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Keywords:

  • Embryonic stem cell;
  • Differentiation;
  • Thymic epithelial progenitors;
  • T cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Thymopoiesisis regulated by the thymic microenvironment, of which epithelial cells are the major components. Both cortical and medullary thymic epithelial cells (TECs) have been shown to arise from a common progenitor cell. Here we show for the first time that mouse embryonic stem cells (mESCs) can be selectively induced in vitro to differentiate into cells that have the phenotype of thymic epithelial progenitors (TEPs). When placed in vivo, these mESC-derived TEPs self-renew, develop into TECs, and reconstitute the normal thymic architecture. Functionally, these ESC-derived TEPs enhanced thymocyte regeneration after bone marrow transplantation and increased the number of functional naive splenic T cells. In addition to providing a model to study the molecular events underlying thymic epithelial cell development, the ability to selectively induce the development of TEPs in vitro from mESCs has important implications regarding the prevention and/or treatment of primary and secondary T-cell immunodeficiencies. STEM CELLS 2009;27:3012–3020


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

T lymphocytes (T cells) play a central role in the adaptive immune system by protecting against bacterial, viral, fungal, and parasitic infections and neoplasia. T-cell development in the thymus is dependent on the thymic microenvironment, in which epithelial cells are the major components [1, 2]. The importance of thymic epithelial cells (TECs) has been demonstrated in patients and in animals in which genetic mutations or deletions affect thymic epithelial cell development or function. Such defects have dramatic effects on intrathymic T-cell development, leading to severe immunodeficiency [1–3]. Restriction of the number of thymic epithelial cells has been shown to result in a reduced number of T cells in the thymus [4–6]. This, along with changing TEC function, is believed to be one of the major factors in age-dependent thymic involution [2].

Many efforts have been made to restore the thymic microenvironment for T-cell regeneration [7]. For example, keratinocyte growth factor and sex steroid ablation have been reported to improve thymopoiesis by restoration of the functions of thymic epithelial cells [8–13]. Transplantation of cultured thymus fragments has been used to provide the thymic microenvironment for T-cell regeneration in patients and experimental animals with T-cell deficiencies or as a method for the induction of tolerance in organ transplantation [7, 14–17]. Studies on thymus organogenesis in the murine embryo have indicated that both the cortical and medullary epithelial cells arise from a common progenitor population [18, 19]. Several groups have identified thymic epithelial progenitors (TEPs) in the embryonic thymus, and have shown that they can reconstitute a functional thymic microenvironment capable of supporting T-cell development in vivo [20–22]. Recently, Rossi et al. reported that a single EpCAM1+ cell isolated from the embryonic thymus could develop into cortical and medullary epithelial cells in vivo [22]. The identification of TEPs has important clinical implications for restoring thymus function. However, use of TEPs for this purpose has been restricted by the limited availability of such cells.

Given that embryonic stem cells (ESCs) have the dual ability to propagate indefinitely in vitro in an undifferentiated state and to differentiate into all three germ layers [23], it is possible that ESCs can be induced to generate large numbers of TEPs. Here we report that murine ESCs (mESCs) can be induced to differentiate into EpCAM1+ cells, some of which concomitantly express the markers for both thymic cortical (keratin [K] 8+) and medullary (K5+) epithelial cells, a characteristic of TEPs. The mESC-derived EpCAM1+ cells could form normal thymic architecture in vivo, and a single EpCAM1+ cell, when injected into an irradiated thymus implanted under the kidney capsule, developed into three types of donor-origin cells, K5+K8+ TEPs, K5K8+ cortical epithelial cells, and K5+K8 medullary epithelia cells. Furthermore, the ESC-derived EpCAM1+ cells enhanced thymocyte and peripheral T-cell regeneration when injected directly into the thymus of lethally irradiated mice reconstituted with T-cell deleted (TCD) bone marrow (BM). These results indicate that functional TEPs can be selectively generated from mESCs in vitro, and that these TEPs can restore the thymic microenvironment and support T-cell development in vivo.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Mice

Six- to 12-week-old 129SVEVTac, 129Ola/Hsd, C57BL/6 (B6)-Ly5.1, B6 (Ly5.2), and B6-129S2-H2dlAb1−Ea/J mice were purchased from Taconic (Hudson, NY, http://www.taconic.com), Harlan (Indianapolis, IN, http://www.harlan.com), National Cancer Institute (Frederick, MD, http://www.cancer.gov), and The Jackson Laboratory (Bar Harbor, ME, http://www.jax.org), respectively. Mice were housed, treated, and handled in accordance with the guidelines set forth by the University of Connecticut Health Center Animal Care Committee.

Cell Culture

TC-1 and E14TG2a-Tau-GFP mESCs (derived from 129SVEVTac and 129 Ola mice, respectively) were maintained on irradiated murine embryonic fibroblasts in Dulbecco's modified Eagle's medium with 15% fetal calf serum and 103 U/ml leukemia inhibitory factor (LIF) [24, 25]. For embryoid body (EB) differentiation, mESCs were plated in hanging drops in an inverted bacterial Petri dish [26]. EBs were collected from the hanging drops at day 2 and transferred into 6-well culture plates containing differentiation medium (mESC medium without LIF) and growth factors. For monolayer differentiation, undifferentiated mESCs were cultured in collagen IV (10 μg/ml; from R&D Systems Inc., Minneapolis, http://www.rndsystems.com)-precoated 6-well culture plates [27] containing differentiation medium and growth factors. The medium and growth factors were changed every 3–4 days.

Flow Cytometry Analysis

Thymocytes, splenic cells, and single-cell suspensions from mESC-derived cells were stained with the fluorochrome-conjugated antibodies as described [28]. For intracellular staining, the cells were first permeabilized with a BD Cytofix/Cytoperm solution (San Diego, http://www.bdbiosciences.com) for 20 minutes at 4°C. Direct or indirect staining of fluorochrome-conjugated antibodies included: CD4, CD8, CD45RB, CD44, H-2Kb, I-Ab, T-cell receptor (TCR) β, TCR γδ, and interferon (IFN)-γ (BioLegend [San Diego, http://www.biolegend.com] or BD Biosciences, K5, Delta, and CCL25 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), K8 (US Biological, Swampscott, MA, http://www.usbio.net), mouse EpCAM1 and fluorescein isothiocyanate- or phycoerythrin-labeled anti-rat or rabbit IgG (BD Biosciences). The samples were analyzed on a FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) machine with CellQuest acquisition software. Data analysis was done using FlowJo software (TreeStar, Inc., Ashland, OR, http://www.treestar.com).

Real-Time Quantitative Reverse-Transcription Polymerase Chain Reaction

RNA was extracted from cells using the Qiagen RNAeasy Kit (Valencia, CA, http://www1.qiagen.com). Reverse-transcription (RT) and polymerase chain reactions (PCRs) were done using iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad Laboratories, Hercules, CA, http://www.bio-rad.com) according to the manufacturer's instructions. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA using the Δ cycle threshold method [29]. The primer sequences used are shown in supporting information Table 1.

Immunomagnetic Cell Separation

Single-cell suspensions from differentiated mESCs were harvested after the cells were treated with 2 mg/ml collagenase IV. The cells were stained with rat anti-mouse EpCAM1 antibody, washed, and stained with anti-rat IgG MicroBeads (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com). EpCAM1+ cells were positively selected using a magnetic-activated cell sorter immunomagnetic separation system (Miltenyi Biotec).

Kidney Capsule Grafting

Purified mESC-derived EpCAM1+ or EpCAM1 cells (1 × 105) were mixed with CD4CD8CD45+ (1 × 105) thymocytes from syngeneic mice, and subjected to reaggregate cultures for 24–48 hours, as described [20, 21, 30]. The solidified reaggregate was grafted under the kidney capsule of syngeneic mice [20–22]. For single EpCAM1+ cell injection, enhanced green fluorescent protein (EGFP)+ mESC-derived EpCAM1+ cells were diluted to one cell in 10 μl phosphate-buffered saline (PBS) that was injected into a thymic fragment from lethally irradiated mice. The thymic fragment was then transplanted under the kidney capsule. Six weeks after implantation, the transplants were harvested and analyzed by immunohistology.

Immunohistology and Confocal Microscopy

Immunohistological analysis of grafted thymus tissues was performed according to a modified protocol [22, 31]. Briefly, tissues were incubated in 4% paraformaldehyde for 4 hours followed by incubation in 30% sucrose solution overnight. The tissues were embedded in optimal cutting temperature medium, snap frozen, and subsequently cut into 5-μm sections. The sections were stained with rabbit anti-mouse K5 polyclonal antibody (Covance Research Products, Denver, PA, http://www.covance.com), and rat anti-mouse K8 monoclonal antibody (mAb; Throma I mAb, raised by P. Brulet and R. Kemler and obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), followed by AlexaFluor-488-, 546-, or 647-conjugated goat anti-rabbit IgG, or goat anti-rat IgG (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The cells were observed under a Zeiss LSM510 Meta laser scanning confocal microscope (Carl Zeiss, Thornwood, NY, http://www.zeiss.com).

Intrathymic Injection

The thymus was surgically exposed and one half of the indicated number of mESC-derived cells was injected into the anterior superior portion of each lobe (10 μl/site) using a 1-ml syringe (with attached 28-gauge needle) mounted on a Tridek Stepper (Indicon Inc., Danbury, CT), as described [32, 33]. The sternum was closed with absorbable sutures, and the skin incision was closed with Nexaband Liquid (Veterinary Products Laboratories, Phoenix, AZ, http://www.vpl.com). Control mice were injected intrathymically with PBS alone.

Bone Marrow Transplant Procedure

BM cell suspensions were harvested from mice by flushing the marrow from the femurs and tibias with cold RPMI 1640 (Invitrogen) supplemented with sodium bicarbonate (2 mg/ml) and 1% HEPES (1.5 M). TCD-BM cells were prepared as described [34]. In brief, BM cells were incubated with rat anti-CD4, anti-CD8, and anti-Thy1.2 in saturating amounts at 4°C for 20 minutes, washed, and incubated with goat anti-rat IgG magnetic beads (Miltenyi Biotec) for 20 minutes at 4°C, and run over a depletion column. Recipients received 1,000 cGy total body irradiation (100–110 cGy/minute) from a 137Cs source (Gamma Cell 40 Irradiator; Atomic Energy of Canada, Ottawa, http://www.aecl.ca). Two to four hours later, the mice were injected intravenously (i.v.) with a mixture of mESC-derived cells and TCD-BM, or injected intrathymically with mESC-derived cells followed by i.v. injection of TCD-BM the next day.

Mixed Leukocyte Reactions

Splenocytes (1 × 105 cells/well) from the recipient of TCD bone marrow transplant (BMT) were cultured in the presence or absence of irradiated (2,000 cGy) BALB/c splenocytes as stimulators (2 × 105 cells/well) for 5 days in a 96-well plate. [3H] thymidine (1 μCi) was added to each well for the final 12 hours of culture. Incorporation of [3H] thymidine was measured as counts per minute (CPM) by liquid scintillation spectroscopy. Counts are presented as stimulation index (CPM of splenocytes from mice that received TCD-BM plus EpCAM1+ or EpCAM1 cells/CPM of splenocytes from mice that received TCD-BM alone).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Differentiation of mESCs into Cells Having the Phenotype of TEPs

It has been reported that mesenchymal-epithelial interactions are essential for the expansion but not the differentiation of TEPs in the embryonic thymus [1, 2]. Mesenchymal cells support the proliferation of TEPs, at least in part, via the production of fibroblast growth factor (FGF)-7 or FGF-10 [1, 2]. Bone morphogenetic protein 4 (BMP-4) has been shown not only to enhance epithelial differentiation [35, 36] but also to increases the expression of Foxn1, which plays a crucial role in thymic epithelial development [37–39]. Epithelial growth factor (EGF) and collagen IV also enhance epithelial differentiation from ESCs [27]. Based on these reports, we induced mESCs to differentiate into TEPs in the presence of different combination of these factors. Both three-dimensional (3D) EB formation and two-dimensional (2D) monolayer culture systems were used. After 10 days of culture, the ESC-derived cells were analyzed for the expression of EpCAM1 that has been reported to be expressed by TEPs [22]. As shown in Figure 1A–1C, FGF-7 alone did not increase the absolute numbers of ESC-derived cells in the 3D cultures, but increased the percentage of EpCAM1+ cells. In contrast, addition of EGF increased total cells, but did not increase the percentage of EpCAM1+ cells. The combination of FGF-7, BMP-4, EGF, and FGF-10 increased both the percentage (up to ∼9%) and the number of EpCAM1+ cells significantly.

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Figure 1. Generation of EpCAM1+ cells from mouse ESCs (mESCs) in vitro. (A–C): Three-dimensional (3D) cultures: mESCs were plated in hanging drops at 200 cells per drop for 2 days, and the embryoid bodies (EBs; 20 EBs/well) were then transferred to culture plates in medium containing different combinations of 20 ng/ml FGF-7, FGF-10, BMP-4, and 50 ng/ml EGF, or PBS as control. (D–F): Two-dimensional cultures: mESCs (4,000 cells/well) were inoculated in 6-well culture plates that were precoated with collagen IV and cultured in medium containing the same combinations of growth factors as those in the 3D cultures. Ten days later, the adherent cells were harvested after collagenase IV treatment, and mESC-derived cells were analyzed for the expression of mouse EpCAM1 by flow cytometry. Data show total numbers of the mESCs-derived cells (A, D), and the percentages (B,E) and the numbers (C, F) of EpCAM1+ cells. Mean ± SD from three independent experiments. Abbreviations: BMP4, bone morphogenetic protein 4; EGF, epithelial growth factor; F7, fibroblast growth factor 7; F10, fibroblast growth factor 10; PBS, phosphate-buffered saline.

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In the 2D cultures, EpCAM1+ cells were generated in a pattern similar to that in the 3D cultures (Fig. 1D–1F). However, both the percentages (up to ∼25%) and numbers of EpCAM1+ cells were increased, compared with those in the 3D cultures. We then analyzed the expression of K8 and K5, the markers for cortical and medullary epithelial cells, respectively, by the ESC-derived cells. A range of 68%–82% of EpCAM1+ cells from the cultures containing the four growth factors coexpressed K5 and K8 (Fig. 2A), suggesting that cells with the phenotypes of TEPs [20–22] can be generated from mESCs. To determine whether the EpCAM1+ cells express TEP-associated genes [40, 41], EpCAM1+ and EpCAM1 cells were purified from the cultures (more than 95% purity) and subjected to real-time quantitative RT-PCR analysis for the expression of Pax1, Pax9, FoxN1, and Plet1. As shown in Figure 2B, although the expression levels of these genes in the mESC-derived EpCAM1+ cells were lower than those in EpCAM1+ cells purified from embryonic day (E)-12 thymi, they were significantly higher than those in the mESC-derived EpCAM1 cells, further indicating that the mESC-derived EpCAM1+ cells contained TEPs.

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Figure 2. mESC-derived EpCAM1+ cells contain a large population of cells that coexpress K5 and K8, and express thymic epithelial progenitor-associated genes. (A): TC-1 mESC-derived cells from cultures containing fibroblast growth factor (FGF)-7, FGF-10, bone morphogenetic protein 4 (BMP-4), and epithelial growth factor (EGF) were stained with anti-EpCAM1, anti-K5, and anti-K8 antibodies (or isotype control antibodies), and analyzed by flow cytometry. Shown is a representative analysis on gated EpCAM1+ or EpCAM1 cells. (B): TC-1 mESC-derived EpCAM1+ and EpCAM1 cells were purified from the cultures, and EpCAM1+ cells were purified from embryonic day (E)-12 thymi. The expression of Pax1, Pax9, FoxN1, and Plet1 genes was analyzed by real-time quantitative reverse-transcription polymerase chain reaction. Data are presented as relative levels of expression in mESC-derived EpCAM1+ and E12 thymic EpCAM1+ cells versus mESC-derived EpCAM1 cells. Mean ± SD from three independent experiments. Abbreviations: K, keratin; mESC, mouse ESC; TECs, thymic epithelial cells.

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mESC-Derived EpCAM1+ Cells Can Form Normal Thymic Architecture and Support T-Cell Development In Vivo

To determine whether the mESC-derived EpCAM1+ cells can form normal thymic architecture containing cortical and medullary TECs, purified EpCAM1+ cells were mixed with CD4CD8CD45+ thymocytes and reaggregated in vitro. The cell reaggregates were then transplanted under the kidney capsule of syngeneic mice. Six weeks later, the grafts were harvested, sectioned, and stained for K8 and K5. As shown in Figure 3A, discrete K8+K5 cortical and K8K5+ medullary epithelial areas were present. Some of the cells coexpressed K8 and K5 (Fig. 3 yellow), suggesting they were residual or self-replicating TEPs. In controls, mice injected under the kidney capsule with CD4CD8CD45+ thymocytes, or EpCAM1 and CD4CD8CD45+ cell reaggregates could not form normal thymic architecture, and mice injected with EpCAM1+ cells alone could not efficiently form normal thymic architecture (data not shown). The cells recovered from the grafts were also assessed for T-cell development by flow cytometry. As shown in Figure 3B, CD4 and CD8 double- and single-positive T cells were generated in the EpCAM1+ but not in the EpCAM1 cell grafts. We then examined surface expression of αβ and γδ TCR by the cells recovered from the EpCAM1+ cell grafts. As shown in Figure 3C, more than 90% of the CD3+ cells were αβ TCR T cells. Taken together, the data suggest that the mESC-derived EpCAM1+ cells can form normal thymic architecture that supports T-cell development in vivo.

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Figure 3. Mouse ESC (mESC)-derived EpCAM1+ cells form normal thymic epithelial cell (TEC) architecture and support thymocyte development in vivo. (A): Immunohistochemical analysis of TC-1 mESC-derived EpCAM1+ cell and CD4 CD8CD45+ thymocyte aggregates that were transplanted under the kidney capsule. Six weeks later, K8+K5 cortical TECs (green), K5+K8 medullary TECs (red), and K5+K8+ thymic epithelial progenitors (yellow, arrow) are shown. Scale bar = 50 μm. (B): Flow cytometric analysis of thymocytes recovered from the grafts of EpCAM1 and EpCAM1+ cell aggregates. (C): Expression of αβ and γδ TCR by CD3+ cells from the grafts of EpCAM1+ cell aggregates. Abbreviations: K, keratin; TCR, T-cell receptor.

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A Single mESC-Derived TEP Can Generate Cortical and Medullary Thymic Epithelial Cells In Vivo

The cortical and medullary epithelial cells in the kidney grafts of EpCAM1+ cell reaggregates may have been formed by the differentiation of the mESC-derived TEPs and/or the expansion of small numbers of K8+K5 cortical and K8K5+ medullary epithelial cells among the mESC-derived EpCAM1+ cells. To determine whether the mESC-derived TEPs can give rise to cortical and medullary epithelial cells in vivo, we injected single EGFP+ EpCAM1+ cells into irradiated thymic fragments and implanted these fragments under the kidney capsule. Of 10 injected grafts, 4 were found to contain EGFP+ cells 6 weeks later (Fig. 4A). Immunohistochemical staining showed that the EGFP+ cells in three of the four recovered grafts were composed of three cell subsets: K5+K8+ TEPs, K5K8+ cortical epithelial cells, and K5+K8 medullary epithelial cells (Fig. 4B). Hence, it appears that the mESC-derived TEPs are able to self-renew and to differentiate into thymic cortical and medullary descendants in vivo.

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Figure 4. A single mouse ESC (mESC)-derived EpCAM1+ cell develops into all known thymic epithelial cell types. A single GFP+ EpCAM1+ cell was injected into an irradiated thymus fragment that was subsequently transplanted under the kidney capsule of syngeneic mice. (A): Multiple GFP+ cells are present in the thymus fragment 6 weeks later. Scale bar = 10 μm. (B): K8+K5+ thymic epithelial progenitors (upper panel), K8K5+ medullary epithelial cells (middle panel), and K8+K5 cortical epithelial cells (lower panel) from a single GFP+ EpCAM1+ cell are seen. Each panel displays a single cell. Scale bar = 5 μm. Abbreviations: GFP, green fluorescent protein; K, keratin.

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mESC-Derived EpCAM1+ Cells Enhance Thymocyte Reconstitution

To determine whether the ESC-derived EpCAM1+ cells are functional, lethally irradiated syngeneic mice were injected i.v. with purified mESC-derived EpCAM1+, EpCAM1 cells, or PBS in combination with TCD-BM. Thymic cellularity was analyzed 1 month later. The number of thymocytes in the EpCAM1+ cell-treated mice slightly exceeded that in the EpCAM1 cell- or PBS-treated mice, but the differences did not reach statistical significance (data not shown). To determine whether the EpCAM1+ cells can migrate to the thymus, 5 × 105 green fluorescent protein (GFP)+ EpCAM1+ cells were injected i.v. into irradiated mice, and the thymi were analyzed for GFP+ cells by flow cytometry 1–4 days later. No GFP+ cells were detected in the thymi (data not shown), suggesting that the mESC-derived EpCAM1+ cells could not migrate from the blood to the thymus, although the entrance of very low numbers of cells could not be excluded.

The mESC-derived EpCAM1+ and EpCAM1 cells (or PBS) were then injected intrathymically into irradiated syngeneic mice followed by i.v. injection of TCD-BM the next day. As shown in Figure 5A, 1 month after BMT, the number of thymocytes in the EpCAM1+ cell-treated mice exceeded those in the EpCAM1 cell- or PBS-treated mice by twofold (p < .05) and approximated those in the normal non-BMT control mice. This was also true for the CD4 and CD8 double-positive (DP), and CD4+ and CD8+ single-positive (SP) stages of thymopoiesis (Fig. 5A). Importantly, the number of CD45 major histocompatibility complex (MHC) II+ thymic epithelial cells also was significantly increased in the mice that received EpCAM1+ cells intrathymically, compared with those in the EpCAM1 cell- or PBS-treated mice (Fig. 5A).

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Figure 5. Mouse ESC (mESC)-derived EpCAM1+ cells enhance thymocyte reconstitution after BMT. (A): Lethally irradiated syngeneic mice were injected intrathymically with 5 × 104 mESC-derived EpCAM1+ cells, EpCAM1 cells, or PBS, and injected intravenously (i.v.) with T-cell-depleted bone marrow (TCD-BM; 2 × 106). One month later, the numbers of thymocytes and the numbers of CD4 and CD8 DN, DP, and SP thymocytes were analyzed by flow cytometry. Means ± SD of five mice per group. *p < .05, compared with PBS-treated mice. (B): Lethally irradiated C57BL/c (Ly5.1) mice were injected intrathymically with 5 × 104 green fluorescent protein (GFP)+ mESC-derived EpCAM1+ cells and i.v. with congeneic (Ly5.2) TCD-BM (2 × 106). One month later, the expression levels of CCL25, Delta, MHC I, and MHC II by the GFP+ TECs (solid line) were analyzed, and compared with those by the CD45 EpCAM1+ TECs from normal mice (dotted line). Gray filled line represents isotype controls. (C): Irradiated MHC II knockout mice were injected intrathymically with 5 × 104 TC-1 mESC-derived EpCAM1+ cells or PBS, and injected i.v. with TCD-BM (2 × 106). One month later, the thymocytes were analyzed for the expression of CD4 and CD8 by flow cytometry. Abbreviations: BMT, bone marrow transplant; DN, double negative; DP, double positive; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; SP, single positive.

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To confirm that most of the expanded thymocytes and TECs in the preceding experiments were of donor origin, lethally irradiated C57BL/6 mice were injected intrathymically with GFP+ mESC-derived EpCAM1+ cells and i.v. with congeneic (Ly5.2) TCD-BM. One month later, the thymi were analyzed for the percentages of donor- and host-origin cells. The results showed that 84.4% (±11.3) of the total thymocytes were of donor origin, and 67% (±5) of the thymic epithelial cells were of mESC donor origin. Further analysis revealed that the mESC donor-origin TECs expressed CCL25, Delta-like Notch ligands [42], and MHC I and MHC II molecules (Fig. 5B), all of which are involved in attracting T-cell progenitors to and/or supporting T-cell development in the thymus. The expression levels of these molecules by the mESC-derived TECs were comparable with those by the TECs from normal mice. To determine whether the EpCAM1+ TECs directly mediate thymocyte development, MHC II knockout mice were injected intrathymically with mESC-derived EpCAM1+ cells or PBS, and injected i.v. with TCD-BM. As shown in Figure 5C, only the mice that received an mESC-derived EpCAM1+ cell transplant efficiently generated (and/or promoted survival of) CD4 SP thymocytes, when analyzed 1 month later. Taken together, these data indicate that mESC-derived EpCAM1+ cells increase the number of TECs in the irradiated thymus, and thereby enhance thymocyte reconstitution following BMT.

mESC-Derived EpCAM1+ Cells Increased Peripheral (Splenic) T Cells Derived from Immature Progenitors

To determine whether enhanced thymopoiesis induced by EpCAM1+ cell treatment resulted in higher number of peripheral T cells, 1 month after BMT, the mice that were injected intrathymically with the EpCAM1+ or EpCAM1 cells (or PBS) as described in Figure 5A were analyzed for the numbers of CD4+ and CD8+ T cells in the spleen. As shown in Figure 6A, the numbers of both CD4+ and CD8+ T cells were significantly increased in the EpCAM1+ cell-treated mice, compared with those in the EpCAM1 cell- or PBS-treated mice. Peripheral naive T cells that newly derived from the thymus have been reported to have a phenotype of CD45RBhiCD44lo [8, 9, 43]. We then examined the numbers of CD45RBhiCD44lo cells in the splenic CD4+ T cells from the recipients. As shown in the Figure 6B, the EpCAM1+ cell-treated mice had significantly higher number of CD45RBhiCD44lo cells than the EpCAM1 cell- or PBS-treated mice. The data indicate that mESC-derived EpCAM1+ cells enhanced recovery of naive splenic CD4+ and CD8+ T cells derived from the thymus, in agreement with the data that the EpCAM1+ cell treatment enhanced thymopoiesis.

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Figure 6. Mouse ESC-derived EpCAM1+ cells increase the number of naive splenic CD4+ and CD8+ T cells after BMT. Mice were treated as in Figure 5A and (A) the numbers of total CD4+ and CD8+ T cells and (B) the numbers of naive CD45RBhiCD44loCD4+ T cells in the spleen were determined 1 month later. Means ± SD. *p < .05, compared with PBS-treated group. Abbreviations: BMT, bone marrow transplant; PBS, phosphate-buffered saline.

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Peripheral T Cells from the EpCAM1+ Cell-Treated Mice Are Functional

Functionally mature T cells are characterized by the ability to proliferate and produce cytokines following stimulation with T-cell mitogens and alloantigens [42]. To determine whether the splenic T cells in the mice treated with EpCAM1+ cells followed by TCD-BM transplantation are mature and functional, 1 month after BMT, the splenic CD3+ T cells were stimulated with concanavalin (Con A). As shown in Figure 7A, the cell proliferation after Con A stimulation in the splenic T cells from the EpCAM1+ cell-treated mice was higher than that in the T cells from the EpCAM1 cell- or PBS-treated mice. Similarly, in a third-party mixed lymphocyte reaction, the cell proliferation in response to alloantigens was higher in the splenocytes from the EpCAM1+ cell-treated mice than those from the EpCAM1 cell- or PBS-treated mice (Fig. 7B). We also analyzed cytokine response and found that a significantly greater fraction of splenic CD4+ and CD8+ T cells from the EpCAM1+ cell-treated mice was able to produce IFN-γ than those from the EpCAM1 cell- or PBS-treated mice (Fig. 7C). These data indicate that the peripheral T cells in the EpCAM1+ cell-treated mice are mature and functional.

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Figure 7. The increased numbers of peripheral T cells from EpCAM1+ cell-treated mice are functional. Mice were treated as in Figure 5A and the splenic T cells were stimulated with (A) concanavalin (4 μg/ml) and (B) alloantigens (from irradiated BALB/c splenocytes), respectively. Cells were pulsed with 3H-thymidine (1 μCi/ml) 12 hours before harvesting, and the cell proliferation was determined by [3H]-thymidine incorporation. Data are shown as stimulation index. (C): Splenic cells were incubated with phorbol 12-myristate 13-acetate (10 ng/ml) and ionomycin (2 μm) for 5 hours and Brefeldin A (10 μg/ml) was added at 2 hours. The percentages of CD4 and CD8 T cells that expressed IFN-γ were determined. Abbreviations: IFN-γ, interferon γ; PBS, phosphate-buffered saline.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

We have demonstrated that mESCs can be induced to differentiate into EpCAM1+ cells that contain a population of cells expressing markers for both thymic cortical and medullary epithelial cells, a characteristic of TEPs [20–22]. Purified mESC-derived EpCAM1+ cells could reconstitute the normal thymic architecture in vivo, forming both cortical and medullary areas; and a single mESC-derived EpCAM1+ cell could give rise to three types of epithelial cells: K5+K8+ TEPs, K5K8+ thymic cortical cells, and K5+K thymic medullary cells. After being transplanted into the thymus of irradiated mice, followed by TCD-BMT, mESC-derived EpCAM1+ cells reconstituted the thymus epithelium, thereby enhancing thymopoiesis and increasing the numbers of functional peripheral T cells. Taken together, these data indicated that mESCs can be induced to differentiate in vitro into TEPs, which can self-replicate and differentiate into cortical and medullary epithelial cells capable of supporting T-cell development when placed in vivo. To our knowledge, this is the first report of the selective generation of TEPs by ESCs, although ESCs have been reported to differentiate into various types of progenitors and cells [23].

Although the same combination and concentration of growth factors could be used to induce the differentiation of mESCs into EpCAM1+ cells, we found that significantly larger numbers of EpCAM1+ cells were generated in monolayer cultures than in cultures of embryoid bodies, possibly because the growth factors could more efficiently reach the mESCs in the former cultures. Another difference between the two culture systems was that the plates used for the monolayer culture were precoated with collagen IV. It may be significant, as extracellular matrix has been reported to enhance epithelial differentiation from ESCs [44, 45]. Therefore, it will be important to determine whether other extracellular matrix constituents, such as laminin, fibronectin, collagen I, and gelatin, can further enhance the differentiation of mESCs into TEPs.

TEPs have been proposed to have a phenotype of K5+K8+. However, keratins are intracellular antigens, and cells have to be permeabilized before the antibody staining. Therefore, the patterns of the keratins allow only phenotypic analysis, and do not allow isolating live cells for functional studies. Recently, Rossi et al. [22] used the cell surface marker EpCAM1 to isolate cells from the embryonic thymus and showed that a single EpCAM1+ cell could develop into cortical and medullary epithelial cells in vivo. We show here that a large proportion of mESC-derived EpCAM1+ cells coexpress K5+K8+, whereas smaller proportions have K5K8+ and K5+K8 phenotypes, characteristic of cortical and medullary TECs. These results are consistent with reports that initial TEC development from TEPs can occur without thymocyte-derived signals [46]. However, it has also been shown that the interaction between T cells and TECs is reciprocal. TECs support T-cell development. In return, subsets of thymocytes also play a role in the development and maintenance of the cortical and medullary TECs [1, 2, 46–48]. It has been suggested that CD4CD8 early thymocytes affect the formation of the thymic cortex, whereas mature αβ TCR+ thymocytes affect the development of the medulla [40, 49, 50]. We have observed that mESC-derived EpCAM1+ cell aggregates did not efficiently form normal thymic architecture when implanted under the kidney capsule in the absence of CD4CD8CD45+ thymocytes. Therefore, the presence of immature T cells may significantly enhance the differentiation of the mESC-derived TEPs. It is also possible the mESC-derived TECs induce the differentiation of the T cells that in turn affect the further differentiation of the mESC-derived cells. Thus, the in vitro generation of mESC-derived TEPs should provide a useful system with which to study the developmental interactions between thymocytes and TECs in detail. Toward this end, we have demonstrated that the EpCAM1+-derived TECs express CCL25, Delta-like Notch ligands, and MHC I and MHC II molecules, and it will be important in future studies to trace the patterns of expression of adhesion molecules, chemokines, cytokines, and other molecules in the stromal cell niches that regulated the complex process of thymopoiesis [1, 2, 32–34, 51–58].

In addition to providing insights into the molecular events underlying TEC and early T-cell development, the ability to selectively induce the development of TEPs in vitro from mESCs has important clinical implications regarding the prevention and/or treatment of primary and secondary T-cell immunodeficiencies. For example, degeneration of TECs is one of the major factors in the procession of “physiological” thymic involution, resulting in decreased number, TCR diversity, and functional activities of T cells in elderly patients [2]. Various genetic and infectious diseases (such as AIDS) are associated with T-cell deficiencies, as is intensive chemotherapy or radiotherapy of cancer. Such T-cell deficiencies contribute to increased morbidity and mortality from opportunistic infections, and contribute to the occurrence and relapse of cancers [7]. Similarly, preparative regimens for foreign tissue or organ transplants often result in severe lymphopenia, and the recovery of T cells after these treatments or after hematopoietic stem cell transplant is slow and incomplete. Consequently, our demonstration that the injection of mESC-derived EpCAM1+ cells intrathymically or under the kidney capsule can increase the number of cortical and medullary TECs and enhance thymopoiesis and the generation of peripheral T cells holds promise for correcting these immunodeficiency syndromes. Furthermore, such ESC-derived TECs might also prove useful in the induction of tolerance to ESC-derived organ or tissue transplants [59, 60]. It would be especially useful in this regard if the mESC-derived EpCAM1+ cells could be engineered to migrate to the thymus.

Summary

We have demonstrated that mESCs can be induced to differentiate into cells with the phenotypes of TEPs that could form normal thymic architecture and develop into all types of thymic epithelial cells in vivo. These ESC-derived cells, when injected into the thymus of lethally irradiated mice followed by bone marrow transplantation, enhanced thymocyte reconstitution and increased the number of functional naive splenic T cells derived from the thymus. Therefore, mESCs can be selectively induced in vitro to generate TEPs that are able to further differentiate into cortical and medullary TECs and support T-cell development in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

We thank Dr. C Guo (Gene Targeting & Transgenic Facility in the University of Connecticut Health Center) and Dr. Q Ying (University of Southern California) for kindly providing TC-1 and E14TG2a-Tau-GFP mESCs, respectively.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  9. References
  10. Supporting Information

Additional supporting information available online.

FilenameFormatSizeDescription
STEM_238_sm_suppinfotable1.doc31KTable 1. Primer sequences used for qRT-PCR

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