Development of a protein marker panel for characterization of human induced pluripotent stem cells (hiPSCs) using global quantitative proteome analysis

Abstract

The emergence of new methods for reprogramming of adult somatic cells into induced pluripotent stem cells (iPSC) led to the development of new approaches in drug discovery and regenerative medicine. Investigation of the molecular mechanisms underlying the self-renewal, expansion and differentiation of human iPSC (hiPSC) should lead to improvements in the manufacture of safe and reliable cell therapy products. The goal of our study was qualitative and quantitative proteomic characterizations of hiPSC by means of electrospray ionization (ESI)-MS(e) and MALDI-TOF/TOF mass spectrometry (MS). Proteomes of hiPSCs of different somatic origins: fibroblasts and peripheral blood CD34(+) cells, reprogrammed by the same technique, were compared with the original somatic cells and hESC. Quantitative proteomic comparison revealed approximately 220 proteins commonly up-regulated in all three pluripotent stem cell lines compared to the primary cells. Expression of 21 proteins previously reported as pluripotency markers was up-regulated in both hiPSCs (8 were confirmed by Western blot). A number of novel candidate marker proteins with the highest fold-change difference between hiPSCs/hESC and somatic cells discovered by MS were confirmed by Western blot. A panel of 22 candidate marker proteins of hiPSC was developed and expression of these proteins was confirmed in 8 additional hiPSC lines.

Figure 1

Characteristics of proteins up- or down-regulated in hiPSCs compared to somatic cells. (A): Example of Ingenuity Pathway Analysis results: biological processes affected by the proteins up- or down-regulated in SB5-MP1 vs. fibroblasts. The p-value cut-off is <0.05 (>1.3 in −log₁₀). Biological processes affected by up-regulated proteins exclusively in hiPSC or in somatic cells are shown in red. (B): Top Ingenuity canonical pathways up-regulated in both hiPSCs compared to primary parental cells (p-value < 0.01). (C): Subcellular localization of the proteins up-regulated in both hiPSCs vs. primary cells.

Figure 2

Validation of quantification by ESI-MS^e of the previously described pluripotency markers by Western blot. (A): Selected previously described markers of pluripotency quantified by ESI-MS^e (TOF). Absolute quantity (fmol/10 µg) in each cell line is shown in the chart. The absolute quantity of GAPDH in each cell line estimated by ESI-MS^e was found to be unchanged in all 5 cell lines. (B): Western blot analysis of three pluripotent (hESC-H9, SB5-MP1 and iNC-01) and two parental primary (PBMC and fibroblasts) cell lines. Protein quantity was normalized against GAPDH. * denotes markers also detected by MALDI-TOF/TOF. (C): Known markers of pluripotency not identified by any of MS techniques in this study but detected by Western blot.

Figure 3

Candidates to hESC/hiPSC markers found in this study by comparative quantification of five cell lines using ESI-MS^e. (A): Quantification of 12 candidates to markers by ESI-MS^e (TOF). Absolute quantity (fmol/10 µg) in each cell line is shown in the chart. Protein quantity was normalized against GAPDH. Absolute quantity for GAPDH measured by ESI-MS^e was the same as in Fig. 2. (B): Western blot detection of the 12 candidates to markers in three pluripotent (hESC-H9, SB5-MP1 and iNC-01) and two parental primary (PBMC and fibroblasts) cell lines. Protein quantity was normalized against GAPDH. (C): Quantification of 4 candidates to “contrasting” markers by ESI-MS^e (TOF). (D): Western blot detection of the candidates to “contrasting” markers. * denotes markers also detected by MALDI-TOF/TOF.

Figure 4

Results of MALDI-TOF/TOF analysis of three pluripotent stem cell lines: hESC-H9, SB5-MP1 and iNC-01. (A): Number of proteins detected by MALDI-TOF/TOF exclusively, by ESI-MS^e exclusively or by both techniques in each cell line in at least one out of two biological experiments. (B): Number of common proteins between three cell lines identified in at least one out of two biological experiments exclusively by MALDI-TOF/TOF (not detected by ESI-MS^e). (C): One previously described (*) and 5 novel markers of hESC/hiPSC detected by MALDI-TOF/TOF exclusively were validated by Western blot analysis in three pluripotent and two parental primary (PBMC and fibroblasts) cell lines. Protein quantity was normalized against GAPDH.

Figure 5

Qualification of the panel of 22 previously described and novel hESC/hiPSC protein markers and two opposed (somatic cell) markers in 9 hiPSC lines, primary fibroblasts and PBMC by Western blot. (A): Well-known markers (*) and proteins that were previously described in a literature as possible hESC/hiPSC markers. (B): Novel hiPSC/hESC markers found in this study by ESI-MS^e quantification. (C): Novel markers detected in hESC/hiPSC in this study exclusively by MALDI-TOF/TOF. (D): “Contrasting” markers found in this study by ESI-MS^e quantification. Nine hiPSC lines used in analysis (origin): 1 — iNC-06s-2E — P16 (CD34⁺), 2 — iNC-01 — P31 (CD34⁺), 3 — iM6-3-2 — P19 (adult fibroblasts), 4 — iM6-1-5 — P18 (CD34⁺), 5 — iPS(IMR90)-1 — P30 (fetal fibroblasts), 6 — NC1-P38 (adult fibroblasts), 7 — NC3-P16 (human umbilical vein endothelial cells (HUVECs)), 8 — NC8-P8 (adult fibroblasts), 9 — 80-4-P20 (adult fibroblasts).