A Systematic Evaluation of Integration Free Reprogramming Methods for Deriving Clinically Relevant Patient Specific Induced Pluripotent Stem (iPS) Cells

<div><p>A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells. </p> </div

Comparison of different culture conditions during plasmid based reprogramming.

<p>A) Genomic integration of episomal plasmids. EBNA-1 and OriP sequences could not be detected in established iPS lines by passage 10. B) Morphology of RDP2-iPS cell lines derived in E8 medium, using different extracellular matrices. Scale bar is 400µm. C) Flow cytometric analysis of SSEA-4, TRA-1-81 and TRA-1-60 expression in RDP2-iPS cell lines derived on different matrices. Grey shaded areas denote the secondary antibody alone control. D) Whole well alkaline phosphatase staining of BJ, RDP2, PD1 and RP2 fibroblasts at day 30 of reprogramming using E8 medium and vitronectin.</p

Pluripotency of established iPS lines and detecting the genomic integration of episomal plasmids.

<p>A) Representative images of embryoid bodies (EBs) generated from BJ-pla-iPS (left) and BJ-mRNA-iPS (right). B) Repsentative image of karyotype 46, XY. BJ-pla-iPS cells at passage 15 is shown. C) H&E stained slides of teratomas formed from injections of iPS cells into the testis capsule of NOD-SCID mice. Tissue derivatives indicative of the three germ lineages were observed. D) Human pluripotent stem cell scorecard assay results comparing BJ-pla-iPS and BJ-mRNA-iPS lines.</p

Characterisation of established iPS lines derived from BJ fibroblasts using retrovirus, plasmids and mRNA.

<p>A) Representative phase images of established iPS and H1 cell lines. Immunostaining with pluripotency markers (green) and counter staining with DAPI (blue). Scale bar is 200µm. B) Alkaline phosphatase staining of whole plates/wells at days 30 (retrovirus and plasmid) and day 20 (mRNA). C) Flow cytometry analysis with pluripotency markers SSEA-4 and TRA-1-81. SSEA-1 is a negative marker of human pluripotent stem cells. Green line denotes H1, red line denotes BJ-RV-iPS, green line denotes BJ-Pla-iPS and black line denotes BJ-mRNA-iPS. D) RT-PCR analysis for expression of key pluripotency genes.</p

Depletion of B cells using anti-CD20.

<p><b>A.</b> Treatment schedule. Hemophilia A (HA) mice in “αCD20” groups received a dose of 10 mg/kg IgG2a αCD20 i.v. on day 0 and day 21. One week following the first αCD20 injection, mice in “AAV8” groups received 10¹¹ vg/mouse of AAV8-hFVIII. Blood samples were collected at indicated time points. Mice were challenged with intravenous hFVIII (1 IU/mouse, weekly for 4 weeks) at indicated time points. <b>B.</b> Representative examples of B cell depletion in different lymphoid organs of BALB/c-HA mice 1 day and 7 weeks after the second injection second αCD20 injection. Numbers in each histogram represent percent CD19⁺ lymphocytes (as shown by forward and side scatter gating in left panel) for both untreated control (in black) and αCD20-treated animals (red). Cells were stained with anti-CD19 antibody conjugated to V450 fluorochrome at 1 day post second injection, or to APC-Cy7 for the 7-weeks post-αCD20 time point.</p

Codon-optimized hFVIII to induce tolerance and correction.

<p>BALB/c-HA and BL/6-129/sv-HA mice were injected with 10¹¹vg/mouse of an AAV8 vector expressing codon-optimized hFVIII. <b>A.</b> Coagulation times (aPTT in sec) and <b>B.</b> HFVIIII activity were measured as a function of time after vector administration. Data are averages ±SD for n = 4/group. Mice were challenged (starting at week 10 after gene transfer) with hFVIII at the same dose and schedule as in previous experiments. Anti-hFVIII formation was measured <b>C.</b> as HFVIIII-specific IgG1 titers, and <b>D.</b> by Bethesda assay. Values in panels C and D are shown for individual animals and as averages ± SD and plotted on the same scale as in Fig. 2 to compare magnitude of responses.</p

Anti-CD20 treatment to prevent antibody formation in hFVIII protein replacement therapy.

<p>BL/6-129/sv-HA and BALB/c-HA mice were treated with αCD20 antibody as outlined in Fig. 1A (indicated with large arrows) followed by 4 weeks of hFVIII challenge (indicated by small arrows) beginning at 4 weeks after the second αCD20 administration. Mice were treated with hFVIII twice more following the same schedule. Antibody formation against HFVIIII was measured by Bethesda assay (<b>A</b>) and anti-hFVIII IgG1 ELISA (<b>B</b>) two weeks after each 4-week challenge. Control mice did not receive αCD20. Gray triangles represent B cell recovery. Data are averages ±<u>SD</u> for n = 3–5/group.</p

T cell responses in BALB/c-HA mice.

<p><b>A.</b> Following the final tail vein bleed (Fig. 3D), mice were sacrificed and spleens collected. Splenocyte cultures for individual mice (n = 4 per group) were stimulated <i>in vitro</i> with 10 µg/mL of hFVIII for 48 h. Subsequently, CD3⁺CD4⁺ T cells were purified by flow cytometry and analyzed by quantitative RT-PCR for expression of several immune-regulatory genes. Shown are data for indicated groups (averages ±<u>SD</u>; “fold increase” is change in RNA transcripts of hFVIIII- vs. mock-stimulated). The dotted horizontal line indicates the minimally required increase of 2.5-fold for a statistically significant difference. <b>B.</b> Evidence for Treg induction in mice that had received gene transfer or a combination of αCD20 and gene transfer. Following one round of hFVIII challenge, CD4⁺CD25⁺ splenocytes were purified from each treatment group via magnetic sorting, and 10⁶ cells/mouse were adoptively transferred to naïve BALB/c-HA recipients via tail vein injection. Control cells were from <u>unchallenged</u> naïve mice of the same strain. Twenty-four hours later, all recipient mice (n = 3 per group) were challenged with 1 IU hFVIII in adjuvant. Anti-FVIII IgG titers were measured 1 month later by ELISA. Data are averages ±<u>SD</u>.</p

Depletion of B cells combined with gene therapy in BL/6-129/sv-HA mice.

<p><b>A.</b> Activated partial thromboplastin time (aPTT) of mice receiving 10¹¹vg/mouse of AAV8-hFVIII either alone (“AAV8-F8”) or in combination with αCD20 therapy (“AAV8-F8+CD20”). “FVIII challenge” indicates a period of weekly HFVIII injections (see Fig. 1A). “AAV8-unchallenged” group is mice that received vector but were not challenged with hFVIII protein. Range of aPTT for untreated mice and coagulation time for HA mouse plasma corrected to 1% HFVIII activity are also shown. Data are average values ±<u>SD</u> for n = 5 per experimental group. Two weeks after completion of the FVIII challenge, antibody formation against FVIII was measured: <b>B.</b> Total FVIII-specific IgG1 levels as determined by ELISA. <b>C</b>. Inhibitory antibody titers as measured by Bethesda assay (in BU). Values in panels B and C are shown for individual animals and as averages ±<u>SD</u>. Statistically significant differences between groups are indicated.</p