Choice of Feeders Is Important When First Establishing iPSCs Derived From Primarily Cultured Human Deciduous Tooth Dental Pulp Cells

Abstract

Feeder cells are generally required to maintain embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs). Mouse embryonic fibroblasts (MEFs) isolated from fetuses and STO mouse stromal cell line are the most widely used feeder cells. The aim of this study was to determine which cells are suitable for establishing iPSCs from human deciduous tooth dental pulp cells (HDDPCs). Primary cultures of HDDPCs were cotransfected with three plasmids containing human OCT3/4, SOX2/KLF4, or LMYC/LIN28 and pmaxGFP by using a novel electroporation method, and then cultured in an ESC qualified medium for 15 days. Emerging colonies were reseeded onto mitomycin C-treated MEFs or STO cells. The colonies were serially passaged for up to 26 passages. During this period, colony morphology was assessed to determine whether cells exhibited ESC-like morphology and alkaline phosphatase activity to evaluate the state of cellular reprogramming. HDDPCs maintained on MEFs were successfully reprogrammed into iPSCs, whereas those maintained on STO cells were not. Once established, the iPSCs were maintained on STO cells without loss of pluripotency. Our results indicate that MEFs are better feeder cells than STO cells for establishing iPSCs. Feeder choice is a key factor enabling efficient generation of iPSCs.

Keywords: Deciduous tooth; Dental pulp; Feeder cell; Induced pluripotent stem cells (iPSCs); Mouse embryonic fibroblasts (MEFs); STO cells.

Figure 1

Generation of human deciduous teeth dental pulp cell induced pluripotent stem cells (HDDPC-iPSCs). (a) Plasmid vectors used for reprogramming. The location of each primer is denoted above the construct. shRNA(shp53): short hairpin RNA for tumor protein 53 (p53); CAG, cytomegalovirus enhancer + chicken β-actin promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; PA, poly(A) sites; EBNA-1, Epstein–Barr nuclear antigen 1. (b) Reprogramming protocol of HDDPCs. HDDPCs were plated (day −10) in the absence of a feeder layer and transfected (day −7) with three different plasmid vectors. Seven days after transfection, the emerging colonies were then reseeded onto mouse embryonic fibroblasts (MEFs) or STO cells (an immortalized line established from mouse Santos inbred mouse (SIM) embryonic fibroblasts resistant to 6-thioguanine and ouabain). This first passage of embryonic stem cell (ESC)-like colonies was designated as passage 1 (P1). Staining for alkaline phosphatase (ALP) activity was performed on P3, P10, P18, P20, and P26. NOD-Scid, nonobese diabetic-severe combined immunodeficient. (c, d) Determination of colony formation efficiency. Approximately 17 days after transfection, several foci were observed. The number of growing colonies appeared to be the same regardless of feeder type, but after P1, the number of ESC-like colonies seeded onto MEFs increased at least up to P5; however, the number of cells seeded onto STO cells did not (c). In contrast, the number of growing colonies appeared to be the same between those continuously grown on MEFs and those grown on MEFs and then grown on STO cells at P10 (d). (e, f) HDDPCs before (e) and after (f) transfection. When enhanced green fluorescent protein (EGFP)-derived fluorescence was inspected 1 day after transfection, more than 70% of the cells were successfully transfected, as shown in (f). Bars: 50 µm.

Figure 2

ALP activity of ESC-like colonies. Seven days after transfection with Yamanaka factors, HDDPCs were reseeded onto MEFs or STO cells. Emerging ESC-like colonies grown on MEFs were then serially passaged onto the same feeder cells (a–f). Similarly, ESC-like colonies grown on STO cells were subjected to serial passage (g–j). In some cases, the ESC-like colonies grown on MEFs were seeded onto STO cells at P10 and then were serially passaged using the same feeder (k, l). (a–c, g–i, k) Photographed under phase contrast microscopy; (d–f, j, l) cytochemical staining for ALP activity. Scale bars: 50 µm.

Figure 3

Immunostaining of ESC-like colonies. Immunocytochemical staining (ICC) of ESC-like colonies showed reactivity against octamer-binding transcription factor 3/4 (OCT3/4), stage-specific embryonic antigen 4 (SSEA-4), and TRA-1-60, but not against SSEA-1. Nuclei were stained with 6-diamidino-2-phenylindole (DAPI). Phase, photographs taken using a phase-contrast microscope. Scale bars: 50 μm.

Figure 4

Gene expression profiling of ESC-like colonies. (a) RT-PCR analysis performed to detect the expression of endogenous genes [OCT3/4, sex-determining region Y box 2 (SOX2), Krüppel-like factor 4 (KLF4), NANOG, and ALP] in HDDPC-iPSCs, HDFa (negative control), and PA-1 (positive control). Expression of OCT3/4 and SOX2 mRNA from the exogenous construct was also assessed using primer sets βA-1/OCT3/4-AS (for OCT3/4) and βA-1/SOX2-RV (for SOX2). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. −RT indicates the negative control (PCR with no RT product). M, 100-bp ladder markers. (b) PCR analysis of genomic DNA to detect the presence of integrated transgenes using the primer sets β-gl-1S/OCT3/4-AS (for pCXLE-hOCT3/4-shp53) and β-gl-1S/SOX2-RV (for pCXLE-hSK). As positive controls, plasmid DNA (5 ng) was concomitantly subjected to PCR and loaded.

Figure 5

Teratoma formation. (a, b) Embryoid bodies formed from the ESC-like cells 2 (a) and 10 (b) days after floating culture. Note the formation of differentiated cells (arrows) at the surface of embryoid bodies. (c, d) In vitro outgrowth of embryoid bodies 2 (c) and 10 (d) days after transfer of floating embryoid bodies to the adhesive substratum. Note that extensive outgrowth of differentiated cells (arrows) from the center of a colony is observed along with extension of cultural period. (e–g) Immunocytochemical staining of cells 10 days after in vitro differentiation of embryoid bodies from HDDPC-iPSCs at P25 by typical endodermal (forkhead box protein A2; FOXA2, e), ectodermal (βIII tubulin; antibody clone TUJ1, f) and mesodermal (smooth muscle actin; SMA, g) markers. (h–k) Hematoxylin–eosin staining of sections of solid tumors generated after transplantation of HDDPC-iPSCs at P18 into immunocompromised mice. Bars: 50 µm.

Figure 6

Karyotype analysis. (a) Abnormal karyotypes in HDDPC-iPSCs. A total of 20 cells were examined. Arrows indicate chromosomal abnormality including addition or deletion of chromosomal fragments. (b) Distribution pattern of chromosomal number obtained after karyotype analysis.