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.
This article contains data related to the research article entitled "Expression of FBN1 duri... more 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 highlig...
Fibrillin-1 is a large glycoprotein encoded by the FBN1 gene in humans. It provides strength and ... more 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 TGFβ. 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 ...
The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate dem... more The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and ...
The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now b... more 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 heterozygos...
There has been great progress in Huntington’s disease (HD) research. Yet, effective treatments to... more There has been great progress in Huntington’s disease (HD) research. Yet, effective treatments to halt disease before the onset of disabling symptoms are still unavailable. Scientific breakthroughs require an active and lasting commitment from families. However, they are traditionally less involved and heard in studies. Accordingly, the European Huntington Association (EHA) surveyed individuals at risk (HDRisk) and with premanifest HD (PreHD) to determine which factors affect their willingness to participate in research. Questions assessed research experience and knowledge, information sources, reasons for involvement and noninvolvement, and factors preventing and facilitating participation. The survey included 525 individuals, of which 68.8% never participated in studies and 38.6% reported limited research knowledge. Furthermore, 52% trusted patient organizations to get research information. Reasons for involvement were altruistic and more important than reasons for noninvolvement,...
<p>(A) Table showing the number of methylated CGIs (Me-CGI) in each hESC line, and for adul... more <p>(A) Table showing the number of methylated CGIs (Me-CGI) in each hESC line, and for adult somatic tissues (Illingworth et al., 2008), the percentage of methylated CGIs (% Me-CGI), number of methylated gene-associated CGIs (Me-GA-CGI) and the percentage of methylated CGIs that are gene-associated (% Me-GA-CGI). Me-CGIs are given in the supplementary file <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131102#pone.0131102.s018" target="_blank">S1 Table</a>. (B) Venn Diagram shows heterogeneity of hESC CGI methylation. 1111 CGIs are methylated in all 4 lines. (C-G) Genome maps depicting locations of various CGI groups: (C) Me-CGIs in hESC lines (red, gene-associated; green, not gene-associated). (D) 201 GA-CGIs methylated in hESCs but unmethylated in somatic tissues. (E) 98 GA-CGIs unmethylated in hESCs but methylated in somatic tissues. (F) hESC-expressed genes whose associated CGIs are hESC-methylated and unmethylated in somatic tissues. (G) hESC-expressed genes whose associated CGIs are hESC-unmethylated and methylated in somatic tissues. Autosomes ordered 1–22, 23 = X, 24 = Y.</p
Effects of superovulated heifer diet type and quantity on relative mRNA abundance and pyruvate me... more Effects of superovulated heifer diet type and quantity on relative mRNA abundance and pyruvate metabolism in recovered embryos
For a long time it has been assumed that the only role of sperm at fertilization is to introduce ... more For a long time it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo1,2. However conflicting reports have challenged the existence of epigenetic marking of sperm genes3,4, and there are no functional test supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of spermand spermatid-derived frog embryos we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marking. Experimental removal of these epigenetic marks, at fertilization, deregulate gene expression in the resulting embryo...
hypoxia-inducible 2-oxoglutarate dioxygenases to block chemical oxidative stress-induced differen... more hypoxia-inducible 2-oxoglutarate dioxygenases to block chemical oxidative stress-induced differentiation of human embryonic stem cells', Stem Cell Research.
The inadvertent transmission of long incubating, untreatable and fatal neurodegenerative prionopa... more 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.
Methods in molecular biology (Clifton, N.J.), 2018
Cell transplantation therapy aspires to repair and restore lost function while minimizing the ris... more 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).
Methods in molecular biology (Clifton, N.J.), 2017
The promise of human pluripotent stem cells to serve as a scalable and renewable starting materia... more The promise of human pluripotent stem cells to serve as a scalable and renewable starting material for "off the shelf" therapeutic cell products to repair or replace cells and tissues damaged by disease or injury is unparalleled. Whether originating from embryos or the genetic manipulation of adult tissue-derived cells, this prospective impact dictates a comprehensive yet practicable standard of quality assured characterization, blending existing and bespoke standards and considerations. Here, we provide a guide to qualifying the suitability of this resource for human clinical application.
A fast track "Hot Start" process was implemented to launch the European Bank for Induced Pluripot... more 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.
This article contains data related to the research article entitled "Expression of FBN1 duri... more 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 highlig...
Fibrillin-1 is a large glycoprotein encoded by the FBN1 gene in humans. It provides strength and ... more 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 TGFβ. 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 ...
The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate dem... more The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and ...
The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now b... more 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 heterozygos...
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Papers by Paul De Sousa