Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes

Abstract

Background: Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.

Methodology/principal findings: Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.

Conclusion/significance: The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.

Figure 1. Lentiviral transduction and FACS enrichment of hESCs.

(A) Lentiviral constructs used in this study. Structural details are provided in Supplemental Figures S3, S4, S5, S6, S7, S8, S9, S10 and Methods. (B,C) Merged brightfield phase contrast micrographs of mCherry-positive cells overlayed with fluorescence images illustrate the variegated expression in a colony before (b) and reduced variability after (c) FACS enrichment (see Materials and Methods). hESCs were maintained on unlabeled MEFs.

Figure 2. Nuclear labeling and DNA content tracking with Histone-2B fluorescent fusion protein.

(A) Fluorescence and brightfield micrographs showing uniform PGK-H2BeGFP fluorescence in nuclei of cardiomyocytes derived from a FACS-enriched stable line. Spontaneous contractile activity is apparent in Supplemental Movie S1. H2B eGFP expression correlated with DAPI by flow cytometry (R² = 0.733, see text). (B) Examples of tracking of PGK-H2BmCherry hESCs cells, also from a FACS-enriched stable line, undergoing cytokinesis at the border of an undifferentiated colony. Lower left inset shows fluorescence intensity of 10 images before and 10 images after cell division of the individual cells in the larger figure (n = 5). Upper right inset shows heatmap representation of H2BmCherry fluorescence intensity of a single dividing cell. (C) Tracks calculated automatically using ImageJ Plugin from time-lapse images of H2BmCherry fluorescence at the border of undifferentiated ESC colony over a 2 day period (Methods). Yellow boxes indicate frames of movie clips in Supplemental Movie S2. (D) Time frames showing a single track of the H2Bmcherry centroid (blue).

Figure 3. Single colony expansion of hESCs.

(A) Schematic diagram of the procedure. Parental hESCs were gamma-irradiated and used as metabolically active but replication defective feeder cells. Lentivirally-transduced hESCs expressing a fluorescent reporter protein were enriched by FACS and seeded at low density on to the feeders, expanded as single colonies, and manually selected for serial passage as indicated (Methods). (B) FACS-isolated hESCs plated onto irradiated hESC feeder layer at low dilution after FACS. (C–F) Fluorescence micrographs showing examples of the sorted cells at different times after plating. Individual cells seen one day after sorting (C) grow into small colonies as imaged at days 7, 9 and 16 (D–F) (different colonies). (G) H2BmCherry-positive differentiated cells did not proliferate under the expansion conditions. (H,I) Phase contrast (H) and fluorescence (I) micrographs of clonal H2BmCherry hESCs originated from a single cell at day 7 following passage 1.

Figure 4. Pluripotency of clonal hESC reporter lines.

(A–C) Outgrowths of differentiated cells from EBs derived from a clonal PGK-H2BmCherry hESC line showed coincident H2BmCherry (A) and DAPI (B) immunostaining visible in the merged fluorescent image (C). (D–I) Clonal PGK-H2BmCherry hESC-derived EBs developed endothelial (D–F) and neuronal (G–I) lineages. (J) Expression of endodermal genes alpha fetoprotein (AFP) and hPDX-1 by day 20 EBs of clonal PGK-H2BmCherry hESCs (lane 2) compared to undifferentiated cells (lane 1) and isolated human pancreatic islet tissue as positive control for hPDX-1 (lane 3) and day 20 parental H9 EBs as control for AFP (lane 3). (K) Pluripotency markers expressed by clonal PGK-H2BmCherry hESCs (lane 2) compared to no reverse transcriptase control (lane 1) and undifferentiated parental H9 cells (lane 3).

Figure 5. T/Brachyury eGFP hESC line.

(A–D) Phase contrast (A), eGFP reporter fluorescence (B) anti-T/Brachyury immunostaining fluorescence (C) and merged fluorescence (D) micrographs of T/Brachyury-eGFP hESCs showing overlapping expression at the edge of hESCs colonies and in surrounding mesenchymal cell outgrowths. (E,F) Phase contrast (E) and eGFP reporter fluorescence (F) of day 4 differentiating EBs.

Figure 6. Drug resistance selection of hESC-derived cardiomyocytes.

(A) Schematic diagram of the drug selection regimen for isolation of cardiomyocytes (see Methods). Puromycin selection is typically done for a 1.5–2 day window between day 12 and 120 days (latest attempted) after EB formation. (B,C) Phase contrast micrographs of hESC colonies at day −2 (b) and again at day −1, after 24 hours exposure to G418 (C). (D,E) Beating cardiomyocyte clusters at day 12 (D) and again following Puromycin treatment at day 13.5 (E). Dark color is due to opacity of the tissue. Supplemental Movie S4 shows brightfield and fluorescence images of CSs at day 16. (F) Expression of αMHC and βMHC mRNA during differentiation in EBs (day 0–4) and in Puro^r CSs (day 18–90), by quantitative RT-PCR (error bars represent standard deviation) showing that αMHC transcripts are evident as cardiomyocytes emerge and persist for at least 90 days, consistent with efficacy of Puro^r selection when performed from day 12 and up to 150 days, the latest attempted (see text). β-MHC mRNA, in contrast, was first detected at day 90. (G,H) Histological sections of a day 18 EB (G) and day 20 CS (H) showing immunostaining of Troponin-I (red) and DAPI (blue). Cardiomyocytes were enriched in CSs, comprising 96.0%±8.6, as quantified from multiple sections of 10 independent biological replicates. (I) 3D confocal reconstruction of a 16 µm section through a CS isolated from a day 100 EB by the Neo^r, Puro^r selection protocol from a three-vector hESC line (αMHC-Puro^r_Rex-1-Neo^r, αMHC-mCherry_Rex-Bla^r, and PGK-H2BeGFP) showing cytoplasmic mCherry (magenta) and nuclear H2BeGFP (green). Numerous nuclei are not apparent since they are inside the reconstructed tissue.

Figure 7. Comparison of day 40 cardiomyocyte spheroids and adult heart gene expression profiles.

(A) Human Affymetrix exon microarray data for Neo^r undifferentiated hESCs and Puro^r CSs (isolated as in Figure 6a and analyzed at day 40 of culture) were combined with a previously described neural differentiation dataset (Cythera, HUES6 and fetal hCNS stem cells) in addition to adult tissue samples using the RMA algorithm (ExpressionConsole 1.0). A non-clustered heatmap of well-characterized cardiac and embryonic stem cell pluripotency genes are displayed for all combined differentiation and adult tissues, relative to their respective undifferentiated hESC controls (adult tissue compared to Neo^r hESCs from this study). (B) Individual array fold changes for day 40 CS and adult heart, relative to Neo^r undifferentiated hESCs were clustered using the HOPACH algorithm (Bioconductor), for 3030 genes up or down-regulated in the day 40 CS comparison. Genes clustered into one of nine top-level clusters (indicated), where red and green indicate gene up- and down-regulation, respectively, relative to undifferentiated hESCs. (C–E) Pathway over-representation analysis with the tool GO-Elite of the 1466 and 2502 upregulated genes in day 40 CS and adult heart samples, respectively, relative to undifferentiated hESCs as a function of the percentage of the genes changed per GO term. Cellular cardiac differentiation and complex tissue developmental pathways were enriched in both samples (C). Genes disproportionately enriched in adult heart aligned with in vivo tissue processes typical of whole heart and endothelial function (D). Genes disproportionately enriched in day 40 CS samples (E) included stem cell and early developmental processes.

Figure 8. Electrophysiological properties of Neo^r, Puro^r-selected, hESC-derived cardiomyocyte spheroids at day 20 of differentiation.

(A) The dominant electrophysiological phenotypes of action potentials (APs) of cardiomyocytes recorded from day 20 CSs. (B) Some cardiomyocytes displayed more hyperpolarized MDP and faster Vmax. Left panels (a and b) show expanded time scale and right panels show 3 APs. (C) The summary table of electrophysiological parameters of action potentials.

Figure 9. Force generation of individual day 50 Neo^r, Puro^r-selected cardiomyocytes.

CSs were isolated at day 12–13.5 and cultured until day 48 when they were dispersed and deposited onto gelatin-functionalized surfaces of polyacrylamide cast with fluorescent beads and analyzed for force generation at day 50. (A) Diagram of the apparatus containing the polyacrylamide. Platinum electrodes were used to electrically pace the cardiomyocytes at 0.5 Hz with 0.8 ms pulses of 50 volts. Inset shows micrograph of fluorescent beads. Scale bar represents 10 µm. (B) α-actinin immunostaining of hESC-derived cardiomyocytes on the functionalized surface shows characteristic striations. Scale bar represents 10 µm. (C) Bead displacements near individual cardiomyocytes (Supplemental Figures S2A,B and Movie S4) were tracked using a cross-correlation-based optical flow algorithm in order to map deformations (red arrows) or stresses (blue arrows) across the face of the gel corresponding to individual cardiomyocytes . Red arrows mark local bead displacement length is as per red scale bar (1 µm), which is expanded 20× relative to that of the image (white bar represents 10 µm) to permit visualization. Blue arrows indicate force magnitude as per blue scale bar [1 nN/µm2 (equivalent to 1 kPa)]. (D) Sample plot of total, transverse and longitudinal force versus time for a cardiomyocyte as in (C).