Ignacio Sancho-Martinez

Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.

Yes and PI3K bind CD95 to signal invasion of glioblastoma.

Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-beta pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

Directed differentiation of human pluripotent cells to ureteric bud kidney progenitor-like cells

Diseases affecting the kidney constitute a major health issue worldwide. Their incidence and poor prognosis affirm the urgent need for the development of new therapeutic strategies. Recently, differentiation of pluripotent cells to somatic lineages has emerged as a promising approach for disease modelling and cell transplantation. Unfortunately, differentiation of pluripotent cells into renal lineages has demonstrated limited success. Here we report on the differentiation of human pluripotent cells into ureteric-bud-committed renal progenitor-like cells. The generated cells demonstrated rapid and specific expression of renal progenitor markers on 4-day exposure to defined media conditions. Further maturation into ureteric bud structures was accomplished on establishment of a three-dimensional culture system in which differentiated human cells assembled and integrated alongside murine cells for the formation of chimeric ureteric buds. Altogether, our results provide a new platform for the study of kidney diseases and lineage commitment, and open new avenues for the future application of regenerative strategies in the clinic.

Targeted Gene Correction of Laminopathy-Associated LMNA Mutations in Patient-Specific iPSCs

Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics.

Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019), which is associated with familial and sporadic Parkinson’s disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson’s disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization, clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson’s disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson’s disease patients. Together, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s disease pathology and may help to open new avenues for Parkinson’s disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure.

Reprogramming of Human Fibroblasts to Pluripotency with Lineage Specifiers

Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.

Conversion of human fibroblasts to angioblast-like progenitor cells

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.

Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Lebers hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

CD95-Ligand on Peripheral Myeloid Cells Activates Syk Kinase to Trigger Their Recruitment to the Inflammatory Site

Injury to the central nervous system initiates an uncontrolled inflammatory response that results in both tissue repair and destruction. Here, we showed that, in rodents and humans, injury to the spinal cord triggered surface expression of CD95 ligand (CD95L, FasL) on peripheral blood myeloid cells. CD95L stimulation of CD95 on these cells activated phosphoinositide 3-kinase (PI3K) and metalloproteinase-9 (MMP-9) via recruitment and activation of Syk kinase, ultimately leading to increased migration. Exclusive CD95L deletion in myeloid cells greatly decreased the number of neutrophils and macrophages infiltrating the injured spinal cord or the inflamed peritoneum after thioglycollate injection. Importantly, deletion of myeloid CD95L, but not of CD95 on neural cells, led to functional recovery of spinal injured animals. Our results indicate that CD95L acts on peripheral myeloid cells to induce tissue damage. Thus, neutralization of CD95L should be considered as a means to create a controlled beneficial inflammatory response.

Lineage conversion methodologies meet the reprogramming toolbox

Lineage conversion has recently attracted increasing attention as a potential alternative to the directed differentiation of pluripotent cells to obtain cells of a given lineage. Different means allowing for cell identity switch have been reported. Lineage conversion relied initially on the discovery of specific transcription factors generally enriched and characteristic of the target cell, and their forced expression in cells of a different fate. This approach has been successful in various cases, from cells of the hematopoietic systems to neurons and cardiomyocytes. Furthermore, recent reports have suggested the possibility of establishing a general lineage conversion approach bypassing pluripotency. This requires a first phase of epigenetic erasure achieved by short overexpression of the factors used to reprogram cells to a pluripotent state (such as a combination of Sox2, Klf4, c-Myc and Oct4), followed by exposure to specific developmental cues. Here we present these different direct conversion methodologies and discuss their potential as alternatives to using induced pluripotent stem cells and differentiation protocols to generate cell populations of a given fate.