Isolation and characterization of UC-MSCs

The umbilical cords were obtained from local maternity hospitals with donors' informed consent. Human tissue collection for research was approved by the institutional review board of the Alliancells Institute of Stem Cells and Translational Regenerative Medicine. UC-MSCs were isolated and cultured from umbilical cord tissue by a tissue explant method according to a previous protocol.¹⁶ The UC-MSCs were incubated in DMEM-F12 medium containing 10% fetal bovine serum (FBS) in a humidified incubator maintained at 37 °C in a 5% CO₂ humidified atmosphere, and once 70%–80% confluence had been reached, the cells were detached with 0.1% trypsin-EDTA.

The phenotype of MSC was analyzed by flow cytometry using the following monoclonal antibodies: CD34-FITC, CD45-PE, HLA-DR-PE, CD90-PE CD73-PE (Becton Dickinson Biosciences, San Jose, CA, USA) and CD105-PE (Tianjin Sungene Biotech, Tianjin, China). A nonspecific IgG1 isotype was used as a control. For CD248 analysis, MSCs were incubated with rabbit anti-human CD248 polyclonal antibody (Abnova, Taipei, Taiwan) for 30 min and then stained with goat anti-rabbit IgG-FITC (Invitrogen) for 30 min. Samples were analyzed using a FACS Calibur flowcytometer (Becton Dickinson Biosciences).

For osteogenic and adipogenic differentiation, MSCs were seeded in 24-well tissue culture plates (Corning Incorporated, Corning City, NY, USA), the culture medium was changed with a StemPro Osteogenesis/Adipogenesis Differentiation Kit (Invitrogen), and the cells were cultured for 2–3 weeks. Osteogenesis and adipogenesis differentiation was analyzed by Alizarin Red-S and Oil-Red-O (Sigma, St Louis, MO, USA), respectively.

Histology and immunofluorescence staining

Freshly removed thymus tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan), frozen in a freezing microtome (Leica microsystems, NuBloch, Germany) and then cut into 4 µm-thick frozen sections. Serial sections were air dried and fixed in ice-cold acetone for 10 min and were then stained by hematoxylin and eosin staining for histological analysis. Freshly cut frozen sections were fixed in ice-cold acetone for 10 min, then washed using 1× PBS for 5 min three times. Five percent bovine serum albumin (BSA) was used as a blocking buffer, and then the specimens were incubated overnight at 4 °C with the following primary antibodies: anti-mouse Keratin 5 (Covance Research Products, Berkeley, CA, USA), Troma-1 mAb (rat anti-Keratin 8; Rolf kemler, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), PE ant-mouse CD4 (BD Pharmingen, San Diego, CA, USA) and FITC anti-mouse CD8 (Abcam, Cambridge, MA, USA) as well as biotinylated UEA-1 (Vector Laboratories, Burlingame, CA, USA)with strepavidin-PE (BD). Control slides were incubated with the corresponding isotype antibodies PE-rat IgG1 and FITC-rat IgG1 (BD). After washing, sections were incubated for 30 min with directly conjugated secondary reagents. DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride) was used in the final step for nuclear staining (Vector Laboratories, Burlingame, CA, USA). Microscopic analysis was performed with an Olympus IX51 microscope (Olympus, Center Valley, PA, USA).

Flow cytometry

Freshly isolated suspension thymocytes, splenocytes or peripheral blood cells (10⁶) were used for each sample. Cells were blocked for 15 min at 4 °C with purified anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA) and then stained using the following antibodies: PE anti-mouse CD4 (BD), FITC anti-mouse CD8a (BD), FITC anti-mouse CD4 (BD), APC anti-mouse CD25 (BD), PE anti-mouse Foxp3 (eBioscience) and Foxp3 Staining Buffer Set (eBioscience). Cells were analyzed on a flow cytometer (FACS Calibur; BD).

Thymic stromal cells were isolated by digesting thymic lobes with collagenase I (0.25 mg/ml+1 mg/ml)+NB4 (Serva, Heidelberg, Germany) and DNase I (10 µg/ml) for 45 min and were then centrifuged at 1000 r.p.m. for 5 min. Trophoblast stem cell were washed and stained with anti-CD45–APC (BD), anti-MHC class II-FITC (eBioscience), and anti-UEA-1–biotin (Vector Laboratories,) followed by streptavidin-PE (BD) for flow cytometry analysis.

T-cell receptor excision circle (TREC) analysis

Spleens were weighed and frozen at −80 °C for later analysis. Murine Rec-Jα-TRECs were determined using real time PCRas described previously.¹⁷ Total DNA was extracted and purified with the DNeasy Blood & Tissue Kit (Qiagen, Milan, Italy). The sequences of the primers are as follows: mRec primer(5′-GGGCACACAGCAGCTGTG-3′), Jαprimer (5′-GCAGGTTTTTGTAAAGGTGCTCA-3′) and mRec-Jα fluorescent probe (5′-FAM-CACAAGCACCTGCACCCTGTGCA-TAMRA-3′) (Invitrogen).¹⁸ The PCR reaction contained, 1500 ng DNA, 0.5 µM of each primer, 0.1 µM fluorescent probe and Taqman mix (Roche Applied Science, Penzberg, Upper Bavaria, Germany). Amplifications were performed in triplicate on a LightCycler 2.0 (Roche) at 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 35 s, 72 °C for 1 s and a final step at 40 °C for 30 s.

Thymus development-associated cytokine expression in UC-MSCs

Total RNA was extracted using Trizol reagents, and cDNA was synthesized by using ReverTra Ace qPCR Reverse Transcriptase MIX Kit (Toyobo Co., Ltd., Osaka, Janpan).The primers were synthesized by Invitrogen as follows: KGF forward primer: 5′-AAGAACTGCCCTTCCTCA-3′, reverse primer: 5′-AAGTCCTCCTCTGCCCTC-3′ ghrelin¹⁹ forward primer: 5′-CCATGCCCTCCCCAGGGACC-3′, reverse primer: 5′-ATCACTTGTCGGCTGGGGCCTC-3′ IL-15²⁰ forward primer: 5′-ATGGGTTCTTGGTCTCAC-3′, reverse primer: 5′-ACTCAAAGCCACGGTAAA-3′ growth hormone-1 forward primer: 5′-CCTACCAGGAGTTTGAAGAA- 3′, reverse primer: 5′-CAGGCTGTTGGCGAAGAC-3′ growth hormone receptor forward primer: 5′-TAGTATTTCCCATTTATCGC-3′, reverse primer: 5′- TGTTGTCAGGCTGTTGTG-3′ and IL-7 forward primer: 5′-CCCCTTGGGATGGATGAA-3′, reverse primer: 5′-ATGATGACCGCAACTGGA-3c. The first-strand cDNA was amplified using PCR in 50 µl reactions with the following program: denaturation for 5 min at 95 °C, followed by 30 amplification cycles of 1 min at 95 °C, 30 s at 55 °C and 30 s at 72 °C, and a 5 min final extension at 72 °C. RT-PCR products were separated on 1.5% agarose gels and visualized by staining with NA-Red (Beyotime Biotechnology, Nantong, JiangSu, China) and photographed using a ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA). The relative mRNA expression of each gene was normalized to β-actin (forward primer: 5′-CCTGGCACCCAGCACAAT-3′, forward primer: 5′-GGGCCGGACTCGTCATAC-3′).

UC-MSCs derived from four different donors were seeded onto Nunc Lab-Tek II Chamber Slide System (ThermoFisher Scientific, Waltham, Massachusetts), 2×10⁴ cells/well. After 24 h, cells and frozen sections of the thymic tissues were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% normal donkey serum, and were then incubated overnight at 4 °C with goat anti-human KGF/FGF7 antibody (R&D Systems, Minneapolis, MN, USA) at 10 µg/ml. After washing, sections were incubated for 60 min with an Alexa Fluor 488 donkey anti-goat secondary antibody (Invitrogen). DAPI was used in the last step for nuclear staining. Microscopic analysis was performed with a spinning-disk confocal microscope (Nikon Imaging, Tokyo, Japan).

CellTracker CM-DiI-labeled UC-MSCs injection into Foxn1^−/− mice

CellTracker CM-DiI (Invitrogen, Carlsbad, CA, USA) was used to label UC-MSCs according to the labeling procedure.²¹ Foxn1^−/− mice were injected intraperitoneally with CM-Dil-labeled UC-MSCs, 2×10⁶ cells/mice. The mice were then killed at 24 h, 72 h and 168 h after injection, respectively. Thymic tissues were taken and immediately frozen to obtain frozen sections. DAPI was used to stain nuclear and microscopic analysis was performed with an Olympus IX51 microscope.

UC-MSCs promote the enlargement of the thymic rudiment in Foxn1^−/− mice

The Foxn1^−/−gene mutation results in abnormalities of thymic development, and the thymus-like organ in Foxn1^−/− mice is composed of condensed lymphatic-like tissue. The structure is surrounded by a thin fibrous capsule and a poorly demarcated sinusoidal space underneath the capsule with mature adipose tissue on the medial side.¹⁵ To study whether MSCs promote thymic development, we analyzed thymic tissue in terms of size and histological morphology with H&E staining in mice from both the treated and untreated groups at different time points. There were no significant differences in the appearances and weights of thyme between the groups. However, H&E staining revealed that a larger area of full normal thymic structure and a more organized corticomedullary architecture was clearly observed in the treated mice compared to the untreated group (Figure 2). These data strongly indicate that MSCs can rebuild the thymic architecture of the Foxn1^−/− mice at as early as 6 weeks after their administration and continued to improve after repetitive cell infusion.

Open in a separate window

Figure 2

UC-MSCs promote thymic enlargement in Foxn1^−/− mice. Hematoxylin and eosin-stained frozen sections of thymi from untreated, treated and normal BALB/c mice at different time points. Untreated nude mice thymi showed completely disorganized CMJs. After UC-MSC administration, thymi were greatly enlarged, and the corticomedullary architecture became organized in the treated thymus. CMJ, corticomedullary junction; US-MSC, umbilical cord-derived mesenchymal stem cell.

UC-MSC administration increases thymopoiesis and enhances thymic T-cell export

Because the thymic architecture clearly improved with time after the UC-MSCs infusions (Figure 2), the effect of UC-MSCs on thymopoietic capacity was further investigated. Although in normal thymic tissue the majority of thymocytes are CD4/CD8 double positive, in Foxn1^−/− mice, the double-positive T cells were almost absent due to a lack of interactions between the thymic epithelium and the thymocyte progenitors. After UC-MSC administration, however, the CD4⁺CD8⁺ thymocytes expanded with only a few single-positive cells appearing in the medulla (Figure 3a). Additionally, the total thymic cellularity of the treated Foxn1^−/− mice increased by 2.5-fold compared to the untreated mice by 8 weeks after UC-MSC administration (Figure 3b). An additional flow cytometric analysis revealed that the absolute numbers of thymic T-cell subtypes also increased significantly after treatment (Figure 3c–e).

Open in a separate window

Figure 3

UC-MSCs increase thymocyte numbers in Foxn1^−/− mice. (a) The distribution of CD4⁺ (red) and CD8⁺ (green) thymocytes in thymi. The orange area represents double-positive cells (CD4⁺CD8⁺). Single-positive cells appeared in the medulla. (b–e) Thymi were analyzed by flow cytometry 8 weeks after administration. (b) Total thymocyte numbers (cells×10⁶). (c–e) Total numbers of CD4⁺CD8⁻, CD4⁻CD8⁺, and CD4⁺CD8⁺cells per thymus, respectively. Mean±s.d.; *P<0.05 versus untreated controls, with five mice per group for each time point. US-MSC, umbilical cord-derived mesenchymal stem cell.

TRECs are generated during T-cell receptor gene recombination and can be used as a quantitative measurement of the output of newly generated naive T cells.²² To further evaluate the thymocyte efferent from the newly developed thymi, we analyzed the expression of TRECs in the splenocytes, peripheral blood cells and thymic stromal cells. As shown in Figure 4a–c, the relative expression levels of TRECs in thesplenocytes and peripheral blood cells were significantly increased in the treated Foxn1^−/− mice compared to the untreated group, although the TREC expression levels in the thymic stromal cells did not change significantly. These results demonstrated that administration of UC-MSCs could increase thymopoiesis and the emigration of recently generated T cells.

Open in a separate window

Figure 4

UC-MSCs enhance thymic T-cell export in Foxn1^−/− mice. (a–c) Relative expression of TRECs was enumerated after treatment: in spleens (a), in peripheral blood (b) or in thyme (c). There were significantly more TREC⁺ splenocytes in the 8-week treated group than in the untreated group, 6 mice/group, *P<0.05. (d–e) The percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells within CD4⁺ T cells in the thymi (d) or in the peripheral blood (e). The surface phenotype of cells gated on CD4 expression was analyzed. The percentage of Tregs increased after UC-MSC treatment in the 8-week treatment group, 6 mice/group, *P<0.05. TREC, T-cell receptor excision circle; Treg, regulatory T cells; US-MSC, umbilical cord-derived mesenchymal stem cell.

UC-MSCs increase Treg cells in the thymi and peripheral blood of Foxn1^−/− mice

CD4⁺CD25⁺Foxp3⁺ Treg cells, which represent 5%–10% of peripheral CD4⁺ T cells in healthy adult mice,²³ play a key role in maintaining self-tolerance and immune balance by suppressing the effector functions of CD4⁺ T cells.²⁴ Accumulating data have demonstrated that the deficiency of CD4⁺CD25⁺Foxp3⁺ Treg cells is closely correlated with the development of autoimmune diseases.²⁵ To assess the variation of Treg cells before and after MSC infusion, we analyzed the percentage of Treg cells in the peripheral blood and the absolute numbers of Treg cells in the thymus. As shown in Figure 4d–e, both the percentage of Treg cells in the peripheral blood and the absolute numbers of Treg cells in the thymus were significantly increased in treated Foxn1^−/− mice compared to the corresponding untreated group.

The reconstitution of the cortex/medulla and the differentiation of TECs in the Foxn1^−/− thymus

Foxn1^−/− mice have very small thymi with an abnormal architecture that lacks cortical and medullary domains, and in which most TECs display an intermediate progenitor phenotype.^26,27 Keratin 8 (K8) and keratin 5 (K5) are markers of the cortical and medullary areas of TECs, respectively, and can be used to characterize TEC localization and organization. In the normal thymus, medullary TECs form compact medullary compartments (K5⁺) that are surrounded by K8⁺ cells in the cortex. In our study, we analyzed thymic organization over periods of 6, 8 or 10 weeks after UC-MSCs infusions. Anti-keratin staining demonstrated that the untreated thymi fromFoxn1^−/− mice consisted of clusters of epithelial cells. In contrast, the thymi of the treated groups showed well-organized cortexes (K5⁻K8⁺ TECs) and distinct medullary regions (K5⁺K8⁻ TECs). In addition, in the treated thymi, TECs displayed a dense mesh-like structure in both the cortical and medullary regions, and the structure in the corticomedullary junction was close to normal (Figure 5). This finding indicated that MSCs may promote TEC differentiation and regulate their proliferation to rebuild the thymic architecture of Foxn1^−/− mice.

Open in a separate window

Figure 5

Progressive thymic epithelium cells in the groups. Cryosections were stained for keratin 8 (red) and keratin 5 (green). Scale bar=100 µm. In the 6-week groups, treated thymi showed more keratin 8⁺ and keratin 5⁺ thymic epithelial cells than the untreated thymi, but no organization in the cortical or medullary regions was observed. In the 8-week groups, treated thymi revealed a tendency towards an organized corticomedullary architecture. In the 10-week groups, treated thymi display an organized corticomedullary architecture and a corticomedullary junction similar to normal thymi.

UC-MSCs lead to the maturation of medullary epithelial cells in Foxn1^−/− mice

Because UEA-1⁺ TECs, which represent the mature medullary TEC subset, were reported to be associated with medullary compartment organization,²⁸ and UEA-1⁺ TECs never developed in Foxn1^−/− mice,²⁷ we further analyzed the expression of UEA-1⁺ TEC cells to assess whether UC-MSCs altered the thymic microenvironment at the cellular level. Well-organized UEA-1⁺ cells appeared in the medulla of the treated thymi that appeared similar to normal thymi, whereas only a few UEA-1⁺ cells were observed scattered in the untreated Foxn1^−/−thymus (Figure 6a). Because MHC class IIis regulated by Foxn1andrepresents functional maturation for TECs,²⁹ we further analyzed two stromal cell subsets defined as UEA-1⁺MHCII^hi and UEA-1⁺MHCII^lowcells. Although a previous study demonstrated that Foxn1^−/− stromal cells were MHC class II-negative/low,³⁰ in our study, both subsets of UEA-1⁺MHCII^low and UEA-1⁺MHCII^hi cells were detected by flow cytometry after MSC infusions (Figure 6b). Taken together, those data highly suggested that MSC administration may affect the differentiation of epithelial progenitor cells leading to an expansion of both mature and immature TECs.

Open in a separate window

Figure 6

UC-MSCs increase UEA-1⁺ TECs in Foxn1^−/− mice. (a) Frozen thymic sections were stained with DAPI (blue) and UEA-1 (red). Scale bar=100 µm. The treated thymus has more UEA-1⁺ TECs within medullary regions than that of the untreated 6-week group. (b) Summary of the percentage of UEA-1⁺MHC^lo and UEA-1⁺MHC^hi TECs within CD45⁻ stromal cells analyzed by flow cytometry. Gated CD45⁻ stromal cells were analyzed and revealed that both subpopulations significantly increased in the treated thymi compared to the untreated thymi at 8 weeks post UC-MSC administration (6 mice/group), *P<0.05. DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; TEC, thymic epithelial cell; US-MSC, umbilical cord-derived mesenchymal stem cell.

We thank Professor Dr Martin John Evans (University of Cardiff, UK) for constructive instruction and all members of the laboratory for their valuable discussion. The authors declare no competing financial interests. This work was supported by the National Natural Science Foundation of China (Grant: 30872618) and State High-tech Research and Development Plans (Grants: 2011AA020103 and 2011AA020109).