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.

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.

New Bicistronic TALENs Greatly Improve Genome Editing.

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. Basic Protocol 1: Generation of new TALENs Basic Protocol 2: Genome editing using TALEN-F Alternate Protocol 1: Generation of TALEN-M Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector Alternate Protocol 2: Editing of primary T cells using TALEN-M Basic Protocol 3: Verifying gene editing Support Protocol 2: Rapid expansion protocol for edited T-cells.

Generation of one iPSC line (IMEDEAi006-A) from an early-onset familial Alzheimer's Disease (fAD) patient carrying the E280A mutation in the PSEN1 gene.

The mutation E280A in PSEN1 (presenilin-1) is the most common cause of early-onset familial Alzheimer's Disease (fAD). It presents autosomal dominant inheritance and frequently leads to the manifestation of the disease in relatively young individuals. Here we report the generation of one PSEN1 E280A iPSC line derived from an early-onset patient. OriP/EBNA1-based episomal plasmids containing OCT3/4, SOX2, KLF4, L-MYC, LIN28, BCL-xL and shp53 were used to reprogram oral mucosa fibroblasts. The iPSC line generated has normal karyotype, carry the E280A mutation, is free of plasmid integration, express high levels of pluripotency markers and can differentiate into all three germ layers.

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: 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.