Generation of a bank of clinical-grade, HLA-homozygous iPSC lines with high coverage of the Spanish population

BackgroundInduced pluripotent stem cell (iPSC)-derived cell therapies are an interesting new area in the field of regenerative medicine. One of the approaches to decrease the costs of iPSC-derived therapies is the use of allogenic homozygous human leukocyte antigen (HLA)-matched donors to generate iPSC lines and to build a clinical-grade iPSC bank covering a high percentage of the Spanish population.MethodsThe Spanish Stem Cell Transplantation Registry was screened for cord blood units (CBUs) homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes. Seven donors were selected with haplotypes covering 21.37% of the haplotypes of the Spanish population. CD34-positive hematopoietic progenitors were isolated from the mononuclear cell fraction of frozen cord blood units from each donor by density gradient centrifugation and further by immune magnetic labeling and separation using purification columns. Purified CD34 + cells were reprogrammed to iPSCs by transduction with the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit.ResultsThe iPSCs generated from the 7 donors were expanded, characterized, banked and registered. Master cell banks (MCBs) and working cell banks (WCBs) from the iPSCs of each donor were produced under GMP conditions in qualified clean rooms.ConclusionsHere, we present the first clinical-grade, iPSC haplobank in Spain made from CD34 + cells from seven cord blood units homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes within the Spanish population. We describe their generation by transduction with Sendai viral vectors and their GMP-compliant expansion and banking. These haplolines will constitute starting materials for advanced therapy medicinal product development (ATMP)

Access to stem cell data and registration of pluripotent cell lines: the Human Pluripotent Stem Cell Registry (hPSCreg)

The value of human pluripotent stem cells (hPSC) in regenerative medicine has yet to reach its full potential. The road from basic research tool to clinically validated PSC-derived cell therapy products is a long and winding one, leading researchers, clinicians, industry and regulators alike into undiscovered territory. All stakeholders must work together to ensure the development of safe and effective cell therapies. Similarly, utilization of hPSC in meaningful and controlled disease modeling and drug screening applications requires information on the quality and suitability of the applied cell lines. Central to these common goals is the complete documentation of hPSC data, including the ethical provenance of the source material, the hPSC line derivation, culture conditions and genetic constitution of the lines. Data surrounding hPSC is scattered amongst diverse sources, including publications, supplemental data, researcher lab books, accredited lab reports, certificates of analyses and public data repositories. Not all of these data sources are publicly accessible nor associated with metadata nor stored in a standard manner, such that data can be easily found and retrieved. The Human Pluripotent Stem Cell Registry (hPSCreg; https://hpscreg.eu/) was started in 2007 to impart provenance and transparency towards hPSC research by registering and collecting standard properties of hPSC lines. In this chapter, we present a short primer on the history of stem cell-based products, summarize the ethical and regulatory issues introduced in the course of working with hPSC-derived products and their associated data, and finally present the Human Pluripotent Stem Cell Registry as a valuable resource for all stakeholders in therapies and disease modeling based on hPSC-derived cells

Derivació de línies de cèl·lules mare embrionàries humanes

Les cèl·lules mare embrionàries (CME) representen una font potencial de cèl·lules per a ús terapèutic en algunes malalties produïdes per la pèrdua de la funció cel·lular. Normalment, aquestes cèl·lules procedeixen d'embrions donats per les parelles sotmeses a tècniques de reproducció assistida (TRA). Es presenten els resultats obtinguts en el Banc de Línies Cel·lulars del Centre de Medicina Regenerativa de Barcelona (CMRB), on s'han descongelat 254 embrions donats per parelles sotmeses al programa de fecundació in vitro (FIV) de l'Institut Universitari Dexeus. La taxa de supervivència va ser del 51,9 %. S'han obtingut cinc línies de CME. Tres d'aquestes línies procedeixen d'embrions de mala qualitat i presenten la capacitat d'autorenovació, pluripotència i diferenciació característiques d'aquestes cèl·lules. Els embrions de mala qualitat, sovint descartats en els centres de RA, poden ser una font útil per a la derivació de CME.Human embryonic stem cells (hESC) represent a potential source for cell therapy for many degenerative diseases. Usually hESC lines are derived from surplus embryos donated from couples undergoing In Vitro Fertilisation (IVF). We here present the results obtained in the Stem Cell Bank at the Center of Regenerative Medicine in Barcelona. 254 embryos have been thawed. The embryos were donated from couples from the IVF programme at the Institut Universitari Dexeus. The embryo survival rate was 51.9%. Five ESC lines were obtained. Three of these lines came from poor quality embryos. The cell lines present self-renewal, pluripotency, and differentiation properties characteristic of these cells Poor quality embryos, usually discarded in assisted reproduction centres, could be useful for ESC derivation

Generation of an induced pluripotent stem cell line from a healthy Caucasian male

The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC

Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2

Mouse and human fibroblasts were the first cell types successfully reprog- rammed by ectopic expression of OCT4, SOX2, KLF4, and c-MYC (OSKM) (Lowry et al., 2008; Maherali et al., 2007; Park et al., 2008; Takahashi et al., 2007; Taka- hashi and Yamanaka, 2006; Yu et al., 2007). Further studies have shown that the age, origin, and cell type used have a deep impact on the reprogramming effi- ciency, eventually requiring the expres- sion of fewer factors and/or reducing the timing of the whole process. In general, stem cells are rare and difficult to access and isolate in large numbers (neural stem cells, for instance [Kim et al., 2008, 2009c]) and, therefore, represent a com- plicated target for reprogramming. How- ever, Cord Blood (CB) could represent an alternative and readily accessible source of stem cells. Here, we describe reprog- ramming of CB cells to pluripotency by retroviral transduction of four (OSKM), three (OSK), and as few as two (OS) tran- scription factors, without the need for additional chemical compounds

Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy

Background: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA. Keywords: age-related macular degeneration (AMD); geographic atrophy; pig; animal model; stem cells; iPSC; RPE; retina; regenerative medicine; advanced cell therap

Cèl·lules mare embrionàries: què en sabem després de trenta anys d'investigació?

L'aïllament i derivació de les cèl·lules mare embrionàries humanes (CME) l'any 1998 va generar grans expectatives en el camp de la teràpia cel·lular. Les CME presentaven la capacitat de poder dividir-se infinitament i el potencial necessari per diferenciar-se a qualsevol tipus cel·lular del nostre cos. Malauradament, després de més de vint anys d'investigació, l'aplicació clínica encara presenta problemes relacionats amb la utilització d'embrions humans, el rebuig immunitari després del trasplantament i la formació de tumors. Així doncs, un dels principals objectius en el camp de la medicina regenerativa és l'obtenció de CME a partir de cèl·lules somàtiques de pacients. L'any 2006 es va descriure, per primera vegada, que cèl·lules totalment diferenciades es podien reprogramar a cèl·lules semblants a les CME. Aquestes CME resultants presentaven les característiques típiques de les CME i, finalment, evitaven els problemes relacionats amb el rebuig immunitari i la utilització d'embrions humans. Tot i així, les tècniques utilitzades en els últims cinc anys no són encara les adequades per a l'ús immediat d'aquestes cèl·lules en aplicacions clíniques. Els propers anys seran fonamentals per al desenvolupament d'estratègies que en permetin l'obtenció de manera segura, i esdevinguin l'alternativa ideal en les teràpies de substitució cel·lular i el modelatge de malalties in vitro.Human embryonic stem cells: What do we known after thirty years of research? The isolation and derivation of human embryonic stem cells (hESCs) in 1998 attracted significant attention in the regenerative medicine field. Among other properties, these cells possessed the dual ability to self-renew and differentiate into all the cell types of the body. These characteristics suggest that hESCs hold great potential for application in regenerative medicine. However, after more than twenty years of intense research, their clinical application still shows many concerns regarding the use of human embryos, tissue rejection after transplantation, and tumour formation. Thus, one of the ultimate goals in regenerative medicine is the generation of pluripotent hESCs directly from somatic cells obtained from patients. A breakthrough appeared when fully differentiated cells were reprogrammed for the first time in 2006 into ES-like cells. The resulting ES-like cells exerts all the characteristics intrinsic to ESC biology and finally could overcome the issue related with immune rejection after transplantation and the use of human embryos for research. Nevertheless, most of the techniques used to reprogram human somatic cells are still not adaptable for immediate clinical applications. Improvements in their safe derivation and validation will bring them one step closer for becoming the ideal alternative in cell-replacement therapies and disease modelling in vitro

Derivació de línies de cèl·lules mare embrionàries humanes

Les cèl·lules mare embrionàries (CME) representen una font potencial de cèl·lules per a ús terapèutic en algunes malalties produïdes per la pèrdua de la funció cel·lular. Normalment, aquestes cèl·lules procedeixen d'embrions donats per les parelles sotmeses a tècniques de reproducció assistida (TRA). Es presenten els resultats obtinguts en el Banc de Línies Cel·lulars del Centre de Medicina Regenerativa de Barcelona (CMRB), on s'han descongelat 254 embrions donats per parelles sotmeses al programa de fecundació in vitro (FIV) de l'Institut Universitari Dexeus. La taxa de supervivència va ser del 51,9 %. S'han obtingut cinc línies de CME. Tres d'aquestes línies procedeixen d'embrions de mala qualitat i presenten la capacitat d'autorenovació, pluripotència i diferenciació característiques d'aquestes cèl·lules. Els embrions de mala qualitat, sovint descartats en els centres de RA, poden ser una font útil per a la derivació de CME.Human embryonic stem cells (hESC) represent a potential source for cell therapy for many degenerative diseases. Usually hESC lines are derived from surplus embryos donated from couples undergoing In Vitro Fertilisation (IVF). We here present the results obtained in the Stem Cell Bank at the Center of Regenerative Medicine in Barcelona. 254 embryos have been thawed. The embryos were donated from couples from the IVF programme at the Institut Universitari Dexeus. The embryo survival rate was 51.9%. Five ESC lines were obtained. Three of these lines came from poor quality embryos. The cell lines present self-renewal, pluripotency, and differentiation properties characteristic of these cells Poor quality embryos, usually discarded in assisted reproduction centres, could be useful for ESC derivation