Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette

Abstract

The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral "stem cell cassette" vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1. Human iPSC generation using a humanized, floxed single lentiviral stem cell cassette (hSTEMCCA-loxP)

A) Vector schematic illustrating the polycistronic lentiviral backbone encoding either 4 reprogramming factors or 3 factors plus mCherry. A loxP site inserted in the viral 3′LTR is duplicated to the 5′LTR during viral infection and reverse transcription. The resulting floxed vector integrated in the host mammalian genome can then be excised upon exposure to Cre recombinase. LTR=long terminal repeats. dU3=deleted U3 region of viral LTR. EF1α=Elongation Factor 1 alpha constitutive promoter. WPRE=Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element. B) Representative micrographs of human foreskin fibroblasts (HFF) in culture (left panel) and reprogrammed human iPSC colonies (right panel). Multiple alkaline phosphatase (AP) positive colonies are observed 30 days after reprogramming 50,000 human fibroblasts with the hSTEMCCA-loxP virus. C and D) Characterization of four independent iPSC clones, generated with either 4 factor or 3 factor hSTEMCCA-loxP, showing expression by RT-PCR (C) and immunostaining (D) of typical pluripotent stem cell markers. E) Representative normal 46XY karyotype of HFF-derived iPSC clone. F) Southern blot of BamHI digested gDNA from representative HFF-derived iPSC clones, probed against WPRE, demonstrates a single viral integration in all clones. HFF=human foreskin fibroblasts. hESC=H9 human embryonic stem cells. Bars= 200 μm (B); 250 μm (D).

Figure 2. Human 3 factor reprogramming with hSTEMCCA-RedLight-loxP vector

A) Flourescence microscopy shows mCherry fluorescence in an early passage (P1) iPSC clone, which becomes undetectable in later passages (P6). B) Flow cytometry of uninfected control hESC as well as hESC 72 hours after hSTEMCCA-RedLight-loxP lentiviral infection (infected+), compared to two individual late passage (P6) iPSC clones (hiPS R #6 and #7) showing absence of detectable mCherry gene expression. Bars=200 μm.

Figure 3. Generation of disease-specific iPSC

A) Human fibroblasts in culture and their reprogrammed iPSC progeny, derived from individuals with alpha-1 antitrypsin deficiency, cystic fibrosis, sickle cell disease, and scleroderma (SSc). B and C) characterization of lung disease-specific cystic fibrosis iPSC by RT-PCR (B), immunostaining (C), and real time qRT-PCR (D) demonstrating expression of stem cell markers, quantified as molecules of RNA of the indicated gene per molecules of RNA of the housekeeping gene, Cyclophilin. H1, H9, HES2= 3 control lines of human embryonic stem cells. CL=clone #. DF508= ΔF508 homozygous cystic fibrosis iPSC clones. E) Southern blot of BamHI digested gDNA demonstrating 18 of 20 individual iPSC clones (made from 4 individuals with either inherited AAT deficiency or CF) have been reprogrammed with a single integrated vector copy. Arrowheads indicate lanes with clones reprogrammed with 2 vector copies as well as a known 2 copy clone (far right lane). Blot has been probed against WPRE sequence of the lentiviral vector backbone. hiPS=human induced pluripotent stem cell. Clone # represents human volunteer (100–102) followed by clone number after hyphen (e.g. 100-2). PiZZ=homozygous Z alleles of the alpha-1 antitrypsin (AAT) protease inhibitor (Pi). Bars=200 μm.

Figure 4. Functional in vivo pluripotency of disease-specific human iPSC assessed by teratoma formation assay

Transplantation of a representative cystic fibrosis-specific iPSC (clone 202) into immunodeficient mice gave rise to a teratoma containing differentiated lineages representing the three primary germ layers, mesoderm, ectoderm, and endoderm. A) low power magnification (4×) of hematoxylin and eosin-stained tissue section of teratoma, illustrating formation of multiple glandular and cystic structures. B) Mesodermal differentiation demonstrated by bone formation (Bn) and ectoderm differentiation demonstrated by pigmented neuroepithelium (arrow), 40× magnification. C) Cartilage formation (*) indicative of mesodermal differentiation. D, E) glandular (endodermal) epithelium. F) pigmented ectodermal differentiation reminiscent of retinal pigmented epithelium (arrow). Bars= 200 μm (A), 100 μm (B,C,E,F), and 50 μm (D).

Figure 5. Cre-mediated excision of hSTEMCCA-loxP to generate transgene-free lung disease-specific iPSC

A) Schematic summarizing experimental approach for generating transgene-free iPSC. B) Southern blot of BamHI digested gDNA probed against the lentiviral WPRE fragment (top), demonstrating a representative parental iPSC line (AAT #100-3) and successful Cre-mediated excision of the single copy hSTEMCCA vector in 3 iPSC subclones (AAT #100-3-Cr1, -Cr5, and –Cr6). Equal DNA loading in each lane is evident by ethidium bromide (bottom) staining of the gel prior to transfer. C) PCR of gDNA confirming vector excision of the clones shown in B. D) Micrographs of iPSC showing colony morphologies after transfection of Cre-IRES-PuroR plasmid and 48 hours of antibiotic selection of excised colonies, at different time points after selection. Arrowheads denote emerging iPSC colonies. E) Karyotypic stability (46XX) by G-banding analysis of iPSC clone AAT #100-3 both before Cre-mediated vector excision, as well as in the subclones after vector excision. F) Pyrosequencing analysis of bisulfite-treated gDNA provides quantitation of methylation of the human NANOG promoter across 6 CpG islands in dermal fibroblasts prior to reprogramming, as well as in the AAT iPSC clone before and after Cre-mediated excision (pre-Cre vs. post-Cre) of the hSTEMCCA vector. H9 ES=control human embryonic stem cells. Bar=200 μm.

Figure 6. Directed differentiation of disease-specific iPSC into definitive endoderm after activin stimulation

A) Representative undifferentiated iPSC clone from an individual with alpha-1 antitrypsin (AAT) deficiency, assessed both before (AAT pre-Cre) and after (AAT post-Cre) excision of the hSTEMCCA reprogramming vector. Left panel shows FACS dot plot analyses of SSEA4 and TRA 1-81 cell surface markers. Right panel shows FACS analysis after intracellular staining for the stem cell marker, OCT4 (black line) vs. isotype control staining (grey histogram). B and C) Directed differentiation of the iPSC clones shown in A, in cultures designed to induce definitive endoderm vs. extra-embryonic endoderm (ExE) over 6 days. FACS (B) was used to quantify induction of the definitive endodermal markers CXCR4, CD117 (C-KIT), and FOXA1. Grey histogram indicates undifferentiated cells on day 0 (d0). Quantitative RT-PCR (C) shows induction of endodermal transcriptional regulators FOXA2, SOX17, and HNF4A on day 6 (d6), or the extra-embryonic marker SOX7. Gene expression levels are represented as molecules of RNA of the indicated gene per molecules of RNA of the housekeeping gene, Cyclophilin.