Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells

<div><p>Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and <i>in vitro</i> spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.</p></div

Generation of iPSC pool by MACS from Sendai virus reprogrammed cells.

<p>(<b>A</b>) Cell populations from before (Pre-sort) and immediately after (Post-sort) MACS, and from the flow through fraction, were analyzed by FACS for enrichment of TRA-1-60 or SSEA4 positive stem cells. Three rounds of MACS were performed. Sort 1 was performed with TRA-1-60 antibody; TRA-1-60 positive cells were then used for sort 2 with TRA-1-60 antibody again; and the resulting cells were used for sort 3 with SSEA4 antibody. (<b>B</b>) Phase contrast pictures of newly reprogrammed colonies before MACS and sorted cells cultured for 4 days after each round of antibody sorting (Post-sort). FACS plot of p4 Sendai virus reprogrammed cell pool after 3 rounds of sorting showing percentage of SSEA4+TRA-1-60+ cells (right panel).</p

Characterization of MACS purified iPSC pool and clones.

<p>(<b>A</b>) FACS analysis showing pool selected iPSCs (SVPool) stably express pluripotency markers TRA-1-60 and SSEA4 through long-term passaging up to p74, similar to three clonally derived iPSC lines (SV7, SV10, SV20) from the same donor. (<b>B</b>) Immunocytochemistry and (<b>C</b>) qRT-PCR analyses of mRNA levels from iPSCs showing SVPool and iPSC clones (p15–20) express similar levels of a panel of pluripotency markers tested. (<b>D</b>) SVPool and iPSC clones both show Sendai viral reprogramming vector clearance at p15–20 by RT-PCR analysis. (<b>E</b>) G-banded karyotyping was performed on SVPool and clones at p28–33 and all cell lines were found to be karyotypically normal. The data presented are chromosomal banding patterns of SVPool and one of the clones (SV10) for comparison.</p

Selective uptake of cholesteryl ester in plasma and liver.

<p>[A] Uptake of [¹²⁵I] <i>upper panel</i> and [³H] <i>lower panel</i> in plasma over 24 hrs and in tissues: liver, adrenals, testis and kidney at 24 hrs. *Indicates a significant difference (p<0.01) between <i>Scarb1^I179</i> and control mice in uptake of [³H] in liver (means +/−SEM). [B] Uptake in plasma. Data is presented as means +/−SEM. Selective uptake determined from the clearance of double radio-labeled HDL in plasma over 24 hrs. FCR indicates fractional catabolic rate.</p

Generation of iPSC pool by MACS from hSTEMCCA reprogrammed cells.

<p>Cell populations from before (Pre-sort) and immediately after (Post-sort) MACS, and from the flow through fraction, were analyzed by FACS for enrichment of TRA-1-60 or SSEA4 positive stem cells. Two rounds of MACS were performed. Sort 1 was performed with TRA-1-60 or SSEA-4 antibodies in parallel and sort 2 was performed with either TRA-1-60 or SSEA4 antibody on cells previously sorted with TRA-1-60 antibody.</p

Comparison of <i>in vitro</i> differentiation potential of iPSC pool and clones.

<p>(<b>A</b>) iPSC pool (SVPool) and clones (SV7, SV10, SV20) were induced to differentiate by embryoid body (EB) formation assay for 14 days. Multiple differentiations were performed for each cell line between p20–30. Total RNA isolated from the EBs was used for hPSC Scorecard analysis for tri-lineage differentiation potential. Scores for expression of pluripotency, ectoderm, mesoderm and endoderm lineage genes were calculated relative to a set of reference hESC and iPSC lines. Scores for the hESC line H9 was used for comparison. SVPool and all iPSC clones show similar differentiation potential. (<b>B</b>) SVPool and clones were induced to differentiate into hepatocyte-like cells. The iPSC line K3 was used as a positive control. Immunocytochemistry of day 21 differentiated hepatocytes show comparable expression of hepatocyte markers HNF4α and Albumin in all cell lines. (<b>C</b>) Taqman qRT-PCR analysis of mature hepatocyte markers show similar gene expression levels among hepatocytes derived from SVPool and iPSC clones. mRNA levels were expressed as fold changes relative to those of human primary hepatocytes. The iPSCs were induced for endothelial cell differentiation. At day 14 of differentiation, cells were sorted for CD31⁺CD144⁺ cells using FACS. A representative FACS plot of the percentage of CD31⁺CD144⁺ cells in the differentiation culture (<b>D</b>) and similar differentiation efficiencies (percentages) amongst SVPool and clones from two independent experiments (<b>E</b>) are shown.</p

<i>In vitro</i> analysis of <i>Scarb1^I179</i>.

<p>[A] Co-transfected <i>Scarb1</i> wildtype and mutant with <i>Pdzk1</i>: Null indicates cells were treated as with other groups but without plasmid added. PDZK1 primary antibody in HuH7 cells was diluted 1∶5,000 to reduced endogenous detection. All other conditions are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006521#s4" target="_blank">materials and methods</a>. [B] Means and SE of SCARB1 expression levels from N = 3 wells per group of one experiment, representative of three experiments.</p

Effects of chloroquine and ALLN on <i>Scarb1</i> wildtype and mutant expression.

<p>Wild-type (WT), the I179N variant of <i>Scarb1</i>, and GFP were transiently expressed using HEK293 cells. 36 h post-transfection, cells were incubated with ±75 µM chloroquine (Chlor, to inhibit lysosomal degradation) or ±100 µM <i>N</i>-acetyl-leucinyl-leucinyl-norleucinal (ALLN, to inhibit proteosomal degradation) for 6 hr. [A] Cell samples were collected and SCARB1 proteins were visualized by immunoblotting. Data are representative of three experiments. [B] Means and SE of SCARB1 expression levels from N = 3 wells per group of one experiment. Null indicates no plasmids; cells were treated as with other groups but without plasmid added.</p

Plasma cholesterol analysis for <i>Scarb1^I179N</i> and control mice.

<p>[A] FPLC analysis of plasma lipoproteins: pooled plasma from female mice (N = 6). [B] Western analysis of lipoproteins: 14 ul of respective FPLC fractions and probed for ApoE and ApoAI.</p

Mapping <i>ScarbI^179N</i>.

<p>[A] QTL/mutant mapping analysis (B6-HLB398xD2) for HDL. Dashed line indicates 99% significance threshold after 1000 permutations. Vertical marks above the x-axis indicate location of markers, additional markers were added to Chr 5 after initial analysis indicated chromosome 5 as the only significant locus with B6 alleles giving high HDL levels. [B] Recombinant map and fine mapping on D2 background. Initial QTL mapping located the critical region between 109 and 138 Mb (grey region). Further backcrossing to D2 generated a number of recombinant congenic lines (A–F) narrowing the region to approximately 2 Mb at 124–126 Mb (D5Mit60 - D5Mit214). Triangles indicate locations of markers within the initial fine-mapped region. [C] Distribution of HDL in B6 x D2 mapping population segregating at the <i>Scarb1</i> locus showing that the allele effect is additive. HDL mean +/− SEM given for each sex separately and N for each genotype-sex group. Genotypes at the <i>Sr-b1</i> locus: +/+ indicates mice with no mutant B6 allele, +/ENU indicates mice with a single mutant B6 allele, and ENU/ENU indicates mice with two B6 mutant alleles. Within sex analysis reveals a significant difference between each genotype group (p<0.001).</p