A Blueprint for Translational Regenerative Medicine

The last few decades have produced a large number of proof-of-concept studies in regenerative medicine. However, the route to clinical adoption is fraught with technical and translational obstacles that frequently consign promising academic solutions to the so-called “valley of death.” This review is intended to serve as a blueprint for translational regenerative medicine: we suggest principles to help guide cell and material selection, present key in vivo imaging modalities and argue that the host immune response should be considered throughout therapeutic development. Finally, we suggest a pathway to navigate the often complex regulatory and manufacturing landscape of translational regenerative medicine.J.P.K.A. acknowledges support from an Arthritis Research U.K. Foundation Fellowship (21138) and the Medical Research Council (MRC) (MR/S00551X/1). T.J.K. acknowledges the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Individual European Fellowship (Grant Agreement No. 746980). A.C.R. acknowledges support from the Wellcome Trust Institutional Translational Partnership Award (208858/Z/17/Z). P.S.P. acknowledges funding from the UK Regenerative Medicine Platform (MR/K026739/1), the MRC (MR/R026416/1), and the EPSRC (EP/R511638/1). C.M.M. was supported by the MRC-funded UK Regenerative Medicine Platform Immunomodulation Hub award (MR/L022699/1). W.L.K. is supported by the MRC/UKRI Innovate Fellowship (MR/S005528/1). V.P. acknowledges funding from the UK Regenerative Medicine Platform (MR/R015724/1). R.O.C.O acknowledges support from the UK Regenerative Medicine Platform “Acellular / Smart Materials – 3D Architecture” (MR/R015651/1), the Rosetrees Trust, Wessex Medical Research and Biotechnology and Biological Sciences Research Council (BBSRC) (BB/P017711/1). D.J.S. acknowledges support from a British Heart Foundation (BHF) Intermediate Basic Science Research Fellowship (FS/15/33/31608), the BHF Centre for Regenerative Medicine (RM/17/1/33377), and an MRC Project Grant (MR/R026416/1). F.M.W. acknowledges support from the UK Regenerative Medicine Platform (MR/LO22699/1, MR/R015635/1, MR/K026666/1). S.J.F. acknowledges support from the UK Regenerative Medicine Platform Engineered Cell Environment Hub and the MRC. R.A.B. acknowledges MRC-WT funding of the Cambridge Stem Cell Institute and MRC funding of the UK Regenerative Medicine Platform Pluripotent Stem and Engineered Cell hub (MR/R015724/1) along with funding from the Rosetrees Trust (A1519 M654) and Cure Parkinson's Trust. R.A.B. is also supported by NIHR funding of Biomedical Research Centre Cambridge (146281). R.A.B. is an NIHR Senior Investigator (NF-SI-0616-10011) and a 23 P.I. in the MRC/WT Stem Cell Institute (203151/Z/16/Z). M.M.S. acknowledges support from the grant from the UK Regenerative Medicine Platform “Acellular / Smart Materials – 3D Architecture” (MR/R015651/1), the European Research Council (ERC) Seventh Framework Programme Consolidator grant “Naturale CG” (616417), the Rosetrees Trust and the Wellcome Trust Senior Investigator Award (098411/Z/12/Z). The authors would also like to thank David Pan (MRC) for his input into this manuscript during the course of Phases 1 and 2 of the UK Regenerative Medicine Platform

Biofunctionalised bacterial cellulose scaffold supports the patterning and expansion of human embryonic stem cell-derived dopaminergic progenitor cells.

BACKGROUND: Stem cell-based therapies for neurodegenerative diseases like Parkinson's disease are a promising approach in regenerative medicine and are now moving towards early stage clinical trials. However, a number of challenges remain including the ability to grow stem cells in vitro on a 3-dimensional scaffold, as well as their loss, by leakage or cell death, post-implantation. These issues could, however, be helped through the use of scaffolds that support the growth and differentiation of stem cells both in vitro and in vivo. The present study focuses on the use of bacterial cellulose as an in vitro scaffold to promote the growth of different stem cell-derived cell types. Bacterial cellulose was used because of its remarkable properties such as its wettability, ability to retain water and low stiffness, all of which is similar to that found in brain tissue. METHODS: We cultured human embryonic stem cell-derived progenitor cells on bacterial cellulose with growth factors that were covalently functionalised to the surface via silanisation. Epifluorescence microscopy and immunofluorescence were used to detect the differentiation of stem cells into dopaminergic ventral midbrain progenitor cells. We then quantified the proportion of cells that differentiated into progenitor cells and compared the effect of growing cells on biofunctionalised cellulose versus standard cellulose. RESULTS: We show that the covalent functionalisation of bacterial cellulose sheets with bioactive peptides improves the growth and differentiation of human pluripotent stem cells into dopaminergic neuronal progenitors. CONCLUSIONS: This study suggests that the biocompatible material, bacterial cellulose, has potential applications in cell therapy approaches as a means to repair damage to the central nervous system, such as in Parkinson's disease but also in tissue engineering

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization.

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2(D2723H), and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2(D2723H) decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation

Kruppel-associated Box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1β (HP1-β) mobilization and DNA repair in heterochromatin

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair

Germline Brca2 heterozygosity promotes Kras(G12D) -driven carcinogenesis in a murine model of familial pancreatic cancer

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldInherited heterozygous BRCA2 mutations predispose carriers to tissue-specific cancers, but somatic deletion of the wild-type allele is considered essential for carcinogenesis. We find in a murine model of familial pancreatic cancer that germline heterozygosity for a pathogenic Brca2 truncation suffices to promote pancreatic ductal adenocarcinomas (PDACs) driven by Kras(G12D), irrespective of Trp53 status. Unexpectedly, tumor cells retain a functional Brca2 allele. Correspondingly, three out of four PDACs from patients inheriting BRCA2(999del5) did not exhibit loss-of-heterozygosity (LOH). Three tumors from these patients displaying LOH were acinar carcinomas, which also developed only in mice with biallelic Brca2 inactivation. We suggest a revised model for tumor suppression by BRCA2 with implications for the therapeutic strategy targeting BRCA2 mutant cancer cells

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization

Germline mis-sense mutations affecting a single BRCA2 allele predispose humans to cancer. Here, we identify a protein-targeting mechanism disrupted by the cancer-associated mutation, BRCA2(D2723H) that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mis-localization of mutant BRCA2 inhibits the nuclear retention of RAD51, by exposing a similar NES in RAD51 usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Interestingly, BRCA2(D2723H) decreases RAD51 nuclear retention even when wildtype BRCA2 is present. Our findings suggest a mechanism for regulation of the nucleo-cytoplasmic distribution of BRCA2 and RAD51, and for its impairment by a heterozygous disease-associated mutation

The GDI-like solubilizing factor PDE delta sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.Chan Zuckerberg Initiative Neurodegeneration Challenge Network, New York Stem Cell Foundatio