Derivación y caracterización de un transgen libre Humano madre pluripotentes inducidas Línea Celular y Conversión en condiciones definidas de calidad clínica

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

Las células madre pluripotentes inducidas humanas (hiPSCs) se pueden generar con las metodologías de reprogramación a base de lentivirus. Sin embargo, los rastros de genes potencialmente oncogénicos restantes en regiones activamente transcrito del genoma, limitan su potencial para su uso en aplicaciones terapéuticas humanas ^1. Además, los antígenos no humanos derivados de la reprogramación de células madre o la diferenciación en derivados terapéuticamente relevantes se oponen a estas hiPSCs se utilicen en un contexto clínico humano ^2. En este vídeo, se presenta un procedimiento para la reprogramación y analizar hiPSCs factoriales libre libres de transgenes exógenos. Estos hiPSCs se pueden analizar para detectar anomalías de expresión génica en el intrón específico que contiene el lentivirus. Este análisis puede llevarse a cabo utilizando la reacción en cadena de la polimerasa cuantitativa sensible (PCR), que tiene una ventaja sobre técnicas menos sensibles utilizado anteriormente para detectar diferencias de expresión génica ^3. La conversión total enbuenas prácticas de fabricación de grado clínico (GMP), permite relevancia clínica humana. Nuestro protocolo ofrece otra metodología, siempre que los criterios actuales de salvaguarda ampliarán e incluir los derivados basados ​​en hiPSC caracterizados factoriales libre para aplicaciones fines terapéuticos humanos derivadas hiPSCs GMP-grado, que debe eliminar cualquier riesgo inmunogenicidad debido a antígenos no humanos. Este protocolo es ampliamente aplicable a lentiviral reprogramado células de cualquier tipo y proporciona un método reproducible para la conversión de células reprogramadas en condiciones GMP grado.

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 factors^4,5. 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 transduction^3,9. 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

NOTA: Este método se utilizó en la investigación publicada en Awe et al 10.. 1. Reprogramación Humana Adulto fibroblastos dérmicos con STEMCCA Fibroblastos dérmicos Descongele adultos humanos (HUFS), paso 4 o inferior, en un 37 ° C baño de agua durante 2 minutos, teniendo cuidado de no sumergir la parte superior del vial con agua. Coloque la suspensión 1 ml de fibroblastos humanos en un vial cónico de 15 ml y colocar lentamente 4 ml de medio…

Representative Results

Se presenta un protocolo para derivar hiPSCs factor libre de grado clínico utilizando el enfoque basado reprogramación lentiviral-STEMCCA. La figura 1A muestra una imagen representativa de tres diferentes líneas hiPSC previamente extirpada, después de la reprogramación con el enfoque STEMCCA sobre una capa de MEFs. La principal ventaja del enfoque de reprogramación STEMCCA radica en el éxito reprogramación consistente alcanzado por múltiples científicos, en diferentes grupos y las ubicaciones …

Discussion

Se describe una metodología de derivar hiPSCs factor libre y hacerlos clínicamente relevante mediante la conversión de estas células en condiciones GMP grado de diferenciación celular aguas abajo en futuras terapias humanas. Aunque este protocolo es ampliamente aplicable a una variedad de tipos de células, que elegimos para reprogramar fibroblastos dérmicos humanos, debido a la facilidad de extracción de la paciente y su aplicabilidad a la terapéutica humana personalizados. Una vez que se subsanen las limitacio…

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.