Intervention with metabolites emulating endogenous cell transitions accelerates muscle regeneration in young and aged mice.

Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.

Wiskott-Aldrich syndrome protein forms nuclear condensates and regulates alternative splicing.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Cross-species metabolomic analysis identifies uridine as a potent regeneration promoting factor.

Regenerative capacity declines throughout evolution and with age. In this study, we asked whether metabolic programs underlying regenerative capability might be conserved across species, and if so, whether such metabolic drivers might be harnessed to promote tissue repair. To this end, we conducted metabolomic analyses in two vertebrate organ regeneration models: the axolotl limb blastema and antler stem cells. To further reveal why young individuals have higher regenerative capacity than the elderly, we also constructed metabolic profiles for primate juvenile and aged tissues, as well as young and aged human stem cells. In joint analyses, we uncovered that active pyrimidine metabolism and fatty acid metabolism correlated with higher regenerative capacity. Furthermore, we identified a set of regeneration-related metabolite effectors conserved across species. One such metabolite is uridine, a pyrimidine nucleoside, which can rejuvenate aged human stem cells and promote regeneration of various tissues in vivo. These observations will open new avenues for metabolic intervention in tissue repair and regeneration.

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction.

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

Single-dose CRISPR-Cas9 therapy extends lifespan of mice with Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare lethal genetic disorder characterized by symptoms reminiscent of accelerated aging. The major underlying genetic cause is a substitution mutation in the gene coding for lamin A, causing the production of a toxic isoform called progerin. Here we show that reduction of lamin A/progerin by a single-dose systemic administration of adeno-associated virus-delivered CRISPR-Cas9 components suppresses HGPS in a mouse model.

In vivo reprogramming of wound-resident cells generates skin epithelial tissue.

Large cutaneous ulcers are, in severe cases, life threatening1,2. As the global population ages, non-healing ulcers are becoming increasingly common1,2. Treatment currently requires the transplantation of pre-existing epithelial components, such as skin grafts, or therapy using cultured cells2. Here we develop alternative supplies of epidermal coverage for the treatment of these kinds of wounds. We generated expandable epithelial tissues using in vivo reprogramming of wound-resident mesenchymal cells. Transduction of four transcription factors that specify the skin-cell lineage enabled efficient and rapid de novo epithelialization from the surface of cutaneous ulcers in mice. Our findings may provide a new therapeutic avenue for treating skin wounds and could be extended to other disease situations in which tissue homeostasis and repair are impaired.

Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells.

CpG islands (CGIs) are primarily promoter-associated genomic regions and are mostly unmethylated within highly methylated mammalian genomes. The mechanisms by which CGIs are protected from de novo methylation remain elusive. Here we show that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation. By targeting the DNA mismatch repair gene MLH1 CGI, we could generate a PSC model of a cancer-related epimutation. Furthermore, we successfully corrected aberrant imprinting in induced PSCs derived from an Angelman syndrome patient. Our results provide insights into how CpG-free DNA induces de novo CGI methylation and broaden the application of targeted epigenome editing for a better understanding of human development and disease.

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Multiple mechanisms mediate the taurine-induced proliferation of neural stem/progenitor cells from the subventricular zone of the adult mouse.

Taurine was previously reported to increase the proliferation of neural precursor cells (NPCs) from subventricular zone of the mouse brain. The results of a study that aimed to understand the mechanisms of this effect are presented here. Because taurine was not found in NPC nuclei, direct interactions with nuclear elements seem unlikely. A gene expression profile analysis indicated that genes that are regulated by taurine have roles in i) proliferation, including the Shh and Wnt pathways; ii) cellular adhesion; iii) cell survival; and iv) mitochondrial functioning. Cell cycle analysis of propidium iodide and CFSE-labeled cells using flow cytometry revealed an increase in the number of cells in the S-phase and a decrease in those in the G0/G1 phase in taurine-treated cultures. No changes in the length of the cell cycle were observed. Quantification of the viable, apoptotic, and necrotic cells in cultures using flow cytometry and calcein-AM, annexin-V, and propidium iodide staining showed reductions in the number of apoptotic and necrotic cells (18% to 11% and 13% to 10%, respectively) and increases in the number of viable cells (61% to 69%) in the taurine-treated cultures. Examination of the relative mitochondrial potential values by flow cytometry and rhodamine123 or JC-1 staining showed a 44% increase in the number of cells with higher mitochondrial potential and a 38% increase in the mitochondrial membrane potential in taurine cultures compared with those of controls. Taken together, the results suggest that taurine provides more favorable conditions for cell proliferation by improving mitochondrial functioning.

Regulatory volume decrease in neural precursor cells: taurine efflux and gene microarray analysis.

BACKGROUND/AIMS: Neural stem/ progenitor cells (NPCs) endure important changes in cell volume during growth, proliferation and migration. As a first approach to know about NPC response to cell volume changes, the Regulatory Volume Decrease (RVD) subsequent to hypotonic swelling was investigated. METHODS: NPCs obtained from the mesencephalon and the subventricular zone of embryonic and adult mice, respectively, were grown and cultured as neurospheres. Cell volume changes were measured by large-angle light-scattering and taurine efflux by [(3)H]-taurine. Expression of genes encoding molecules related to RVD was analysed using a DNA microarray obtained from NPC samples. RESULTS: Embryonic and adult NPCs exposed to osmolarity reduction (H15, H30, H40) exhibited rapid swelling followed by RVD. The magnitude, efficiency and pharmacological profile, of RVD and of [(3)H]-taurine osmosensitive efflux were comparable to those found in cultured brain cells, astrocytes and neurons. The relative expression of genes encoding molecules related to volume regulation, i.e. K(+) and Cl(-) channels, cotransporters, exchangers and aquaporins were identified in NPCs. CONCLUSION: NPCs show the ability to respond to hypotonic-evoked volume changes by adaptative recovery processes, similar to those found in other cultured brain cells. Genes related to molecules involved in RVD were found expressed in NPCs.

Taurine enhances the growth of neural precursors derived from fetal human brain and promotes neuronal specification.

Taurine is present at high concentrations in the fetal brain and is required for optimal brain development. Recent studies have reported that taurine causes increased proliferation of neural stem/progenitor neural cells (neural precursor cells, NPCs) obtained from embryonic and adult rodent brain. The present study is the first to show that taurine markedly increases cell numbers in cultures and neuronal generation from human NPCs (hNPCs). hNPCs obtained from 3 fetal brains (14-15 weeks of gestation) were cultured and expanded as neurospheres, which contained 76.3% nestin-positive cells. Taurine (5-20 mM) increased the number of hNPCs in culture, with maximal effect found at 10 mM and 4 days of culture. The taurine-induced increase ranged from 57 to 188% in the 3 brains examined. Taurine significantly enhanced the percentage of neurons formed from hNPCs under differentiating conditions, with increases ranging from 172 to 480% over controls without taurine. Taurine also increased the cell number and neuronal generation in cultures of the immortalized human cell line ReNcell VM. These results suggest that taurine has a positive influence on hNPC growth and neuronal formation.

Taurine stimulates proliferation and promotes neurogenesis of mouse adult cultured neural stem/progenitor cells.

This study reports an effect of taurine (1-10 mM) increasing markedly (120%) the number of neural precursor cells (NPCs) from adult mouse subventricular zone, cultured as neurospheres. This effect is one of the highest reported for adult neural precursor cells. Taurine-containing cultures showed 73-120% more cells than controls, after 24 and 96 h in culture, respectively. Taurine effect is due to enhanced proliferation as assessed by BrdU incorporation assays. In taurine cultures BrdU incorporation was markedly higher than controls from 1.5 to 48 h, with the maximal difference found at 1.5 h. This effect of taurine reproduced at every passage with the same window time. Taurine effects are not mimicked by glycine, alanine or GABA. Clonal efficiency values of 3.6% for taurine cultures and 1.3% for control cultures suggest a taurine influence on both, progenitor and stem cells. Upon differentiation, the proportion of neurons in control and taurine cultures was 3.1% (±0.5) and 10.2% (±0.8), respectively. These results are relevant for taurine implication in brain development as well as in adult neurogenesis. Possible mechanisms underlying taurine effects on cell proliferation are discussed.