Mesenchymal stromal cells express GARP/LRRC32 on their surface: Effects on their biology and immunomodulatory capacity

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-β1 to the cell surface of activated Foxp3+ regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-β1 and reduced their proliferative capacity in a TGF-β1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. Stem Cells 2015;33:183-19

Síndrome de Wiskott - Aldrich: Modelos celulares humanos para esudios preclínicos de vectores lentivirales para terapia génica

El síndrome de Wiskott-Aldrich es una inmunodeficiencia primaria ligada al cromosoma X causada por mutaciones en el gen WAS, afectando a todo el linaje hematopoyético. La expresión de WAS está regulada por dos promotores; promotor proximal, de 1.6Kb, situado justo en el sitio de inicio de la transcripción y el promotor proximal o distal, de 0.6Kb, situado a 6Kb aguas arriba del promotor proximal. El gen WAS, de expresión específica de linaje hematopoyético, codifica una proteína multimodular citoplasmática (WASP) cuya función más estudiada es la integración de señales extracelulares con la nucleación de la actina y la reorganización del citoesqueleto celular. Sin embargo el rol de WASP en el linaje megacariocítico resulta controvertido con evidencias que indican una función más especializada de WASP en plaquetas. Actualmente el único tratamiento curativo para pacientes con WAS es el trasplante de médula ósea. Sin embargo, este tratamiento no está siempre disponible ni está exento de efectos secundarios severos. Por ello, la terapia génica (TG) emerge como una alternativa real al trasplante alogénico de progenitores hematopoyéticos. En la actualidad, se están llevando a cabo diferentes ensayos clínicos de TG para el tratamiento de pacientes con WAS utilizando células madre hematopoyéticas (HSCs) modificadas genéticamente con un vector lentiviral (VL) que expresa el gen WAS a través del promotor proximal del propio gen WAS (el vector lentiviral WW1.6). A pesar de que la gran mayoría de los pacientes se han recuperado de la inmunodeficiencia y manifestaciones autoinmunes, en muchos casos persiste la microtrombocitopenia y en algunos casos también los sangrados y hemorragias. Las razones de que esta estrategia repare con éxito los defectos funcionales de células T, B y macrófagos (causantes de la inmunodeficiencia), pero no tenga el mismo éxito en plaquetas son desconocidas. Una hipótesis es que los vectores lentivirales utilizados (WW1.6), consiguen una expresión adecuada de WASP en células linfoides y mieloides pero no en las células del linaje megacariocítico. Esto produciría una “peor” expresión de WASP en megacariocitos y plaquetas y, por tanto, unos peores resultados en cuanto a reparación de los defectos funcionales de las mismas. Estos problemas de eficiencia en la restauración de la trombocitopenia no fueron detectados en los modelos animales utilizados en los ensayos preclínicos, reflejando la necesidad de desarrollar nuevos modelos para estos estudios. En base a lo mencionado anteriormente, la presente tesis se ha centrado en los siguientes objetivos: 1- Estudiar el papel de WASP durante la megacariopoyesis y a la trombopoyesis en células humanas. Esto permitirá entender los resultados de los ensayos clínicos actuales y los requerimientos para mejorar dichos resultados, 2- Analizar si el patrón de expresión de los vectores terapéuticos mimetizan la expresión endógena de WASP durante estos dos procesos. De esta manera se podrá investigar donde está el problema de los vectores actuales y como mejorarlos y 3– Desarrollar nuevos vectores terapéuticos e investigar la capacidad de los mismos para reparar los defectos funcionales de los megacariocitos y plaquetas, así como de otros tipos celulares linfoides y mieloides. La idea final es proponer una nueva estrategia de TG para WAS basada en nuevos VLs que muestren mejores perfiles de expresión en el linaje megacariocítico y mantengan la capacidad de restauración de los defectos funcionales encontrados en las células hematopoyéticas de los pacientes con WAS.Tesis Univ. Granada. Programa Oficial de Doctorado en: Biomedicin

Generation of a human iPSC line (IMEDEAi008-A) derived from natural homozygous CCR5-Δ32 PBMCs enriched in the pro-erythroblast population

A 32 base pair deletion in the C-C chemokine receptor type gene (CCR5-Δ32), the main Human Immunodeficiency Virus (HIV) co-receptor results in a non-functional protein. Individuals homozygous for the CCR5-Δ32 mutation are resistant to HIV infection. Here we report the generation, from pro-erythroblast enriched Peripheral Blood Mononuclear Cells (PBMCs) from a naturally occurring CCR5-Δ32/Δ32 individual, of the fully characterized iPSC line IMEDEAi008-A. The new line has normal karyotype, carry the Δ32 mutation in homozygosity, is free of plasmid integrations, express high levels of pluripotency markers and can differentiate into all three germ layers

Generation of one iPSC line (IMEDEAi007-A) by Sendai Virus transduction of PBMCs from a Psoriasis donor

Psoriasis is a chronic inflammatory skin disease that speeds up the life cycle of skin cells, forming scales and red patches that are itchy and sometimes painful. It is a complex disease of autoimmune origin and genetic predisposition with more than 10 different loci associated. Here we described the production of an iPSC line generated by Sendai Virus (Klf4, Oct3/4, Sox2 and c-Myc) reprogramming of Peripheral Blood Mononuclear Cells (PBMCs) from a Psoriasis patient. The iPSC line generated has normal 46XY karyotype, is free of SeV genome and transgenes insertions, express high levels of pluripotency markers and can differentiate into all three germ layers.Funding was provided by the Spanish Ministry for Science and Innovation (RTC-2016-5324-1). JMMF was a postdoctoral Berrikertu fellow from the Basque government. AF was a recipient of Juan de la Cierva (JCI-2006-2675) and Torres Quevedo (PTQ-16-08496) postdoctoral fellowships from the Spanish Ministry for Science and Innovation

Generation of two induced pluripotent stem cells lines from a Mucopolysaccharydosis IIIB (MPSIIIB) patient

Mucopolysaccharydosis IIIB is the second most frequent form of Sanfilippo syndrome, a degenerative, pediatric lysosomal storage disease (LSD) characterized by severe neurological disorders and death. We have generated two iPSCs lines derived from dermal fibroblast from a MPSIIIB homozygous (P358L) donor. Cells were reprogrammed with OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-MYC, LIN28, BCL-xL and shp53. Both cell lines are homozygous for the P358L mutation of the α-N-acetylglucosaminidase (NAGLU) gene, have normal karyotype, are free of plasmid integration, express high levels of pluripotency-associated markers and can differentiate into the three germ layers. Resource table: Unlabelled TableUnique stem cell lines identifierIMEDEAi005-AIMEDEAi005-BAlternative names of stem cell linesiPS GM02931 CL2 (IMEDEAi005-A)iPS GM02931 CL3 (IMEDEAi005-B)InstitutionIMEDEA - Instituto Mediterráneo de Estudios AvanzadosContact information of distributorDaniel Bachiller; [email protected] of cell linesiPSCsOriginHumanCell SourceOriginal cell type: GM02931 Fibroblast from the Coriell InstituteClonalityClonal cell linesMethod of reprogrammingEpisomal Plasmids (Klf4, Sox2, Oct4, shp53, L-Myc, Lin28, Bcl-xL)Multiline rationaleSame disease, isogenic cell linesGene modificationNOType of modificationN/AAssociated diseaseMucopolysaccharidosis, Type IIIB; MPS3B(MPS IIIB; OMIM entry # 252920)Gene/locusNAGLU 17q21.2Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock date26/09/2018Cell line repository/bankRegistered in the Human Pluripotent Stem Cell Registry (https://hpscreg.eu).Ethical approvalOriginal line (GM02931 Fibroblast) has been obtained by Coriell Institute for Medical Research. Regarding policy of informed consent form, the Coriell Institute for Medical Research, which operates the NIGMS Human Genetic Cell Repository, has obtained a Certificate of Confidentiality from the National Institutes of Health to help ensure patient's privacy. Resource utility: Although the generation of iPSCs has been reported for some lysosomal storage diseases (LSD) in general, and from other mutations of the NAGLU gene in particular (Lemonnier et al., 2011), this is the first time that NAGLU Pro358Leu MPSIIIB-iPSCs lines have been generated and fully characterized demonstrating their quality as iPS cells. Resource details: Mucopolysaccharidosis IIIB (MPSIII, Sanfilippo syndrome type B) is a pediatric neurodegenerative disorder caused by a deficiency in NAGLU, an enzyme required for lysosomal degradation of heparin sulphate (HS). When the enzyme is absent or malfunctioning, HS accumulates in the cells of several tissues, with devastating effects in the brain and central nervous system. MPSIIIB is inherited in an autosomal recessive manner and presents an incidence between 0.03 and 0.78 cases per 1 × 105 live births (Fedele, 2015) depending on the country. Currently there is no therapy available.The NAGLU gene was identified in 1996, is located on chromosome 17q21.1 and contains 6 exons. More than 150 NAGLU mutations have been reported, being most of them missense (Valstar et al., 2010). All of them lead to MPSIIIB but, unlike MPSIIIA, none is predominant.The two iPSCs lines described in this report, IMEDEAi005-A and IMEDEAi005-B, (See Table 1) were generated from skin fibroblast obtained from a clinically affected homozygous donor. The mutant allele consists on a C > T transversion at nucleotide 1073 (1073 > T) resulting in a substitution of leucine for proline at codon 358 (Pro358Leu).Skin fibroblasts were reprogrammed to iPSCs by nucleofection with four OriP/EBNA1 (Epstein-Barr nuclear antigen-1) based episomal plasmids encoding 5 reprogramming genes (OCT3/4, SOX2, KLF4, L-Myc, LIN28 and BCL-xL), in addition to a short hairpin RNA against p53. The iPSCs lines showed morphology (Fig. 1A) and growth behaviour typical of human Embryonic Stem Cells (hESC), as well as normal female karyotype (46, XX) (Fig. 1B). After 12 passages, PCR analysis confirmed that both iPSCs lines had completely lost the episomal vectors (Fig. 1C). The identity of iPS cells and their parental fibroblasts was confirmed by STR analysis (Table 2, data not shown) in addition to the identification of the disease-associated mutation in the NAGLU gene by DNA sequencing (Fig. 1D). Regarding the iPSC phenotype, both lines expressed the pluripotency-associated markers: OCT3/4, NANOG, SOX2 and TRA-1-60 (Fig. 1E), and TRA-1-81 quantified by flow cytometry (Fig. 1G), resulting in 88.17% and 83.4% of TRA-1-81 positive cells in IMEDEAi005-A and IMEDEAi005-B respectively. Finally, the differentiation capacity of iPSCs lines was analyzed by embryoid body (EBs) formation. Expression of markers specific of the three germ layers was observed after at least 10 days of spontaneous differentiation (Fig. 1F). Mycoplasma analysis was negative for both iPSCs lines (Supplementary Fig. S1).Skin fibroblasts were reprogrammed to iPSCs by nucleofection with four OriP/EBNA1 (Epstein-Barr nuclear antigen-1) based episomal plasmids encoding 5 reprogramming genes (OCT3/4, SOX2, KLF4, L-Myc, LIN28 and BCL-xL), in addition to a short hairpin RNA against p53. The iPSCs lines showed morphology (Fig. 1A) and growth behaviour typical of human Embryonic Stem Cells (hESC), as well as normal female karyotype (46, XX) (Fig. 1B). After 12 passages, PCR analysis confirmed that both iPSCs lines had completely lost the episomal vectors (Fig. 1C). The identity of iPS cells and their parental fibroblasts was confirmed by STR analysis (Table 2, data not shown) in addition to the identification of the disease-associated mutation in the NAGLU gene by DNA sequencing (Fig. 1D). Regarding the iPSC phenotype, both lines expressed the pluripotency-associated markers: OCT3/4, NANOG, SOX2 and TRA-1-60 (Fig. 1E), and TRA-1-81 quantified by flow cytometry (Fig. 1G), resulting in 88.17% and 83.4% of TRA-1-81 positive cells in IMEDEAi005-A and IMEDEAi005-B respectively. Finally, the differentiation capacity of iPSCs lines was analyzed by embryoid body (EBs) formation. Expression of markers specific of the three germ layers was observed after at least 10 days of spontaneous differentiation (Fig. 1F). Mycoplasma analysis was negative for both iPSCs lines (Supplementary Fig. S1).In conclusion, we have successfully generated and characterized, for the first time to our knowledge, two human iPSCs lines from a MPSIIIB donor homozygous for the P358L NAGLU mutation. The new lines will complement the existing murine MPS IIIB model, with their potential to be used in a development of a purely human in vitro model of the disease

New Bicistronic TALENs Greatly Improve Genome Editing

This article also appears in: Lineage Tracer and Gene-editing in Stem Cells.Genome editing has become one of the most powerful tools in present‐day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator‐like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN‐F) or membrane receptors (TALEN‐M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence‐assisted cell sorting (FACS) or magnetic‐activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc.This work was supported by grants from the Spanish Ministry for Science and Innovation (PLE2009‐0091, RTC‐2014‐2207‐1 and IPT‐2011‐1402‐900000), ISCIII (PI14/01073 and PI18/00334), co‐funded by ERDF/ESF “Investing in your future”, the Balearic Government (16023/2008), the Spanish Cystic Fibrosis Federation (Pablo Motos grant), Federación ASEM‐Telemaratón “Todos somos raros, todos somos únicos”, Fundación Salud 2000, the European Commission (H2020, PHC‐667079), and an endowment from METROVACESA. J. M. M. F. was a postdoctoral Berrikertu fellow (granted by the Basque Government). A. F. was a recipient of Juan de la Cierva (JCI‐2006‐2675) and Torres Quevedo (PTQ‐16‐08496) postdoctoral fellowships from the Spanish Ministry for Science and Innovation

iPSC-Derived Intestinal Organoids from Cystic Fibrosis Patients Acquire CFTR Activity upon TALEN-Mediated Repair of the p.F508del Mutation

Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809.This work was supported by grants from the Spanish Ministry for Science and Innovation (PLE2009-0091, RTC-2014-2207-1, and IPT-2011-1402-900000); ISCIII PI14/01073, cofunded by ERDF/ESF “Investing in your future”; the Balearic Government (16023/2008); the Spanish Cystic Fibrosis Federation (Pablo Motos Grant); Federación ASEM-Telemaratón “Todos somos raros, todos somos únicos”; Fundación Salud 2000; the European Commission (H2020, PHC-667079); and an endowment from METROVACESA. J.M.M.-F. was a postdoctoral Berrikertu fellow granted by the Basque Government. A.F. was a recipient of Juan de la Cierva (JCI-2006-2675) and Torres Quevedo (PTQ-16-08496) postdoctoral fellowships from the Spanish Ministry for Science and Innovation

Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS