Differential adhesion molecule expression during murine embryonic stem cell commitment to the hematopoietic and endothelial lineages

Abstract

Mouse embryonic stem cells (ESC) make cell fate decisions based on intrinsic and extrinsic factors. The decision of ESC to differentiate to multiple lineages in vitro occurs during the formation of embryoid bodies (EB) and is influenced by cell-environment interactions. However, molecular mechanisms underlying cell-environmental modulation of ESC fate decisions are incompletely understood. Since adhesion molecules (AM) influence proliferation and differentiation in developing and adult tissues, we hypothesized that specific AM interactions influence ESC commitment toward hematopoietic and endothelial lineages. Expression of AM in the adherens, tight and gap junction pathways in ESC subpopulations were quantified. E-cadherin (E-cad), Claudin-4 (Cldn4), Connexin-43 (Cx43), Zona Occludens-1 (ZO-1) and Zona Occludens-2 (ZO-2) transcript levels were differentially expressed during early stages of hematopoietic/endothelial commitment. Stable ESC lines were generated with reduced expression of E-cad, Cldn4, Cx43, ZO-1 and ZO-2 using shRNA technology. Functional and phenotypic consequences of modulating AM expression were assessed using hematopoietic colony forming assays, endothelial sprouting assays and surface protein expression. A decrease in E-cad, Cldn4, Cx43 and ZO-1 expression was associated with less commitment to the hematopoietic lineage and increased endothelial differentiation as evidenced by functional and phenotypic analysis. A reduction in ZO-2 expression did not influence endothelial differentiation, but decreased hematopoietic commitment two-fold. These data indicate that a subset of AM influence ESC decisions to commit to endothelial and hematopoietic lineages. Furthermore, differentially expressed AM may provide novel markers to delineate early stages of ESC commitment to hematopoietic/endothelial lineages.

Figure 1. Expression of markers of early transitional stages of hematopoietic and endothelial differentiation is temporally regulated.

Bry-GFP, D3-ESC and Scl-LacZ ESC were differentiated in EB media for 8 days and analyzed flow cytometrically for expression of Bry, Flk-1 and Scl, respectively. Non-viable cells were excluded based on propidium iodide (PI) fluorescence. Error bars represent standard deviation with n = 3.

Figure 2. AM are differentially expressed in early hematopoietic and endothelial subpopulations of developing EB.

Quantitative analysis of gene expression was performed on populations of ESC differentiated in EB. Cells were sorted by FACS based on expression of Bry for mesodermal commitment, Flk-1 for hematopoietic and endothelial precursors and Scl for early hematopoietic cells. RNA was extracted and quantitative RT-PCR was performed. A) 21 genes were analyzed. Heat map was generated with Heatmap Builder (http://ashleylab.stanford.edu/tools_scripts.html). B) Samples are normalized to GAPDH and relative quantification is reported as percent of control (expression from sorted cell populations negative for expression of Bry, Flk-1 and Scl, respectively). Dashed line represents 2-fold increase/decrease in transcript expression level. (n = 3 for each sample; error bars represent standard deviation).

Figure 3. ESC with knockdown of junction molecules by shRNA maintain ability to differentiate to Flk-1 cells.

A) shRNA constructs efficiently silence endogenous mRNA in mESC. Quantitative RT-PCR was performed on GFP expressing cells generated with lentiviral constructs containing shRNA specific to E-Cadherin, Cx43, ZO1 and ZO2 to analyze transcript expression level. Samples were normalized to GAPDH and expressed relative to transcript levels in untransfected D3-ESC (dashed line). Error bars represent standard deviation of n = 3 samples. B) Flk-1 expression does not significantly differ in knockdown ESC lines. ESC lines stably expressing shRNA sequences specific to E-Cad, Cx43, ZO-1 and ZO-2 and ESC lines with increased knockdown levels of E-Cad and ZO-1 were differentiated in EB for 5 days and Flk-1 expression was analyzed flow cytometrically using anti-Flk-1 conjugated to PE. Non-viable cells were excluded based on fluorescence of DAPI. Paired t-tests with D3-ESC were performed with n = 7 (Kd-E-cad, Kd-Cx43, Kd-ZO-1) or n = 6 (Kd-ZO-2). * p<0.05; *** p<0.0005. Error bars represent standard error of the mean.

Figure 4. Hematopoietic potential decreases in ESC with down-regulation of junction molecules.

A) Knockdown ESC lines were differentiated in EB for 9 days. RNA was extracted from GFP+ cells and quantitative RT-PCR was performed using SYBR Green and primers for hematopoietic transcription factors Gata1, Runx1 and Scl. Error bars represent standard deviation of n = 3 samples. B) ESC lines stably expressing shRNA sequences specific to E-cadherin, Cx43, ZO-1 and ZO-2 were differentiated in EB for 11 days. CD45 expression was analyzed flow cytometrically using anti-CD45 conjugated to PerCP-Cy5.5. Non-viable cells were excluded based on DAPI fluorescence. Paired t-tests were performed with D3-ESC for n = 7 (Kd-Cx43) and n = 6 (Kd-E-cad, Kd-ZO1 and Kd-ZO2) (* p<0.05; ** p<0.005). Error bars represent standard error of mean. C) TF expression level linearly correlates with decreased frequency of CD45+ cells. Gata1: R² = 0.87; Runx1: R² = 0.91; Scl: R² = 0.69. D) Day 5 EB with knockdown of E-cadherin, Cx43, ZO-1 and ZO-2 were dissociated and replated as single cells to determine colony forming potential. Hematopoietic colonies were scored after 12 days in methylcellulose media for presence of granulocyte colony forming units (CFU-G), macrophage colony forming units (CFU-M), erythroid burst forming units (BFU-E), bipotent progenitors of granulocyte and macrophages (CFU-GM) and multipotent progenitors of these myeloid lineages (CFU-mix). Error bars represent standard error of the mean for n = 4 plates.

Figure 5. Endothelial commitment is increased in ESC knockdown lines.

A) ESC lines stably expressing shRNA sequences specific to E-cadherin, Cx-43, ZO-1 and ZO-2 were differentiated in Collagen I with 100 ng/ml bFGF and 50 ng/ml VEGF for 11 days. B) Percentage of sprouting EB and C) number of sprouts per sprouting EB were manually measured on photomicrographs in ESC reduced in expression of junction molecules compared to D3-ESC control. *** p-value<0.0005, ** p-value<0.005; * p-value<0.05.

Figure 6. Adhesion molecules expression and influence in hematopoietic and endothelial differentiation.

A) Knockdown of junction molecules ZO-1, Cx43 and E-Cad in murine ESC results in increased commitment to differentiate along the endothelial lineage with concurrent loss of commitment to hematopoietic cells. These fate decisions occur after the emergence of Flk-1 precursors in EB. Knockdown of ZO-2 in mouse ESC reduces hematopoietic commitment without influencing endothelial differentiation. B) Hematopoietic stages of differentiation can be identified using intracellular and extracellular markers. ESC undergoing mesodermal commitment express the transcription factor Bry. As mesodermal cells become committed to early hematopoietic and endothelial progenitors, the cell surface receptor, Flk-1, is expressed. Scl is a transcription factor expressed as cells commit specifically to the hematopoietic lineage. Expression of E-cad, Cx43, ZO-1 and ZO-2 varies during early stages of hematopoietic and endothelial differentiation of ESC.