Préparation et caractérisation d'un libre-Transgene humain souches pluripotentes induites lignée cellulaire et la conversion en des conditions définies de qualité clinique
Summary
We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.
Abstract
Anthropiques cellules souches pluripotentes (de hiPSCs) peuvent être générés avec des méthodologies de reprogrammation base lentivirus. Toutefois, des traces de gènes potentiellement oncogènes restants dans les régions activement transcrits du génome, limitent leur potentiel pour une utilisation dans des applications thérapeutiques humaines 1. En outre, les antigènes non-humains issus de la reprogrammation de cellules souches ou la différenciation en dérivés thérapeutiques pertinentes empêchent ces hiPSCs d'être utilisé dans un contexte clinique humain 2. Dans cette vidéo, nous vous présentons une procédure pour reprogrammer et analyser hiPSCs gratuitement facteur-libres de transgènes exogènes. Ces hiPSCs peuvent ensuite être analysés pour des anomalies d'expression génique spécifique dans l'intron contenant le lentivirus. Cette analyse peut être effectuée en utilisant sensible réaction en chaîne par polymérase quantitative (PCR), qui a un avantage sur les techniques moins sensibles précédemment utilisé pour détecter l'expression du gène 3 différences. La conversion totale ende qualité clinique bonnes pratiques de fabrication (BPF) des conditions, permet pertinence clinique humaine. Notre protocole offre une autre méthode-à condition que les critères actuels exemptées seront étendre et inclure caractérisés dérivés de libre-facteur-base-hiPSC pour des applications à des fins thérapeutiques humains découlant hiPSCs de qualité GMP, ce qui devrait éliminer tout risque d'immunogénicité due à des antigènes non-humains. Ce protocole est largement applicable aux cellules reprogrammées lentiviraux de tout type et fournit une méthode reproductible pour convertir cellules reprogrammées dans des conditions de qualité GMP.
Introduction
Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions.
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance.
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11.
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.
Protocol
Representative Results
Discussion
Nous décrivons une méthode consistant à dériver hiPSCs libre-facteur et de les rendre cliniquement significative par la conversion de ces cellules dans des conditions de qualité GMP de différenciation cellulaire en aval dans les futurs thérapeutique humaine. Bien que ce protocole est largement applicable à une variété de types de cellules, nous avons choisi de reprogrammer les fibroblastes dermiques humains, en raison de la facilité d'extraction du patient et de leur applicabilité à la thérapeutique hu…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar’s Award to JAB.
Materials
| Name | Company | Catalog Number | Comments |
| Media and reagents | |||
| DMEM/F12 (basal media) | Invitrogen (Carlsbad, CA, USA) | 11330057 | |
| Fetal bovine serum | Invitrogen | 16000044 | |
| Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x | Invitrogen | 11140050 | |
| Glutamax, 100x | Invitrogen | 35050-061 | |
| PenStrep, penicillin-streptomycin, 100x | Invitrogen | 15140-122 | Thaw at 4 °C, aliquot and store at −20 °C |
| Knockout serum replacement | Invitrogen | 10828028 | Thaw at 4 °C, aliquot and store at −20 °C |
| Trypsin/EDTA, 0.5% | Invitrogen | 15400-054 | Dilute stock out to 0.05% in 1x PBS |
| Basic fibroblast growth factor | GlobalStem (Rockville, MD, USA) | GSR-2001 | Reconstitute to 10 µg/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at −80 °C |
| β-mercaptoethanol | Millipore (Billerica, MA, USA) | ES-007-E | |
| Matrigel (basement membrane matrix) | BD Biosciences (San Jose, CA, USA) | 356231 | Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at −20 °C |
| CELLstart (Synthetic Substrate) | Invitrogen | A1014201 | |
| Stemmolecule Y27632 | Stemgent (Cambridge, MA, USA) | 04-0012-02 | |
| Puromycin | Invitrogen | A1113802 | |
| LightCycler 480 Probes Master | Roche (Basel, Switzerland) | 4707494001 | |
| ProFreeze-CDM Medium/freezing medium | Lonza (Basel, Switzerland) | 12-769E | |
| Dimethyl sulfoxide | Sigma-Aldrich (St. Louis, MO, USA) | D8418 | |
| PBS | Invitrogen | 14190-250 | |
| 100 BP DNA Ladder | Invitrogen | 15628019 | |
| SYBR Safe DNA Gel Stain 10000x | Invitrogen | S33102 | |
| Agarose | Bio-Rad Laboratories, Inc. (Hercules, CA, USA) | 161-3101 | |
| Gelatin, from porcine skin | Sigma-Aldrich | G1890-100G | Make stock at 0.2% in PBS, autoclave and store at room temperature |
| mTeSR1 | StemCell Technologies (Vancouver, BC, Canada) | 5850 | Combine Supplement 5X with the basic medium, aliquot and store at 4 °C for up to 2 weeks. |
| Stemedia NutriStem XF/FF Culture Medium | Stemgent | 05-100-1A | Thaw at 4 °C O/N, aliquot and store at 4 °C for up to 2 weeks. |
| Primocin | InvivoGen (San Diego, CA, USA) | ant-pm-1 | |
| Accutase (Dissociation Reagent) | Invitrogen | A1110501 | |
| Donkey anti-Chicken IgG AlexaFluor 488 | Jackson ImmunoResearch (West Grove, PA, USA) | 703-546-155 | |
| Polybrene/transfection agent | Millipore | TR-1003-G | |
| Plasticware | |||
| 12-well plates | VWR (West Chester, PA, USA) | 29442-038 | |
| 6-well plates | VWR | 29442-042 | |
| 10-cm plates | Sigma-Aldrich | Z688819 | |
| 18-gauge needle | Fisher Scientific (Pittsburgh, PA, USA) | 148265D | |
| 21-gauge needle | Fisher Scientific | 14-829-10D | |
| Equipment | |||
| BD LSRII Flow Cytometer | KSystem by Nikon (Tokyo, Japan) | ||
| BD FACSDiva Version 6.1.3 Software | BD Biosciences | ||
| Kits | |||
| PureLink Genomic DNA Mini Kit | Invitrogen | K182000 | |
| KAPA HiFi Hotstart ReadyMix PCR Kit | KAPA Biosystems (Wilmington, MA, USA) | KK2601 | |
| High Pure RNA Isolation Kit | Roche | 11828665001 | |
| Transcriptor First Strand cDNA Synthesis Kit | Roche | 4379012001 | |
| Sialix anti-Neu5Gc Basic Pack Kit | Sialix (Newton, MA, USA) | Basic Pack | |
| Media | |||
| Combined media 1 | StemCell Technologies and Stemgent | Consists of equal parts mTeSR1 and Nutristem | |
| Combined media 2 | StemCell Technologies and Stemgent | Consists of equal parts TeSR2 and Nutristem | |
| HUF Media | Dulbecco’s modified Eagle’s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin | ||
| Human Pluripotent Stem Cell Media | DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x β-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor | ||
| DMEM/F12, Dulbecco’s modified Eagle’s medium/F12; PBS, phosphate-buffered saline. | |||
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