Derivation of iPSCs after Culture of Human Dental Pulp Cells under Defined Conditions

<div><p>Human dental pulp cells (hDPCs) are a promising resource for regenerative medicine and tissue engineering and can be used for derivation of induced pluripotent stem cells (iPSCs). However, current protocols use reagents of animal origin (mainly fetal bovine serum, FBS) that carry the potential risk of infectious diseases and unwanted immunogenicity. Here, we report a chemically defined protocol to isolate and maintain the growth and differentiation potential of hDPCs. hDPCs cultured under these conditions showed significantly less primary colony formation than those with FBS. Cell culture under stringently defined conditions revealed a donor-dependent growth capacity; however, once established, the differentiation capabilities of the hDPCs were comparable to those observed with FBS. DNA array analyses indicated that the culture conditions robustly altered hDPC gene expression patterns but, more importantly, had little effect on neither pluripotent gene expression nor the efficiency of iPSC induction. The chemically defined culture conditions described herein are not perfect serum replacements, but can be used for the safe establishment of iPSCs and will find utility in applications for cell-based regenerative medicine.</p></div

Microarray analysis of gene expression.

<p>Scatter plots compared the embryonic stem cell marker genes expression between chemically defined culture conditions (MSCGM-CD) and normal culture conditions (MSCGM) determined by DNA microarray. The green lines indicate the diagonal and 2-fold changes between the two samples. Black and red circles indicate the expression levels of some embryonic stem cell marker genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115392#pone.0115392.s006" target="_blank">S3 Table</a>).</p

Morphology and growth capability of hDPCs cultured in MSCGM or MSCGM-CD medium.

<p>(A) Images of cultured hDPCs (DP245) 7 days after seeding and passage (P) 2. Cells were obtained from the same donor and cultured with MSCGM or MSCGM-CD, respectively (n = 6 donors). Scale bar = 400 µm. (B) Number of colonies derived from cells cultured in MSCGM or MSCGM-CD. hDPCs grown in MSCGM showed significantly higher primary colony formation than those grown in MSCGM-CD. (C) Curves for long-term growth obtained from 6 cell lines cultured with MSCGM or MSCGM-CD medium. hDPCs cultured in MSCGM medium maintained a high growth rate, whereas those in MSCGM-CD had a lower growth rate with the exception of the DP245 cell line.</p

Characterization of iPSCs derived from cells grown in MSCGM-CD or MSCGM.

<p>(A–J) iPSCs generated from DP264 grown in MSCGM-CD (D264-CD-iPS-2) and MSCGM (D264-M-iPS-5) expressed ALP (A, B) and pluripotency markers SSEA-4 (C, D), TRA1-60 (G, H), and TRA1-81 (I, J), but not SSEA-1 (E, F), as judged by immunostaining. Scale bar = 200 µm. (K) RT-PCR analysis of ESC-marker genes in iPSCs (D264-CD-iPS) generated from DP264 grown in MSCGM-CD, iPSCs (D264-M-iPS) derived from cells in MSCGM, human ESCs, and DP264. Numbers indicate different iPSC clones generated from DP264. Endogenous <i>NANOG</i>, <i>OCT3/4</i>, <i>SOX2</i>, <i>REX1</i>, and <i>KLF4</i> were expressed in two iPSC lines derived from cells grown in MSCGM-CD medium, as well as in human ESCs and iPSCs derived from cells grown in MSCGM, but not in DPCs. Error bars indicate the SD calculated from triplicates. (L–S) To confirm the pluripotency of iPSCs generated from DP264 grown in MSCGM-CD, we injected the cells into the testes of immunodeficient nude mice. Twelve weeks after injection, we observed tumor formation. Hematoxylin and eosin-stained teratoma sections show that the tumor contained various types of tissues, such as gut-like epithelial tissues (L, M, endoderm tissue), cartilage (N, O, mesoderm tissue), neural-tube-like structures (P, Q, ectoderm tissue), and pigment cells (R, S, ectoderm tissue). Scale bar = 100 µm.</p

Efficient generation of iPSCs derived from cells cultured in MSCGM or MSCGM-CD media.

<p>(A) Typical morphology of an iPSC colony generated from DP264 grown in MSCGM (DP264-M-iPS-5) and MSCGM-CD (DP264-CD-iPS-2). Scale bar = 200 µm. (B) Numbers of ESC-like colonies generated from 3×10⁴ hDPCs on day 21 after Sendai viral transduction. We transduced hDPCs from two donors (DP264, and DP265) with Sendai viral expression vectors containing the genes encoding four reprogramming factors (<i>OCT3/4, SOX2, KLF4,</i> and <i>c-MYC</i>), and cultured them in MSCGM or MSCGM-CD for 7 days after virus infection. Cells were then reseeded onto SNL feeder cells and incubated for 21 days. Mean numbers of colonies from two experiments (n = 2) are shown.</p

Differentiation ability of hDPCs cultured in MSCGM or MSCGM-CD media.

<p>(A, B) <i>In vitro</i> differentiation of hDPCs. hDPCs were cultured in MSCGM or MSCGM-CD and then tested for their ability to differentiate into osteo/odontoblastic and adipocyte lineages. (A) Osteo/odontogenesis was assessed by staining for ALP activity. (B) Adipogenesis was assessed by Oil Red O staining. Scale bar = 100 µm. (C, D) <i>In vivo</i> differentiation of hDPCs at P5. Transplants were removed at 12 weeks post-transplantation, sectioned, and stained with hematoxylin and eosin (C). In P5 transplants, the scaffolds (SC) were lined with a layer of dentin-like matrix (D). Scale bar = 200 µm. (D) Image analysis of the regenerated dentin area in subcutaneous composites at 12 weeks post-transplantation. The ratio of dentin regenerated in the hole (n = 7) was determined by histomorphometric analysis of the sections and calculated with ImageJ software.</p

Generation of Tumor Antigen-Specific iPSC-Derived Thymic Emigrants Using a 3D Thymic Culture System

Summary: Induced pluripotent stem cell (iPSC)-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs). iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies. : A barrier for clinical application of iPSC-derived CD8 T cells using OP9/DLL1 is their abnormal biology. Vizcardo et al. show that a 3D thymic culture system enables the generation of a homogeneous antigen-specific T cell subset, named iTEs, which closely mimics naive T cells and exhibits potent anti-tumor activity. Keywords: thymopoiesis, T cell differentiation, iPSC differentiation, adoptive cell transfer, naïve T cell, recent rhymic emigrants, fetal thymus organ culture, immunotherapy, 3D culture, tumor antigen specific T cel