推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件
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
人类诱导多能干细胞（人iPS细胞）可与慢病毒为基础的重编程方法来生成。然而，残留在基因组中的活性转录区潜在的致癌基因的痕迹，限制了其潜在的用于人类治疗应用1使用。此外，来自于干细胞重编程或分化为治疗相关的衍生物的非人类抗原排除来自于一个人的临床背景下2正在使用这些人iPS细胞。在这段视频中，我们提出了一个程序，重新编程和分析因子人iPS细胞无无外源转基因。这些人iPS细胞然后可用于在含有慢病毒的特异性内含子的基因表达异常进行分析。该分析可以使用灵敏的定量聚合酶链反应（PCR），其具有以前用于检测基因表达的3分歧较不敏感的技术的优点进行。全部转化成临床级良好生产规范（GMP）的条件下，使人类的临床意义。我们的协议提供了另一种方法，提供的电流安全港标准将扩大和包括因子无其特征为人类治疗应用，用于导出GMP级人iPS细胞，这应消除任何免疫原性风险因非人抗原hiPSC基衍生物。这个协议是广泛地适用于任何类型的慢病毒重编程的细胞，并提供一个可重复的方法用于将重编程的细胞成GMP级别的条件。
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
我们描述推导因子人iPS细胞无，使他们通过临床未来人类疗法将这些细胞转化为GMP级的条件，下游细胞分化相关的方法论。虽然该协议是广泛地适用于多种细胞类型的，我们选择了重新编程的人皮肤成纤维细胞，因为很容易提取从患者和他们的适用性，以个性化的人类治疗。一旦限制是尽可能补救作为充分分化为临床相关细胞衍生关注图14中，提出了转化为GMP级别的条件将变得更加相关的…
Disclosures
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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