Parathyroid Hormone Enhances Hematopoietic Expansion Via Upregulation of Cadherin-11 in Bone Marrow Mesenchymal Stromal Cells

Authors


Abstract

Parathyroid hormone (PTH) stimulates hematopoiesis in mouse models. The involvement of osteoblasts in this process has been well investigated; however, the effects of PTH on human hematopoiesis and bone marrow mesenchymal stromal cells (BM-MSCs) are unclear. Here, we show that BM-MSCs contribute to the hematopoiesis-stimulating effects of PTH via upregulation of cadherin-11 (CDH11). When culture-expanded human BM-MSCs were stimulated with PTH, their ability to expand cocultured CD34+ hematopoietic progenitor cells (HPCs) was enhanced. Furthermore, when PTH-treated BM-MSCs were subcutaneously implanted into NOD/SCID mice, the induction of hematopoietic cells was enhanced. Culture-expanded human BM-MSCs expressed CDH11, and the level of CDH11 expression increased following PTH stimulation. Depletion of CDH11 expression in BM-MSCs using small interfering RNA abolished the enhancement of HPC expansion by PTH-treated BM-MSCs. In lethally irradiated mice that underwent BM transplantation, CDH11 expression in BM-MSCs was higher and survival was better in PTH-treated mice than in control mice. The number of hematopoietic cells in BM and the number of red blood cells in peripheral blood were higher in PTH-treated mice than in control mice. Our results demonstrate that PTH stimulates hematopoiesis through promoting the upregulation of CDH11 expression in BM-MSCs, at least in part. PTH treatment may be an effective strategy to enhance the ability of BM-MSCs to support hematopoiesis. Stem Cells 2014;32:2245–2255

Introduction

Parathyroid hormone (PTH) is a key molecule responsible for regulating blood calcium concentration. A recombinant fragment of human PTH (amino acids 1–34) is clinically available, and administration of this fragment can be used to treat patients with osteoporosis [1]. PTH stimulates hematopoiesis in mouse models, and osteoblasts (OBs) are involved in the mechanisms underlying this effect [2-5]. Constitutive activation of the PTH/PTH-related protein receptor in OBs increases the number of hematopoietic stem cells (HSCs), and ligand-dependent activation of the PTH receptor by exogenous PTH increases the number of OBs and HSCs [4]. However, PTH has not been shown to effectively stimulate hematopoiesis in humans [6].

Bone marrow mesenchymal stromal cells (BM-MSCs) are nonhematopoietic multipotent cells that are capable of differentiating into a variety of cell types including OBs [7-11]. Preclinical and clinical studies have shown that exogenously administered culture-expanded human BM-MSCs support hematopoietic cell engraftment and reconstitution after HSC transplantation [12-16]. In addition, culture-expanded human BM-MSCs can expand hematopoietic cells ex vivo [17, 18]. These expanded hematopoietic cells improve hematopoietic cell engraftment when they are intravenously transplanted [19]. Thus, culture-expanded human BM-MSCs are expected to be promising for cell therapy to support hematopoiesis.

Given that PTH and BM-MSCs facilitate hematopoiesis, we hypothesized that PTH enhances the ability of BM-MSCs to support hematopoiesis. In this study, the effects of PTH on the expansion of CD34+ hematopoietic progenitor cells (HPCs) by culture-expanded human BM-MSCs were examined. We further identified a molecule, cadherin-11 (CDH11), which contributes to the enhanced ability of BM-MSCs to support hematopoiesis following PTH stimulation.

Materials and Methods

Reagents and Antibodies

A recombinant fragment of human PTH (amino acids 1–34) was purchased from the Peptide Institute (Osaka, Japan, http://www.peptide.co.jp). Stem cell factor (SCF), interleukin (IL)-3, and Flt3-ligand (Flt3-L) were purchased from Wako Chemical Industries (Osaka, Japan, http://www.wako-chem.co.jp). Thrombopoietin (TPO) was provided by Kyowa Hakko Kirin (Tokyo, Japan, http://www.kyowa-kirin.com). Phycoerythrin (PE)-conjugated mouse antibodies against human CD15, CD34, CD44, and CD166, a fluorescein isothiocyanate-conjugated mouse antibody against CD45, and an allophycocyanin (APC)-conjugated mouse antibody against human CD34 were purchased from BD Pharmingen (San Diego, CA, http://www.bdbiosciences.com). PE-conjugated mouse antibodies against human CD3, CD11b, CD14, CD19, CD33, CD41a, CD73, CD90, CD105, CD146, and glycophorin A, and an APC-conjugated mouse antibody against human CD38 were purchased from eBioscience (San Diego, CA, http://www.ebioscience.com). An APC-conjugated mouse antibody against alkaline phosphatase (ALP) was purchased from R&D Systems (Minneapolis, MN, http://www.rndsystems.com/) [20]. An anti-CDH11 antibody was purchased from Invitrogen (Carlsbad, CA, http://www.lifetechnologies.com/) and an anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com). The antibodies used in this study are listed in Supporting Information Table S3.

Culture of Human BM-MSCs

Human BM-MSCs were isolated from BM samples obtained from healthy adult volunteers with informed consent. BM-MSCs were isolated based on our previously published method [21, 22]. A single-cell suspension of 1 × 106 BM mononuclear cells (MNCs) were seeded into 15 cm culture dishes, and adherent cells were cultured in advanced-minimal essential medium (MEM; Invitrogen) supplemented with 5% fetal bovine serum (FBS, Invitrogen), 100 µM ascorbic acid (Wako Chemicals Industries), 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Gibco, Carlsbad, CA, http://www.lifetechnologies.com/). The medium was changed on days 7 and 14. Primary cultures were passaged to disperse the colony-forming cells (CFCs) (passage 1). Cells at passage 1–3 were used as BM-MSCs in this study. For some experiments, human BM-MSCs and normal human dermal fibroblasts (NHDFs) were purchased from LONZA (Allendale, NJ, http://www.lonza.com) and BM samples were purchased from AllCells (Emeryville, CA, http://www.allcells.com). Prior to experiments, flow cytometric analysis was performed in which expression of the surface antigens CD11b, CD19, CD34, CD44, CD45, CD73, CD90, CD105, CD146, and CD166 (Supporting Information Fig. S1) was examined to confirm that these cells expressed mesenchymal stromal/stem cell markers, but did not express hematopoietic cell markers [11]. The study protocol was approved by the ethics committee of Kyoto University Hospital.

Coculture of Human HPCs and BM-MSCs

HPCs were isolated from BM-MNCs using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.co.jp). The purity of the enriched CD34+ cell populations was confirmed by flow cytometric analysis using a PE-conjugated antibody against human CD34. Culture-expanded human BM-MSCs (2 × 104 cells/well) were seeded in a 24-well culture plate. For blocking experiments, BM-MSCs were incubated with 30 µg/ml of an anti-human CDH11 antibody (R&D Systems). HPCs (0.6 × 103 cells/well) were then applied and the cells were cocultured in StemSpan Serum-Free Expansion Medium (STEMCELL Technologies, Vancouver, Canada, http://www.stemcell.com) supplemented with 100 ng/ml SCF, 100 ng/ml Flt3-L, 50 ng/ml TPO, and 20 ng/ml IL-3. After 10 days of coculture, the number and surface marker expression of the expanded hematopoietic cells were examined by flow cytometric analysis. In some experiments, the expanded hematopoietic cells were applied to a methylcellulose-based culture. Transwell culture experiments were performed in a 24-well plate Transwell system (Corning, Corning, NY, http://www.corning.com), which contains 6.5 mm inserts and polycarbonate filters with a pore size of 4 µm. A schema of the coculture experiments is shown in Figure 1A. At the start of coculture with HPCs, BM-MSCs stimulated with 0.1 nM PTH for 2 days (BM-pMSCs) and untreated BM-MSCs (BM-uMSCs) showed no apparent mineralization, and expressed low levels of osteocalcin, osteopontin, and osterix. Furthermore, 7.7% of BM-pMSCs and 8.6% of BM-uMSCs were positive for ALP (Supporting Information Fig. S2).

Figure 1.

PTH enhances the ability of BM-MSCs to expand hematopoietic progenitor cells (HPCs). (A): Schema of the experimental procedure used to analyze the effects of PTH on the ability of culture-expanded BM-MSCs to expand CD34+ HPCs. (B, C): The number of CD45+ hematopoietic cells (B) and CD34+ HPCs (C) expanded after 10 days of culture alone (BM-MSCs−) or coculture with BM-MSCs stimulated with the indicated concentration of PTH (BM-MSCs+), as determined by flow cytometric analysis (n = 5). (D–F): The number of CD34+CD38 cells (D) and CD34+CD38+ cells (E) expanded after 10 days of culture alone (BM-MSCs−) or coculture with BM-MSCs stimulated with the indicated concentration of PTH (BM-MSCs+), as determined by flow cytometric analysis (n = 5). (F) Representative counter-plots. Numbers in each box indicate the percentage of cells. The numbers of CD34+ cells (C), CD34+CD38 cells (D), and CD34+CD38+ cells (E) were calculated by multiplying the number of CD45+ cells (B) by the percentages of the respective cell types in each cell fraction (F). **, p < .01. Abbreviations: BM-MSCs, bone marrow mesenchymal stromal cells; PTH, parathyroid hormone.

In Vivo Hematopoietic Marrow Formation Assay

BM-MSCs (2 × 106) were mixed with 40 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic particles (Zimmer Corporation, Warsaw, IN, http://www.zimmer.com) and then implanted subcutaneously into the dorsal surface of 6–8-week-old female NOD/SCID mice, as previously described [21, 23-26]. BM-MSC implants were harvested 8 weeks after implantation, fixed in 2% paraformaldehyde, and then decalcified with buffered 5% EDTA and 4% sucrose prior to paraffin embedding. Paraffin-embedded sections were deparaffinized, hydrated, and stained with hematoxylin and eosin, or immunostained with an anti-mouse CD45 antibody (eBioscience), an anti-mouse Gr-1 antibody (R&D Systems), or an anti-mouse TER-119 antibody (eBioscience), and then visualized using diaminobenzidine. The level of hematopoietic cell induction in BM-MSC implants was quantitated in fields at ×40 or ×100 magnification using Image J software as previously described [23]. Images were acquired using a DP71 digital camera and DP Controller software in combination with a CX41 microscope and a PlanCN objective lens (×10, 0.25 numerical aperture) (Olympus, Tokyo, Japan, http://www.olympus.co.jp). In each field, the surface areas of hematopoietic marrow and tissue coverage were measured using Image J software. The hematopoietic marrow surface area percentage was calculated as follows: (hematopoietic marrow surface area/tissue coverage surface area) × 100. The numbers of CD45+, Gr-1+, and TER119+ cells were counted in the hematopoietic marrow in each field. The number of cells per area (i.e., the density) was calculated using Image J software. Then, the density of these cells in the PTH-treated sample was divided by that of the control sample.

Small Interfering RNA-Mediated Knockdown of CDH11 in BM-MSCs

Lipofectamine RNAiMax (Invitrogen) was used to transfect BM-MSCs with CDH11-targeting small interfering RNA (siRNA) according to the manufacturer's instructions. Lipofectamine RNAiMax (1 µl) was combined with 6 pmol of siRNA in 100 µl of Opti-MEM media (Gibco) and incubated for 20 minutes. The mixture was then added directly to BM-MSCs in culture, and cells were incubated for 24 hours. siRNAs targeting CDH11 were purchased from Invitrogen (Stealth RNAi). The siRNA sequences were as follows: CDH11 number 1, 5′-AGGAAGUAGGAAGAGUGAAAGCUAA-3′; CDH11 number 2, 5′-CAACAUCACUGUCUUUGCAGCAGAA-3′; and CDH11 number 3, 5′-CAUCGUCAUUCUCCUGGUCAUUGUA-3′. The Stealth RNAi Negative Control Medium GC Duplex (Invitrogen) was used as a negative control [27, 28]. BM-MSCs treated with siRNA were cocultured with HPCs.

BM Transplantation

BM cells (5 × 104/mouse) from C57BL/6 mice were injected via the tail vein into recipient C57BL/6 mice that had received 10 Gy of total body irradiation. PTH (4 µg/kg dissolved in phosphate-buffered saline (PBS)) or PBS (control) was administered to recipient mice by subcutaneous injections on days 0, 1, 3, and 5 after BM transplantation. The survival of mice was observed each day until day 30 after BM transplantation. The survival of mice was analyzed using the log-rank test.

Mice

Specific pathogen-free 6–8-week-old female NOD/SCID mice and C57BL/6 mice (Clea Japan, Tokyo, Japan, http://www.clea-japan.com) were used for the in vivo experiments. These studies were approved by the committee on animal research of the Kyoto University Faculty of Medicine.

In Vitro BM-MSC Differentiation Assays

In vitro osteogenic, adipogenic, and chondrogenic differentiation assays using BM-MSCs were performed according to our published procedures [21, 22]. To induce osteogenic differentiation of BM-MSCs, 1.8 mM potassium dihydrogen phosphate, 100 µM ascorbic acid, and 100 nM dexamethasone were added to the culture media. Mineralization was evaluated by 1% Alizarin Red S staining after 4 weeks of osteogenesis-inducing culture. To induce adipogenic differentiation of BM-MSCs, 0.5 mM isobutyl-methylxanthine, 60 µM indomethacin, 0.5 µM hydrocortisone, and 10 µg/ml insulin were added to the culture media. Oil red O staining was used to assess lipid-laden fat cells after 2 weeks of adipogenesis-inducing culture. The size of mineralized areas and the number of oil red O+ cells were quantitated, as previously described [21]. Chondrogenic differentiation of BM-MSCs was assessed by Alcian blue staining of cartilage matrix deposition in aggregate cultures treated with 100 µM ascorbic acid, 2 mM sodium pyruvate (Wako Chemical Industries), 1% insulin/transferrin/selenous acid mixture (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com), 100 nM dexamethasone, and 10 ng/ml transforming growth factor-β (Wako Chemical Industries) for 4 weeks. Images were acquired using a Biozero BZ-8100 microscope (Keyence, Osaka, Japan, http://www.keyence.co.jp) and BZ Viewer software (Keyence) at a magnification of ×100.

Colony-Forming Unit Fibroblast Assay

Mouse BM nuclear cells (1 × 106) were seeded in T-25 flasks and cultured in alpha-MEM supplemented with 20% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 55 µM 2-mercaptoethanol. After 10–14 days of culture, cells were washed twice with PBS, fixed with 2% paraformaldehyde, and stained with 0.1% toluidine blue O. Aggregates containing more than 50 cells were counted as a colony.

Quantitative Real-Time PCR

Total RNA was extracted using the QIAamp RNA Blood Mini Kit (Qiagen Japan, Tokyo, Japan, http://www.qiagen.com) and subjected to reverse transcription. The 20 µl real-time PCR mixture contained Taqman master mix (Roche Applied Science, Basel, Switzerland, https://www.roche-applied-science.com), cDNA, primer pairs, and the Taqman probe (Universal Probe Library). cDNA was amplified with the LightCycler 3.5 system (Roche) using the following parameters: 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control to normalize any loading differences. The primer sets and universal probes used are listed in Supporting Information Table S1.

Statistical Analysis

The unpaired Student's t test was used for analysis, unless otherwise indicated. Data in bar graphs indicate the mean ± SD, and statistical significance is expressed as follows: *, p < .05; **, p < .01; n.s., not significant.

Results

The Ability of BM-MSCs to Expand HPCs Is Enhanced Following Stimulation with PTH

HPCs are expanded when cocultured with BM-MSCs in vitro [17, 18]. Consistent with this, the expansion of human CD45+ cells and CD34+ HPCs was significantly enhanced when cocultured with human culture-expanded BM-MSCs for 10 days in the presence of SCF, Flt3-L, TPO, and IL-3 in vitro, in comparison to when they were cultured alone (Fig. 1A–1C). Detailed flow cytometric analysis showed that the expansion of CD34+CD38 cells and CD34+CD38+ cells was higher when they were cocultured with BM-pMSCs than when they were cocultured with BM-uMSCs (Fig. 1D–1F). The same effects were observed when BM-MSCs from two other individuals were used (Supporting Information Fig. S3). Around 20% of the expanded CD34+ cells in cocultures with BM-pMSCs expressed the myeloid progenitor marker CD33, whereas almost all were negative for hematopoietic lineage markers including CD11b, CD14, CD15, CD41a, glycophorin A, CD3, and CD19 (Fig. 2A). The expression pattern of these surface markers was similar to those of expanded CD34+ cells in cocultures with BM-uMSCs (Fig. 2A). Methylcellulose-based cultures showed that the number of CFCs that developed from the expanded hematopoietic cells was higher when they were cocultured with BM-pMSCs (Fig. 2B, BM-MSCs+, PTH+) than when they were cocultured with BM-uMSCs (Fig. 2B, BM-MSCs+, PTH−). Importantly, the expansion of HPCs was not enhanced when they were cocultured with PTH-treated NHDFs (Fig. 2C). In addition, the expansion of HPCs was not enhanced by PTH treatment alone, in the absence of BM-MSCs (Fig. 2D), which indicates that PTH does not directly affect the expansion of HPCs. Altogether, these data demonstrate that the ability of culture-expanded human BM-MSCs to expand human HPCs in vitro is enhanced following PTH stimulation.

Figure 2.

Flow cytometric and functional analysis of the expanded hematopoietic cells. (A): Representative counter-plots of the expression of surface markers of expanded CD34+ hematopoietic cells after 10 days of culture alone (left panels) or coculture with BM-MSCs stimulated with 0.1 nM PTH (BM-pMSCs, right panels) or unstimulated BM-MSCs (BM-uMSCs, middle panels), as assessed by flow cytometric analysis. (B): The number of CFCs, as assessed by methylcellulose-based culture, that developed from the expanded hematopoietic cells after 10 days of culture alone (BM-MSCs−) or coculture with BM-uMSCs (BM-MSCs+, PTH−) or BM-pMSCs (BM-MSCs+, PTH+). The number of CFCs was calculated by multiplying the number of CFCs per 2 × 103 hematopoietic cells by the number of expanded cells (n = 5). The ratio of the number of CFCs generated in cocultures with BM-uMSCs or BM-pMSCs relative to the number generated when cultured alone is shown. (C): The number of CD45+ hematopoietic cells expanded after 10 days of coculture with NHDFs stimulated with the indicated concentration of PTH, as determined by flow cytometric analysis (n = 5). (D): The number of HPCs expanded after culture alone for 10 days in the presence of the indicated concentration of PTH, as determined by flow cytometric analysis (n = 5). **, p < .01; n.s., not significant. Abbreviations: BM-MSCs, bone marrow mesenchymal stromal cells; CFC, colony-forming cell; NHDF, normal human dermal fibroblast; PTH, parathyroid hormone.

PTH Stimulation Enhances the Induction of Hematopoietic Cells by BM-MSCs In Vivo

We examined whether BM-pMSCs induced more hematopoietic cells than BM-uMSCs in vivo. We previously showed that culture-expanded human BM-MSCs subcutaneously implanted with HA/TCP into immunocompromised mice induce ectopic host-derived hematopoietic marrow formation in vivo (Fig. 3A), and that the hematopoietic cells induced by the BM-MSCs are functionally similar to the hematopoietic cells in the orthotopic BM [25]. When BM-MSCs were subcutaneously implanted with HA/TCP into NOD/SCID mice, the level of hematopoietic cells induced by BM-pMSCs was significantly higher than that induced by BM-uMSCs (Fig. 3B, 3C). Immunohistochemical analysis showed that the levels of multiple lineages of hematopoietic cells were higher in the implants of BM-pMSCs than in the implants of BM-uMSCs, including CD45+ cells (leukocytes; Fig. 3D), Gr-1+ cells (myeloid cells; Fig. 3E, 3F), and TER119+ cells (erythroid cells; Fig. 3G, 3H). Altogether, these data demonstrate that the ability of culture-expanded human BM-MSCs to induce hematopoietic cells in vivo is enhanced following PTH stimulation.

Figure 3.

PTH enhances the induction of hematopoietic cells by culture-expanded human BM-MSCs in vivo. (A): Schema of the experimental procedure used to analyze the effects of PTH on the ability of culture-expanded BM-MSCs to enhance hematopoietic cell induction. Representative microscopic images (B) and quantitative measurement of hematopoietic marrow areas (C) in sections of BM-MSC implants stained with HE (n = 3). Original magnification ×40. Blue arrows indicate hematopoietic marrow formation (HM). B: bone, HA: hydroxyapatite/tricalcium phosphate, Control: BM-uMSCs implants, PTH: BM-pMSCs implants. (D–H): Immunohistochemical staining of sections of BM-MSC implants for CD45 (D), Gr-1 (E, F), and TER119 (G, H). Blue arrows indicate Gr-1+ cells (E) or TER119+ cells (G) in the hematopoietic marrow (HM). Original magnification, ×40. Dotted lines indicate the border between bone (B) and hematopoietic marrow (HM) (E and G). Quantitative measurement of CD45+ (D), Gr-1+ (F), and TER119+ (H) cells in the immunostained sections of BM-MSC implants. The ratio of cell number/area in BM-pMSC implants relative to that in BM-uMSC implants is plotted (n = 3 in D, F, and H). *, p < .05; **, p < .01. Abbreviations: BM-MSCs, bone marrow mesenchymal stromal cells; HA/TCP, hydroxyapatite/tricalcium phosphate; PTH, parathyroid hormone.

Enhancement of HPC Expansion Following PTH Stimulation of BM-MSCs Requires CDH11

We next sought to identify molecules responsible for the enhancement of HPC expansion mediated by BM-pMSCs. First, BM-pMSCs were cocultured with HPCs in separate compartments of a Transwell system. The enhancement of HPC expansion mediated by BM-pMSCs was almost completely abolished when cells were grown in this system (Fig. 4A). This suggests that a direct interaction between BM-pMSCs and HPCs via adhesion molecules is required for BM-pMSCs to enhance the expansion of HPCs. This conclusion is supported by quantitative real-time PCR analysis showing that multiple hematopoiesis-associated soluble factors were not upregulated in BM-pMSCs (Supporting Information Fig. S4A).

Figure 4.

Enhancement of HPC expansion following PTH stimulation of BM-MSCs requires CDH11. (A): The number of HPCs expanded when cocultured with BM-pMSCs (PTH+) or BM-uMSCs (PTH−) in the presence or absence of a Transwell insert. (B): Immunoblot analysis of CDH11 expression in BM-uMSCs (Control) and BM-pMSCs (PTH). (C): Quantitative real-time PCR analysis showing CDH11 mRNA expression in BM-MSCs treated with three different CDH11-specific siRNAs (numbers 1–3). The level of CDH11 mRNA in samples treated with CDH11-specific siRNA is shown relative to that in the control sample. (D): Immunoblot analysis showing CDH11 expression in BM-MSCs treated with three different CDH11-specific siRNAs (numbers 1–3). (E): The number of HPCs expanded when cocultured with BM-pMSCs or BM-uMSCs treated with CDH11-specific siRNAs (numbers 1–3) (n = 3). (F): Immunoblot analysis of CDH11 expression in HPCs and BM-MNCs. In (B), (D), and (F), the density of the CDH11 signal was measured by densitometry and is shown relative to that of β-actin. *, p < .05; n.s., not significant. Abbreviations: BM-MSCs, bone marrow mesenchymal stromal cells; HPCs, hematopoietic progenitor cells; PTH, parathyroid hormone.

Next, microarray analysis was performed on BM-pMSCs to identify adhesion molecules responsible for the ability of these cells to enhance HPC expansion. Among 24,460 genes analyzed in the microarray, 7,578 genes had a normalized value ≥100, which is considered to indicate significant gene expression. Among these 7,578 genes, when the ratios of their expression in BM-pMSCs to that in BM-uMSCs were ranked from highest to lowest, CDH11 came at position number 466 (Supporting Information Data). Several molecules that belong to the integrin and cadherin families were expressed in BM-pMSCs at relatively high levels; however, CDH11 was identified as a candidate molecule because its expression was highly upregulated in BM-pMSCs compared to BM-uMSCs (Supporting Information Fig. S4C). Immunoblot analysis confirmed that CDH11 was expressed in BM-uMSCs and was upregulated in BM-pMSCs (Fig. 4B). To explore the functional role of CDH11 in the enhancement of HPC expansion, gene knockdown experiments were performed using siRNA targeting CDH11. To exclude potential off-target effects, three different CDH11-specific siRNAs were used. The mRNA and protein expressions of CDH11 in BM-MSCs were efficiently depleted by each of these three CDH11-specific siRNAs (Fig. 4C, 4D, numbers 1–3). The enhancement of HPC expansion mediated by BM-pMSCs was inhibited following treatment with siRNA targeting CDH11 (Fig. 4E). This inhibitory effect was observed following treatment with each of the three CDH11-specific siRNAs (Fig. 4E, numbers 1–3). BM-pMSCs that had been incubated with an anti-CDH11 antibody [29] did not support the enhancement of HPC expansion (Supporting Information Fig. S4D). CDH11 expression was higher in HPCs than in total MNCs (Fig. 4F). Taken together, these results demonstrate that the upregulation of CDH11 in BM-MSCs following PTH stimulation contributes to the enhancement of HPC expansion.

Effects of PTH on BM-MSCs

We examined the effects of PTH on the differentiation of human BM-MSCs and on their expression of mesenchymal stromal/stem cell-associated markers. BM-pMSCs and BM-uMSCs had similar abilities to undergo adipogenic (Fig. 5A, 5B), osteogenic (Fig. 5C, 5D), and chondrogenic (Fig. 5E, 5F) differentiation. Flow cytometric analysis demonstrated that BM-pMSCs and BM-uMSCs expressed similar levels of several mesenchymal stromal/stem cell-associated surface markers (Fig. 5G). Expression of the PTH receptor was similar in BM-pMSCs and BM-uMSCs (Supporting Information Fig. S4B). Expression of N-cadherin was not increased in BM-pMSCs (Supporting Information Fig. S4C). Although PTH is generally considered to be an osteogenesis-inducing agent, BM-pMSCs did not show characteristics of mature OBs. Rather, BM-pMSCs showed similar characteristics to BM-uMSCs.

Figure 5.

Effects of PTH on the differentiation of bone marrow mesenchymal stromal cells (BM-MSCs) and their expression of mesenchymal stromal/stem cell-associated markers. (A, B): Adipogenic differentiation of BM-MSCs, as assessed by oil red O staining. Yellow arrows indicate lipid-laden fat cells (A). Quantitative measurement of the number of oil red O+ cells in five different fields viewed at ×100 magnification (n = 3) (B). (C, D): Osteogenic differentiation of BM-MSCs, as assessed by Alizarin Red S staining. Yellow arrows indicate mineralization (C). Mineralization was quantified in three different fields viewed at ×100 magnification. The ratio of mineralization in BM-pMSCs relative to that in BM-uMSCs is shown (n = 3) (D). (E, F): Chondrogenic differentiation of BM-MSCs, as assessed by Alcian blue staining (Nuclear Fast Red counterstaining) and quantitative real-time PCR analysis of aggrecan mRNA expression. Cartilage matrix deposits are stained blue. PTH: BM-pMSC cultures; Control: BM-uMSC cultures. (G): Expression of various mesenchymal stromal/stem cell-associated surface markers in BM-pMSCs (PTH) and BM-uMSCs (Control), as assessed by flow cytometric analysis. Gray areas indicate isotype controls. n.s., not significant. Abbreviation: PTH, parathyroid hormone.

Effects of PTH on BM-MSCs in Lethally Irradiated Mice that Undergo BM Transplantation

We next examined the effects of PTH on BM-MSCs and hematopoiesis after BM transplantation in vivo. Lethally irradiated (10 Gy) C57BL/6 mice were injected with PTH after BM transplantation from syngenic mice (Fig. 6A). When BM cells (5 × 104) were transplanted into the lethally irradiated mice, PTH administration improved the survival rate from about 30% in control mice (black line) to about 80% (red line) (Fig. 6B). This occurred concomitant with an increase in the number of hematopoietic cells in the BM (Fig. 6C–6E) and in the number of red blood cells in peripheral blood (PB) on day 9 after BM transplantation (Supporting Information Table S2). The numbers of white blood cells and platelets in PB did not significantly differ between the two groups (Supporting Information Table S2). CDH11 expression was upregulated in BM-MSCs from mice treated with PTH after BM transplantation (Fig. 6F, 6G). The number of colony-forming unit fibroblasts was higher in BM from PTH-treated mice than in BM from control mice (Fig. 6H, 6I).

Figure 6.

Effects of PTH on BM-mesenchymal stromal cells (MSCs), hematopoiesis, and survival of lethally irradiated mice after bone marrow transplantation. (A): Schedule of PTH injections. C57BL/6 mice were lethally irradiated with 10 Gy and then received BM cell transplantation (5 × 104 cells) on day 0 (n = 10). Mice received 4 µg/kg PTH on days 0, 1, 3, and 5 after BM transplantation. The survival of mice was observed each day until day 30 after transplantation. (B): Survival curves of mice. On day 30, the survival rate of mice that were not treated with PTH (Control, black line) and PTH-treated mice (red line) was 30% and 80%, respectively. (C, D): Representative microscopic images of hematopoietic marrow in sections of vertebras stained with HE (C) and an anti-Gr-1 antibody (D) (n = 3). Original magnification, ×100. Yellow asterisks indicate hematopoietic marrow areas. Blue arrows indicate Gr-1+ cells. BM cells were harvested from mice on day 9 after BM transplantation (C–E). Control: no PTH treatment; PTH: PTH treatment. (E): The number of ANCs in bilateral femurs of control mice (Control) and PTH-treated mice (PTH). (F, G): Expression of CDH11 in BM-MSCs of control mice and PTH-treated mice after BM transplantation. A representative immunoblot from five experiments is shown (G). BM cells were harvested from mice on day 9 after BM transplantation. Control: no PTH treatment, PTH: PTH treatment. (H, I): CFU-Fs in BM of control mice and PTH-treated mice after BM transplantation (n = 5). Representative images of CFU-F cultures (I). Blue arrows indicate CFU-Fs stained with 0.1% toluidine blue O. BM cells were harvested from mice on day 14 after BM transplantation. Control: no PTH treatment, PTH: PTH treatment. *, p < .05. Abbreviations: ANC, all nucleated hematopoietic cell; BM, bone marrow; CFU-F, colony-forming unit fibroblasts; PTH, parathyroid hormone.

Discussion

Recent studies have revealed that MSCs are involved in hematopoiesis [30, 31] and, more importantly, the ability of culture-expanded human BM-MSCs to support hematopoiesis has been validated in clinical trials [12, 19]. Thus, studying the effects of PTH on BM-MSCs may help to develop cell therapies that can be used to support hematopoiesis using culture-expanded BM-MSCs. Consistent with previous studies [17-19], culture-expanded human BM-MSCs supported the expansion of HPCs in vitro; this effect was enhanced by treatment of human BM-MSCs with PTH in vitro. Our results suggest that PTH could be clinically used to stimulate hematopoiesis mediated by BM-MSCs.

PTH administration improved the survival of lethally irradiated mice following BM transplantation. At day 9, the number of hematopoietic cells in BM and the number of red blood cells in PB were higher in mice that were stimulated with PTH on days 0, 1, 3, and 5 after BM transplantation than in control unstimulated mice. Control mice died soon after irradiation. This suggests that these mice did not die as a result of hematopoiesis failure, but as a result of other effects of the irradiation that were unrelated to hematopoiesis, such as gastrointestinal damage. It is possible that PTH improves the survival of irradiated mice by modulating such hematopoiesis-unrelated effects of the irradiation. Therefore, further studies are required to elucidate the detailed mechanism(s) by which PTH improves the survival of lethally irradiated mice following BM transplantation.

It was recently reported that the administration of 100 µg PTH daily for 28 days does not stimulate hematopoietic recovery after umbilical cord blood transplantation in a clinical trial [6]. To clinically treat osteoporosis, 20 µg of PTH is administered daily. In a previous animal study, PTH (80 µg/kg) was injected into recipient mice five times per week for 4 weeks after BM transplantation, and these mice exhibited increased levels of HSCs and their survival was improved [4]. In this study, PTH (4 µg/kg) was injected into recipient mice four times from day 0 to 5 after BM transplantation. This treatment improved the survival of mice. Thus, short-term administration of PTH might effectively improve survival after BM transplantation. However, administration of PTH in vivo can affect various cell types, and its effects on hematopoiesis are affected by several factors. Several studies have indicated that the effects of PTH on the differentiation and proliferation of OBs vary according to the administration schedule used [32-34]. Further investigation is required to determine the schedule of PTH administration that most effectively improves hematopoiesis and survival following transplantation of HSCs in humans.

With respect to a molecule that underlies how PTH enhances the ability of BM-MSCs to support hematopoiesis, CDH11 expression was upregulated in BM-pMSCs. In addition, depletion of CDH11 expression in BM-pMSCs using siRNA abolished the ability of BM-pMSCs to enhance HPC expansion in coculture experiments. Furthermore, coculture experiments using a Transwell system suggested that the enhancement of HPC expansion was dependent on the direct interaction between HPCs and BM-pMSCs. These results indicate that the upregulation of CDH11 in BM-MSCs by PTH stimulation contributes functionally to the enhancement of HPC expansion. This could be further confirmed by other experiments. For example, CDH11 could be overexpressed in NHDFs and the effects of these cells on the expansion of hematopoietic cells could be determined.

CDH11 is highly expressed in HPCs and mediates cell-cell adhesions [35]. It is possible that CDH11 mediates an interaction between BM-pMSCs and HPCs, and that this interaction contributes to the enhancement of HPC expansion following PTH stimulation. This is supported by a previous study in which HPCs with a high self-renewal capacity expressed a high level of CDH11 [36]. CDH11 interacts with β-catenin [37, 38], and β-catenin signaling is crucial for HPC expansion [39, 40]. Therefore, we speculate that the upregulation of CDH11 in BM-pMSCs enhances β-catenin signaling in HPCs, which in turn enhances HPC expansion. Further investigations are needed to elucidate the precise mechanism underlying CDH11-mediated hematopoietic expansion.

Conclusions

We show that PTH enhances the ability of culture-expanded human BM-MSCs to expand HPCs. CDH11 is expressed in culture-expanded human BM-MSCs and is upregulated following PTH stimulation. In lethally irradiated mice after BM transplantation, upregulation of CDH11 in BM-pMSCs occurs concomitant with improved mouse survival and an increased number of hematopoietic cells in BM and PB. Further elucidation of the mechanisms underlying the ability of PTH to stimulate hematopoiesis might assist in the development of mesenchymal stromal/stem cell-based therapies.

Acknowledgments

We thank Yoko Nakagawa for excellent technical assistance. This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan (H.Y., Y.M., T.I., H.H., and T.M.), a Grant from Project for Development of Innovative Research on Cancer Therapeutics from MEXT in Japan (T.M., H.H.), a Grant-in-Aid from the Ministry of Health, Labour and Welfare in Japan (T.I. and T.M.), the National Cancer Center Research and Development Fund (T.M., 23-A-23), and a Grant-in-Aid from the Japan Science and Technology Agency (Y.M.). This work was also supported by the Japan Leukemia Research Fund (Y.M.), the Kyoto University Translational Research Center (Y.M.), the Cell Science Research Foundation (Y.M.), the Kobayashi Foundation for Cancer Research (T.M.), the Joint Usage/Research Center of Hiroshima University Research Institute for Radiation Biology and Medicine (Y.M. and T.I.), and the Research Grant of the Princess Takamatsu Cancer Research Fund (T.M.).

Author Contributions

H.Y., Y.M., and H.H.: conception and design, financial support, collection of data, data analysis and interpretation, and manuscript writing; S.Y. and M.M.: conception and design, collection of data, data analysis and interpretation, and manuscript writing; Y.H., A.T., E.A., H.K., and A.S.: data analysis and interpretation; M.I.: conception and design, collection of data, and data analysis and interpretation; T.H. and Y.H.: collection of data, and data analysis and interpretation; T.I. and T.M.: conception and design, financial support, data analysis and interpretation, and manuscript writing. All authors listed approve this manuscript.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Ancillary