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. 2016 Jan 26;7(1):e02109-15.
doi: 10.1128/mBio.02109-15.

Human Mesenchymal Stem Cells of Diverse Origins Support Persistent Infection with Kaposi's Sarcoma-Associated Herpesvirus and Manifest Distinct Angiogenic, Invasive, and Transforming Phenotypes

Myung-Shin Lee  1 , Hongfeng Yuan  2 , Hyungtaek Jeon  3 , Ying Zhu  2 , Seungmin Yoo  3 , Songtao Shi  4 , Brian Krueger  5 , Rolf Renne  5 , Chun Lu  6 , Jae U Jung  2 , Shou-Jiang Gao  7
Affiliations

Human Mesenchymal Stem Cells of Diverse Origins Support Persistent Infection with Kaposi's Sarcoma-Associated Herpesvirus and Manifest Distinct Angiogenic, Invasive, and Transforming Phenotypes

Myung-Shin Lee et al. mBio. .

Abstract

Kaposi's sarcoma (KS), a highly angiogenic and invasive tumor often involving different organ sites, including the oral cavity, is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). Diverse cell markers have been identified on KS tumor cells, but their origin remains an enigma. We previously showed that KSHV could efficiently infect, transform, and reprogram rat primary mesenchymal stem cells (MSCs) into KS-like tumor cells. In this study, we showed that human primary MSCs derived from diverse organs, including bone marrow (MSCbm), adipose tissue (MSCa), dental pulp, gingiva tissue (GMSC), and exfoliated deciduous teeth, were permissive to KSHV infection. We successfully established long-term cultures of KSHV-infected MSCa, MSCbm, and GMSC (LTC-KMSCs). While LTC-KMSCs had lower proliferation rates than the uninfected cells, they expressed mixtures of KS markers and displayed differential angiogenic, invasive, and transforming phenotypes. Genetic analysis identified KSHV-derived microRNAs that mediated KSHV-induced angiogenic activity by activating the AKT pathway. These results indicated that human MSCs could be the KSHV target cells in vivo and established valid models for delineating the mechanism of KSHV infection, replication, and malignant transformation in biologically relevant cell types.

Importance: Kaposi's sarcoma is the most common cancer in AIDS patients. While KSHV infection is required for the development of Kaposi's sarcoma, the origin of KSHV target cells remains unclear. We show that KSHV can efficiently infect human primary mesenchymal stem cells of diverse origins and reprogram them to acquire various degrees of Kaposi's sarcoma-like cell makers and angiogenic, invasive, and transforming phenotypes. These results indicate that human mesenchymal stem cells might be the KSHV target cells and establish models for delineating the mechanism of KSHV-induced malignant transformation.

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Figures

FIG 1
FIG 1
Human primary MSCs of different origins are permissive to KSHV infection. (A) Cellular morphology and GFP expression of MSCs following KSHV acute infection at different days postinfection. MSCa, human adipose tissue-derived MSCs; MSCbm, human bone marrow-derived MSCs; GMSC, human gingiva-derived MSCs; DPSC, human dental pulp-derived MSCs; SHED, human exfoliated deciduous tooth-derived MSCs. Magnification, ×100. (B) KSHV infection rates in different MSCs analyzed by flow cytometry based on GFP expression. The percentages of GFP-positive cells are indicated. (C) Cell proliferation of mock-infected and KSHV-infected MSCs.
FIG 2
FIG 2
KSHV has a productive replication program during acute infection in different MSCs. (A) Expression of KSHV latent and lytic transcripts over time in MSCs after KSHV infection analyzed by RT-qPCR. (B) Expression of KSHV latent protein LANA and lytic protein ORF65 in MSCs at day 4 postinfection analyzed by IFA. (C) Quantification of percentages of cells expressing LANA and ORF65 proteins using images taken as described for panel B. (D) Production of infectious virions in acutely KSHV-infected MSCs. HUVEC were infected with supernatants from KSHV-infected MSCs at day 4 postinfection, and the infection was evaluated based on GFP expression at 24 h postinfection. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification, ×400. (E) Quantification of percentages of GFP-positive cells using images taken as described for panel D. (F) Detection of encapsidated KSHV DNA in supernatants of acutely KSHV-infected MSCs. Culture supernatants were collected at day 4 postinfection and concentrated by ultracentrifugation. The pellets were treated with DNase I followed by extraction of DNA. KSHV DNA was quantified using KSHV ORF26 primers. Supernatant from iSLK cells induced with sodium butyrate and doxycycline for 4 days was used as a positive control.
FIG 3
FIG 3
Establishment of long-term cultures (LTC) of KSHV-infected MSCs. (A and B) Cell proliferation of KSHV-infected MSCa (KMSCa) in different culture media with and without the addition of 20% FBS and with and without coating the wells with collagen, shown as cell numbers (A) and GFP images at ×40 magnification (B) at day 4 postseeding. (C) Two different shapes of LTC-KGMSC were observed: type 1 cells, with smaller sizes and millet-like shapes, and type 2 cells, with larger sizes and elongated shapes. Magnification, ×100. (D and E) LTC-KGMSC but not LTC-KDPSC had type 1 cells with millet-like shapes shown at ×40 (D) and ×100 (E) magnifications. (F) Cumulative cell population of uninfected cells and LTC-KMSCa. Uninfected MSCa (Mock) or LTC-KMSCa (KSHV) at 1.25 × 105 cells were seeded in a T25 flask and subcultured every 3 days, and live cell numbers were counted based on trypan blue exclusion.
FIG 4
FIG 4
Expression of KSHV genes in LTC-KMSCs. (A to C) Expression of KSHV latent and lytic genes in LTC-KMSCa (A), LTC-KMSCbm (B), and LTC-KGMSC (C) analyzed by RT-qPCR. Cells infected with KSHV for 4 days were used as controls. β-Actin was used as an internal control. Quantitative values representing the averages ± SDs from three independent experiments are presented. ORF, open reading frame. (D) Expression of KSHV latent and lytic proteins in LTC-KMSCa, LTC-KMSCbm, and LTC-KGMSC analyzed by IFA. iSLK-BAC16 cells induced with doxycycline and sodium butyrate for 2 days were used as positive controls. DAPI, 4′,6-diamidino-2-phenylindole.
FIG 5
FIG 5
Expression of cell surface markers in MSCs and LTC-KMSCs analyzed by flow cytometry. HUVEC were used as controls.
FIG 6
FIG 6
LTC-KMSCs manifest distinct angiogenic, migratory, invasive, and transforming phenotypes. (A) Capillary tube formation of LTC-KMSCs. Equal numbers of uninfected MSCs (Mock) or KMSCs (KSHV) were applied to the top of the Matrigel, and images of tube formation were captured using a phase-contrast microscope at 8 h postseeding (upper panel). Magnification, ×100. An in vitro angiogenic index from the average number of branches per field of view ± SD at ×40 magnification is shown (lower panel). (B and C) Migration and invasiveness of LTC-KMSCs. The migrating (B) or invaded (C) cells were stained with calcein AM. Representative images are shown (upper panel), and the averages ± SDs were calculated based on quantification using a fluorescence microplate reader with 485-nm excitation and 528-nm emission filters. RFU, relative fluorescence units. (D) Colony formation of uninfected MSCs and LTC-KMSCs. Representative cell colonies in soft agar are shown. Colony formation was visualized at day 18 postseeding. Bar, 50 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 7
FIG 7
KSHV miRNAs mediate KSHV-induced angiogenesis by activating the AKT pathway. (A) The miRNA cluster was required for KSHV-induced angiogenesis. Capillary tube formation activity of uninfected MSCa (Mock), LTC-KMSCa, LTC-ΔmiRMSCa, and LTC-ΔmiRMSCa complemented with vector control or the miRNA cluster. Equal numbers of cells were seeded on top of the Matrigel. Images of tube formation were captured at 8 h postseeding (left panel). Magnification, ×100. The average number of branches per field of view ± SD at ×40 magnification was quantified (right panel). WT, wild type. (B) LY294002 inhibited capillary tube formation of LTC-KMSCa. LTC-KMSCa were incubated with different concentrations of LY294002, and tube formation was analyzed at 8 h postseeding. Representative images (left panel) and the average number of branches per field of view ± SD (right panel) are shown. (C) AKT phosphorylation at T308 but not S473 was enhanced in LTC-KMSCa (KSHV) compared to uninfected MSCa (Mock). (D) LY294002 suppressed AKT phosphorylation at T308 in LTC-KMSCa in a dose-dependent manner. (E) The miRNA cluster was required for AKT phosphorylation at T308. AKT phosphorylation was examined by Western blotting in uninfected MSCa (Mock), LTC-KMSCa (KSHV), or LTC-ΔmiRMSCa (ΔmiR) complemented with vector control (vector) or the miRNA cluster (miR cluster). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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