Institut des Cellules Souches pour le Traitement et l'Étude des Maladies Monogéniques

Twenty human colon carcinoma cell lines were studied for their ability to develop some of the characteristics of the normal intestinal epithelium, e.g., epithelial polarity, presence of the actin-binding protein villin, or the occurrence of an enterocytic differentiation either when cultured under standard conditions, as for Caco-2 cells, or when grown in a glucose-free medium, as for HT-29 cells. Except for the regular presence of villin, which can be considered a marker of the colonic origin of the cells, the cell lines of this study could be classified into four types with regard to their differentiation characteristics. In type 1 (only one cell line, i.e., Caco-2) the cells undergo spontaneously an enterocytic differentiation characterized by a polarization of the cell layer with the formation of domes and the presence of an apical brush border the membrane of which is endowed with hydrolases such as sucrase-isomaltase, lactase, amino-peptidase N, dipeptidylpeptidase IV and alkaline phosphatase. In type 2 (three cell lines: HT-29, HCT-EB, and HCT-GEO) the cells are undifferentiated when grown in the presence of glucose but undergo an enterocytic differentiation when grown in the absence of glucose. In type 3 (eight cell lines: HCT-GLY, HCT-FET, HCT-FRI, HCT-CBS, HCT-ALA, Co-115, HRT-18, and SW-1116) the cells are organized into a polarized monolayer with the formation of domes but without any enterocytic differentiation characteristics, whatever the culture conditions. In type 4 (eight cell lines: HCT-116a, HCT-R, HCT-RCA, HCT-Moser, HCT-8R, SW-480, LS-174T, and Vaco-9P) the cells are organized into a multilayer without any feature of epithelial polarity or enterocytic differentiation, whatever the culture conditions.

Considerable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs). Before these cells can be used for cell replacement therapy in Parkinson's disease (PD), it is important to verify their functional properties and efficacy in animal models. Here we provide a comprehensive preclinical assessment of hESC-derived midbrain dopamine neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons. Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for clinical translation of hESC-derived dopamine neurons using approaches similar to those established with fetal cells for the treatment of Parkinson's disease.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34(+) HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 10(10) cRBCs generated under good manufacturing practice conditions and labeled with (51)Cr. The level of these cells in the circulation 26 days after injection was between 41% and 63%, which compares favorably with the reported half-life of 28 ± 2 days for native RBCs. Their survival in vivo testifies globally to their quality and functionality. These data establish the proof of principle for transfusion of in vitro-generated RBCs and path the way toward new developments in transfusion medicine. This study is registered at http://www.clinicaltrials.gov as NCT0929266.

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

Substitutive cell therapy using fetal striatal grafts has demonstrated preliminary clinical success in patients with Huntington's disease, but the logistics required for accessing fetal cells preclude its extension to the relevant population of patients. Human embryonic stem (hES) cells theoretically meet this challenge, because they can be expanded indefinitely and differentiated into any cell type. We have designed an in vitro protocol combining substrates, media, and cytokines to push hES cells along the neural lineage, up to postmitotic neurons expressing striatal markers. The therapeutic potential of such hES-derived cells was further substantiated by their in vivo differentiation into striatal neurons following xenotransplantation into adult rats. Our results open the way toward hES cell therapy for Huntington's disease. Long-term proliferation of human neural progenitors leads, however, to xenograft overgrowth in the rat brain, suggesting that the path to the clinic requires a way to switch them off after grafting.

Significance Human induced pluripotent stem cells (hiPSCs) could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. The production of retinal cells from hiPSCs for personalized therapeutic approaches must comply with certain criteria, such as safety, efficiency, reproducibility, and low production cost. Here, we report a simple and scalable retinal differentiation process for the generation of retinal pigmented epithelial cells and neural retinal tissues containing retinal progenitor cells. These progenitors can be differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors, which could find important applications in regenerative medicine. This method also provides an accessible in vitro model to investigate mechanisms involved in human retinogenesis and retinal diseases.

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.

BACKGROUND: Ethnicity, when it is used to mean shared genetic inheritance within a group, has become one of the most important factors in determining prostate carcinoma risk. Genetic polymorphisms were hypothesized to be the probable explanation for differences in risk among ethnic groups. The authors evaluated the association between polymorphisms in genes involved in the androgen biosynthesis and metabolism pathway and the risk of prostate carcinoma. METHODS: Two hundred twenty-six patients with the pathologic diagnosis of sporadic prostate tumor and 156 healthy matched (age, ethnic group) male controls from a large epidemiologic cohort were genotyped for previously described polymorphisms in the androgen receptor (AR), 5alpha-reductase type II (SRD5A2), p450c17 (CYP17), and aromatase (CYP19) genes. The different polymorphisms in prostate carcinoma patients also were analyzed according to age of onset, preoperative prostate-specific antigen level, tumor stage, and tumor grade. RESULTS: The distribution of the tetranucleotide simple tandem repeat polymorphism (STRP) in intron 4 of CYP19 was significantly different in control and cancer patients (P = 0.012). The 171 allele and the 187 allele were associated with prostate carcinoma risk (P = 0.05 and P = 0.045, respectively). Conversely, no association was observed between prostate carcinoma risk and the other polymorphisms studied as follow: the CAG repeat in exon 1 of AR, the (TA)n dinucleotide repeat polymorphism in the 3' untranslated region, and the A49T or V89L substitutions in SDR5A2, the single base pair (bp) (a T to C transition) polymorphism that creates an additional Sp1-type (CCACC box) promoter site in CYP17. In prostate carcinoma patients, CAG repeats of AR, and TA repeats of SDR5A2 are associated with age of onset (P = 0.05 and P < 0.001, respectively). CONCLUSIONS: The association between the 171-bp allele of CYP19 and prostate carcinoma risk suggests that aromatase could be used as a new indicator for prostate carcinoma prevention in men of White French ethnogeographic origin. Conversely, it is possible that an individual carries both a high- and a low-risk marker (e.g., CYP17 A2 allele and V89L in SRD5A2) resulting in no overall difference in risk observed across the population. For these reasons, the development of a polygenic model, incorporating multiple loci from the individual genes may maximize the chance of identifying individuals with high-risk genotypes.

Recent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a serum-free protocol to differentiate human induced pluripotent stem cells (hiPSCs) into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the 13 hiPSC lines differentiated. Using this model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1β, TNF-α, and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions.

Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.

Among the tools of regenerative medicine, induced pluripotent stem cells (iPSCs) are interesting because the donor genotype can be selected. The construction of banks of iPSC cell lines selected from human leukocyte antigen (HLA) homozygous donors has been proposed to be an effective way to match a maximal number of patients receiving cell therapy from iPSC lines. However, what effort would be required to constitute such a bank for a worldwide application has remained unexplored. We developed a probabilistic model to compute the number of donors to screen for constituting banks of best-chosen iPSC lines with homozygous HLA haplotypes (haplobanks) in four ancestry backgrounds. We estimated what percentage of the patients would be provided with single HLA haplotype matched cell lines. Genetic diversity leads to different outcomes for the four sets in all terms. A bank comprising iPSC lines representing the 20 most frequent haplotypes in each population would request quite different number of donors to screen, between 26,000 for European Americans and 110,000 for African Americans. It would also match different fractions of the recipient population, namely, more than 50% of the European Americans and 22% of African Americans. Conversely, a bank comprising the 100 iPSC lines with the most frequent HLA in each population would leave out only 22% of the European Americans, but 37% of the Asians, 48% of the Hispanics, and 55% of the African Americans. The constitution of a haplobank of iPSC lines is achievable through a large-scale concerted worldwide collaboration.

One puzzling observation in patients affected with Hutchinson-Gilford progeria syndrome (HGPS), who overall exhibit systemic and dramatic premature aging, is the absence of any conspicuous cognitive impairment. Recent studies based on induced pluripotent stem cells derived from HGPS patient cells have revealed a lack of expression in neural derivatives of lamin A, a major isoform of LMNA that is initially produced as a precursor called prelamin A. In HGPS, defective maturation of a mutated prelamin A induces the accumulation of toxic progerin in patient cells. Here, we show that a microRNA, miR-9, negatively controls lamin A and progerin expression in neural cells. This may bear major functional correlates, as alleviation of nuclear blebbing is observed in nonneural cells after miR-9 overexpression. Our results support the hypothesis, recently proposed from analyses in mice, that protection of neural cells from progerin accumulation in HGPS is due to the physiologically restricted expression of miR-9 to that cell lineage.

Human embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES-1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein-2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real-time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and alpha-actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in-scar injections of either culture medium (controls) or BMP2 (+/-SU5402)-treated HES cells. After 2 months, human cells were detected by anti-human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription-PCR and immunofluorescence using an anti-beta myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac-specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.

Abstract Hutchinson–Gilford progeria syndrome (HGPS) is a lethal premature and accelerated aging disease caused by a de novo point mutation in LMNA encoding A‐type lamins. Progerin, a truncated and toxic prelamin A issued from aberrant splicing, accumulates in HGPS cells' nuclei and is a hallmark of the disease. Small amounts of progerin are also produced during normal aging. We show that progerin is sequestered into abnormally shaped promyelocytic nuclear bodies, identified as novel biomarkers in late passage HGPS cell lines. We found that the proteasome inhibitor MG132 induces progerin degradation through macroautophagy and strongly reduces progerin production through downregulation of SRSF‐1 and SRSF‐5 accumulation, controlling prelamin A mRNA aberrant splicing. MG132 treatment improves cellular HGPS phenotypes. MG132 injection in skeletal muscle of Lmna G609G/G609G mice locally reduces SRSF‐1 expression and progerin levels. Altogether, we demonstrate progerin reduction based on MG132 dual action and shed light on a promising class of molecules toward a potential therapy for children with HGPS.

In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes [1], which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications.

Reappraisal of neuropathological studies suggests that pathological hallmarks of Alzheimer's disease and Parkinson's disease (PD) spread progressively along predictable neuronal pathways in the human brain through unknown mechanisms. Although there is much evidence supporting the prion-like propagation and amplification of α-synuclein (α-Syn) in vitro and in rodent models, whether this scenario occurs in the human brain remains to be substantiated. Here we reconstructed in microfluidic devices corticocortical neuronal networks using human induced pluripotent stem cells derived from a healthy donor. We provide unique experimental evidence that different strains of human α-Syn disseminate in "wild-type" human neuronal networks in a prion-like manner. We show that two distinct α-Syn strains we named fibrils and ribbons are transported, traffic between neurons, and trigger to different extents, in a dose- and structure-dependent manner, the progressive accumulation of PD-like pathological hallmarks. We further demonstrate that seeded aggregation of endogenous soluble α-Syn affects synaptic integrity and mitochondria morphology.

transients in fibroblasts from patients with PARK2 mutations. In contrast, a catalytically inactive PD-related Parkin variant had no effect. Overall, our data suggest that Parkin is directly involved in regulating ER-mitochondria contacts and provide new insight into the role of the loss of Parkin function in PD development.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

Cortical neurons of the superficial layers (II-IV) represent a pivotal neuronal population involved in the higher cognitive functions of the human and are particularly affected by psychiatric diseases with developmental manifestations such as schizophrenia and autism. Differentiation protocols of human pluripotent stem cells (PSC) into cortical neurons have been achieved, opening the way to in vitro modeling of neuropsychiatric diseases. However, these protocols commonly result in the asynchronous production of neurons typical for the different layers of the cortex within an extended period of culture, thus precluding the analysis of specific subtypes of neurons in a standardized manner. Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers. The short time course of differentiation into neurons of these progenitors has made them amenable to high-throughput assays. This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules. LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes.

The role of microRNAs (miRNAs) as coordinators of stem cell fate has emerged over the last decade. We have used human embryonic stem cells to identify miRNAs involved in neural lineage commitment induced by the inhibition of TGFβ-like molecule-mediated pathways. Among several candidate miRNAs expressed in the fetal brain, the two isoforms of miR-125 alone were detected in a time window compatible with a role in neural commitment in vitro. Functional analysis indicated that miR-125 isoforms were actively involved in the promotion of pluripotent cell conversion into SOX1-positive neural precursors. miR-125 promotes neural conversion by avoiding the persistence of non-differentiated stem cells and repressing alternative fate choices. This was associated with the regulation by miR-125 of SMAD4, a key regulator of pluripotent stem cell lineage commitment. Activation of miR-125 was directly responsive to the levels of TGFβ-like molecules, placing miR-125 at the core of mechanisms that lead to the irreversible neural lineage commitment of pluripotent stem cells in response to external stimuli.