BMSC co-culture primed efficient non-integrated reprogramming of CB progenitors that required only four episomal Yamanaka factors on a single plasmid.

The role of a brief 3-day BMSC co-culture of GF-activated CD34⁺ CB progenitors following a single pulse of episomal plasmids on Day 0, and the number of episomal factors required for efficient reprogramming. The experimental design is summarized in Fig. S1. (a) Soluble factors from brief BMSC co-culture preserved the viability of GF-activated CB progenitors. Viable CB cells were enumerated via Trypan Blue on Day 3 following nucleofection with four episomal factors (SOX2, OCT4, KLF4, MYC; 4F) on Day 0. Shown are the fold-increases of GF-treated CD34⁺ CB cells from Day 0 to Day 3 that resulted from: no BMSC co-culture (-BMSC), with irradiated BMSC co-culture (+BMSC), or with BMSC co-culture but separated by a Transwell insert (BMSC (T)). The Transwell culture insert prevented cell-cell contact between BMSC and CB cells, but allowed soluble stromal factors to diffuse freely to the cultured CB cells. Averages, SEM, and p values (Students t test) of n = 4 averaged experiments is shown. NS = not significant (p>0.05). (b) Brief co-culture of GF-activated CB cells with irradiated BMSC (+BMSC) did not increase the frequency of cells in G1 or S cell cycle phases at Day 3 (D3) of the reprogramming protocol (see Fig. S1) compared to GF alone (-BMSC). CB cells were nucleofected on Day 0 (D0) with 4F or 7F plasmids, or nucleofected with Amaxa buffer only (Mock). Cell cycle status of stromal-activated D3 CB, DO (pre-nucleofected) control CB cells, and adult fibroblasts (HDF1, HUF5; nucleofected with 7F) was determined on Day 3 of reprogramming protocols via FACS analysis of EdU incorporation and DNA content (7AAD), as described in Methods. Cells in G1 phase were defined as being EdU negative and 7AAD(2n); % cells in S phase were determined by gating on EdU positive 7AAD (2n+) populations. Data shown is the average of 2 experiments from individual batches of pooled CB donors run in triplicate. (c) GF-activated CB cells consist of a relatively homogenous myeloid population. By Day 3 of the reprogramming protocol (following +/− BMSC co-culture), both CD34⁺ and CD34⁻ GF-activated CD45⁺ CB progenitors had differentiated to primarily a myeloid CD33⁺ and CD13⁺ (not shown) phenotype. (d) Episomal reprogramming efficiencies of various somatic targets with either four factors (4F; SOKM, on a single plasmid, pEP4 EO2S EM2K) or seven factors (7F; SOKMNLT, on three separate plasmids), and with (+) or without (−) BMSC priming were determined by rapid AP staining (in triplicate) at 3 weeks following episomal nucleofections for CB progenitors (CB), fetal fibroblasts (FFB), adult fibroblasts (AdFib), and adult keratinocytes (Ker). Reprogramming efficiencies for emergence of Type III hiPSC [45] were quantitated in some experiments with parallel live TRA-1-81 staining, which gave similar results to enumeration of AP⁺ hESC-like colonies (Fig. S3). By 3 weeks post 4F nucleofections, the majority of AP⁺ hESC-like colonies had already converted with extremely high efficiencies to 50–80% fully reprogrammed SSEA4⁺TRA-1-81⁺NANOG⁺ Type III hiPSC (see also Fig. 7a). Each condition presented was repeated at least two to five times, as indicated, and with significance (t test; * = p<0.05) as indicated. 4F SOKM reprogramming of CB progenitors was even more robust than with equimolar DNA quantities of the 7F three-plasmid system (albeit initially less rapid). ND; BMSC co-culture was not done. With omission of the brief BMSC activation step, rarer (∼10–150-fold less) and slower-emerging CB-iPSC were produced with single-plasmid 4F nucleofections, but at frequencies that were still significantly greater than 7F reprogramming of fetal and adult fibroblasts, or adult keratinocytes.