Guan-Hua Qiao, Fei Zhao, Shuang Cheng and Min-Hua Luo. Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection[J]. Virologica Sinica, 2016, 31(3): 219-228. doi: 10.1007/s12250-016-3754-0
Citation: Guan-Hua Qiao, Fei Zhao, Shuang Cheng, Min-Hua Luo. Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection .VIROLOGICA SINICA, 2016, 31(3) : 219-228.  http://dx.doi.org/10.1007/s12250-016-3754-0

间充质干细胞对人巨细胞病毒感染完全容许

  • 通讯作者: 程爽*, chengshuang@wh.iov.cn, ORCID: 0000-0001-9255-0453
    ; 罗敏华*, luomh@wh.iov.cn
  • 收稿日期: 2016-02-24
    录用日期: 2016-04-05
    出版日期: 2016-04-21
  • 先天性HCMV感染是导致新生儿多器官发育异常等出生缺陷的重要病因。间充质干细胞(MSCs)是一类干细胞/前体细胞,具有多分化和自我更新的潜能,在多器官的形成过程中发挥重要作用。而HCMV感染所引起的非神经系统异常是否与MSCs相关尚未见报道。因此,本研究拟从人脐带的华顿氏胶中分离MSCs,分析MSCs对HCMV的易感性。首先,我们分离MSCs,并鉴定其吸附性、表面分子标记的表达和分化特性。然后,通过western blot、间接免疫荧光和噬斑形成试验,分析HCMV在MSCs中的感染特性,如病毒进入、复制、蛋白质表达、释放等,发现MSCs对HCMV感染完全容许。该研究的阐明为揭示先天性HCMV感染所引起的多系统发育异常的机制奠定了基础。

Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection

  • Corresponding author: Shuang Cheng, chengshuang@wh.iov.cn Min-Hua Luo, luomh@wh.iov.cn
  • ORCID: 0000-0001-9255-0453; 
  • Received Date: 24 February 2016
    Accepted Date: 05 April 2016
    Published Date: 21 April 2016
  • Congenital human cytomegalovirus (HCMV) infection is a leading infectious cause of birth defects. Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells (MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multiorgan formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton’s jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry, replication initiation, viral protein expression, and infectious virion release using western blotting, immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

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    Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection

      Corresponding author: Shuang Cheng, chengshuang@wh.iov.cn
      Corresponding author: Min-Hua Luo, luomh@wh.iov.cn
    • State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

    Abstract: Congenital human cytomegalovirus (HCMV) infection is a leading infectious cause of birth defects. Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells (MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multiorgan formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton’s jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry, replication initiation, viral protein expression, and infectious virion release using western blotting, immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

    • Multipotent mesenchymal stromal cells (MSCs) are a group of adult stem cells with self-renewal ability and multi-lineage differentiation potency, and they are found in many tissues, such as bone marrow, adipose tissue, and peripheral blood (Young et al., 1995; Wei et al., 2013). MSCs have the potential to differentiate into various lineages (including adipocytes, osteoblasts, chondrocytes, and myoblasts) (Dominici et al., 2006) and are capable of migrating to tissue injury sites to prevent deleterious remodeling and improve recovery (Pittenger and Martin, 2004). Notably, MSCs have been shown to induce a tolerant immune cell phenotype by altering their inflammatory cytokine secretion pattern and decreasing immune recognition, which could possibly reduce the possibility of developing graft-versus-host disease (Aggarwal and Pittenger, 2005). Therefore, MSCs are generally used for tissue engineering applications (tissue reparation or regeneration) and as a carrier cell in gene therapy, and numerous studies have examined their capability to repair damage to myocardial tissue, bone, tendon, cartilage, and meniscus (Pittenger et al., 1999).

      Human cytomegalovirus (HCMV), a β human herpesvirus, represents the major infectious cause of birth defects, as well as an important pathogen for immunocompromised individuals. After primary infection, HCMV persists latently in the immunocompetent population but leads to significant morbidity and mortality in immunocompromised individuals (such as patients with AIDS and those who underwent solid organ transplantation) and neonates (Bale, 1984; Landolfo et al., 2003). From 5% to 10% of congenitally HCMV-infected neonates present with cytomegalic inclusion disease, including growth retardation, hepatitis, and jaundice, along with brain development disorders in the form of microcephaly, encephalitis, seizures, and focal neurological signs. Approximately 10%-15% of infected infants who are asymptomatic at birth develop sensorineural hearing loss (Landolfo et al., 2003; Noyola et al., 2010). The clear health risks of exposure to HCMV make understanding the pathogenesis mechanism of this virus imperative.

      The HCMV pathogenesis mechanisms center on two aspects: cell permissiveness for HCMV infection and the relationship between HCMV and the host cells (Landolfo et al., 2003). Based on viral protein synthesis and viral particle release, cell permissiveness for HCMV infection is divided into three categories: permissive, semi-permissive, and non-permissive. In permissive cells, the HCMV genome is temporally expressed. HCMV immediate-early (IE) proteins are synthesized immediately after virus entry into cells and activate the expression of early (E) genes and late (L) genes. These events result in the production of large amounts of viral particles and lysis of infected cells. In semi-permissive cells, the kinetics of HCMV protein expression is delayed, and very few viral progeny are produced. In non-permissive cells, viral proteins are not synthesized, and no subsequent viral progeny are produced.

      Evidence has shown that epithelial cells, endothelial cells, and fibroblasts are susceptible to HCMV infection (Sinzger and Jahn, 1996). Our previous study also revealed that neural progenitor cells (NPCs) and their derived neuronal and glial cells were fully permissive for HCMV infection (Luo et al., 2008; Pan et al., 2013b). The monocyte/macrophage lineage in the peripheral blood has been shown to harbor infectious HCMV (Goodrum et al., 2002), and transfusion-mediated HCMV infection can be prevented by removal of the blood leucocytes (Musiani et al., 1984; Bhumbra et al., 1988; Gilbert et al., 1989). However, although HCMV viral transcripts and proteins have been observed in CD34+ hematopoietic stem cells and CD14+ monocytes, viral protein expression is delayed, and very few viral progeny are produced; thus, both cells are semi-permissive for HCMV infection. HCMV can also infect many other types of cells, and the virus has been detected in many organs that are mainly composed of parenchymal and mesenchymal cells and in which MSCs are primarily located. However, whether MSCs support HCMV replication and the infection status of HCMV in MSCs remain unclear.

      To address this issue, MSCs were isolated from Wharton's jelly (WJ) of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, MSCs were infected with HCMV, and the infection status was characterized by assessing viral entry, replication, viral protein expression, and viral particle release using immunofluorescent (IFA), western blotting, and viral titer assays. The results indicated that MSCs were permissive for HCMV infection. The present study demonstrated that MSCs are a novel susceptible cell type for HCMV study and might prove useful to elucidate the pathogenesis mechanism of HCMV infection in non-nervous system diseases.

    • MSCs were isolated from WJ of the human umbilical cord using the tissue explant method (Han et al., 2013). In detail, 10-15-cm segments of umbilical cord tissue were obtained with informed consent from healthy full-term neonates after cesarean section at Zhongnan Hospital of Wuhan University. The segments were transferred to the laboratory in pre-cooled sterile Hank's balanced salt solution containing amphotericin B (5 μg/mL, Gibco), penicillin (200 units/mL, Gibco), and streptomycin (200 μg/mL, Gibco). The blood clots were washed off with ice-cold sterile phosphate buffered saline (PBS), and both ends were removed. The cord segment was then thoroughly washed with sterile PBS to remove the cord blood, dipped in 75% ethanol for 1 minute for sterilization, and washed with PBS twice to remove the residual ethanol. WJ was obtained after thoroughly peeling off the umbilical artery, veins, and amniotic membrane and was cut into 1-mm3 pieces. These small pieces were evenly planted on a cell culture dish (Corning) and maintained on the bench at room temperature for 15 minutes to enhance sticking, and they were then incubated in complete culture medium at 37℃ in a 5% CO2 atmosphere; the medium was changed every 3-4 days. When the MSCs released and attached to the culture surface about 2 weeks later, the pieces of WJ were carefully removed, and complete culture medium was added gently. The isolated MSCs were further cultured, the medium was refreshed every 2-3 days, and the cells were digested (0.25% trypsin-EDTA) and expanded in T75 flasks when at 80% confluence. The complete culture medium was Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F-12) supplemented with 15% (vol/vol) fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL) (all from Life Technologies, California, USA).

    • To verify their multipotent differentiation potential, MSCs were induced to differentiate to adipogenic (Adipo-) and osteogenic (Osteo-) cells as previously described with modifications (Jaiswal et al., 1997; Sibov et al., 2012). Briefly, early-passage MSCs (p4) were cultured in complete medium containing the corresponding stimulators for 21 days, fixed in 10% neutral buffered formalin for 60 minutes after a PBS rinse, and then stained with the corresponding dyes. Images were obtained with an inverted microscope (Nikon Eclipse TS100, Japan) equipped with a camera (Nikon CoolPix P6000, Japan). For adipogenic differentiation, 0.5 μmol/L dexamethasone, 50 μmol/L indomethacin, and 0.5 mmol/L isobutylmethylxanthine (IBMX) were applied as stimulators, and cells were stained with Oil Red O (all from Sigma). For osteogenic differentiation, the induction reagents were 2 mmol/L β-glycerophosphate, 0.1 μmol/L dexamethasone, and 50 μmol/L ascorbic acid, and the dye was Alizarin Red S (all from Sigma, USA). Fresh-cultured MSCs and 21-day cultured MSCs without exposure to stimulators were applied as controls.

    • The HCMV Towne strain was propagated in human embryonic lung fibroblasts (HELs) as previously described (Pan et al., 2013a). Cells were infected with virus at an MOI of 3. After a 3-hour absorption period, the medium was replaced with fresh medium after a brief rinse with PBS. A mock-infection was prepared with an equal volume of HEL culture supernatant mixed with 1% DMSO and used as a negative control.

    • At the indicated time points, cells were collected with a scraper, rinsed with ice-cold PBS, pelleted by centrifugation at 4℃, snap-frozen in liquid nitrogen, and stored at -80℃ until needed. Then, cell pellets were resuspended in RIPA lysis buffer (Luo et al., 2007). After 5 minutes of centrifugation at 12000×g, the supernatant was collected, and the protein concentration was determined using a BCA protein assay kit (Beyotime, China). Equivalent amounts of cell lysate (30 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to PVDF membranes (Millipore, USA). After blocking in 10% skimmed milk, the membranes were incubated with primary antibodies against the indicated target proteins and corresponding secondary antibodies. Signals were visualized using West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, USA), and images were obtained using a FluorChem HD2 System (Alpha Innotech, USA). The applied primary antibodies included mouse monoclonal antibodies (McAbs) against HCMV-IE1/2, HCMV-UL44, HCMV-pp65, HCMV-gB (Virusys, USA), and β-Actin (Santa Cruz, USA). Secondary antibodies were horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (Amersham, Sweden).

    • MSCs were seeded into 100-mm dishes (5×105 cells/dish) containing coverslips and cultured for 6 hours to let the cells equilibrate. At the indicated time points post-infection, the coverslips were collected, rinsed with PBS, and fixed with 4% formaldehyde. The immunofluorescence staining samples were processed as described previously (Duan et al., 2014). The following primary antibodies were used to analyze cell surface markers: mouse McAbs against CD73 (IgG3), CD44 (IgG1), CD105 (IgG2b), CD34 (IgG1), and HLA-DR (IgG1, Santa Cruz Biotechnology); mouse McAbs against HCMV-IE1 (IgG2a), HCMV-UL44 (IgG1), and HCMV-pp65 (IgG1, Virusys) were used for viral protein detection. Secondary antibodies included tetraethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (SouthernBiotech, USA) and Alexa Fluor 488-conjugated goat anti-mouse IgG1 (Invitrogen, USA). Hoechst dye 33342 (Thermo Fisher Scientific, USA) was applied to counterstain the cell nuclei. Images were taken with a Nikon Eclipse 80i fluorescence microscope equipped with a Nikon DS-Ri1 camera and NIS-Elements F3.0 software. For quantification analysis, at least 300 cells from a minimum five random fields were counted. The positive ratio was calculated as the percentage of cells expressing the corresponding viral protein in all cells stained by Hoechst.

    • Cells were infected with HCMV at an MOI of 3. Aliquots of the supernatants were collected at the indicated time points to assess virus shedding. Supernatants were stored at -80℃ in 1% DMSO until used. Virus titer was determined by counting plaque-forming units (PFU) on HELs (Luo and Fortunato, 2007). Briefly, 200 μL of a 10-fold serial dilution of viral supernatant was used to infect HELs. After attachment for 3 hours, cell growth medium containing 1% agarose was added to the inoculum. Plaques were counted at days 7 to 10 post-plating, with multiple wells seeded for each dilution, and an average titer was derived from the repeats.

    • All images shown for the IFA and western blotting analyses are representative results from three independent experiments. Data for virus shedding and positive cell ratio quantification are presented as the mean ± standard deviation (SD) from three independent experiments. A difference was considered to be statistically significant when P < 0.05 with Student's t-tests.

    • Primary MSCs were isolated from WJ of the human umbilical cord and cultured as described in the Materials and methods. According to the criteria proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006), the plastic-adherence of MSCs was first examined. After 7 days, small, rod-like, and irregularly shaped cells grew out of the explant, stuck to the culture dish, and formed colony forming units-fibroblasts (CFU-F) under the explant (data not shown). After 20 days, the primary cultured cells exhibited a fibroblast-like shape and grew around the explant with increased intercellular space (Figure 1A, left). Then, tissue explants were removed, and the culture medium was changed. After reaching 80%-90% confluence, the cells were trypsinized and transferred to a new culture surface. The sub-culture consisted of a mixture of mainly spindle-like or polygonal fibroblast-like shaped cells with a few irregularly shaped cells (Figure 1A, right), which fit the typical morphological description of MSCs.

      Figure 1.  Isolation and characterization of human WJ-MSCs. (A) Morphology of primary cultured MSCs. Primary MSCs isolated from WJ of the human umbilical cord were cultured on a fibronectin-coated surface (left), and MSC sub-cultures were transferred from the primary culture to an uncoated surface (right). A typical plastic adherent fibroblast-like morphology is shown. (B) Identification of WJ-MSC surface markers. The expression levels of CD73, CD105, CD44, CD34, and HLA-DR in MSCs were examined by IFA. SH-N-SH cells were used as negative controls. Scale bar, 10 μm. (C) Adipogenic and osteogenic differentiation of MSCs. Fresh-cultured MSCs (a, d) and 21-day cultured MSCs with (c, f) and without (b, e) induction were stained with Oil Red or Alizarin Red for adipogenic (Adipo-) and osteogenic (Osteo-) cells, respectively. Scale bar, 10 μm. (D) The IFA images were used to calculate of frequencies of MSCs expressing the indicated surface markers. The data shown are the mean ± SD from three independent experiments.

      To further characterize the isolated MSCs, we assessed surface markers, including CD105, CD73, CD44, CD34, and HLA-DR, using IFA. As shown in Figure 1B, the fibroblast-like shaped cells expressed CD73, CD105, and CD44, with positive ratios of 93.13%, 95.4%, and 95.63%, respectively (Figure 1D), while the negative control SH-N-SH cells expressed none of these markers. Neither CD34 nor HLA-DR expression was detected in the MSCs (Figure 1B).

      Then, the multipotent differentiation potential, including adipogenesis (Figure 1C, a-c) and osteogenesis (Figure 1C, d-f), was assessed using different inductors to examine the differentiation ability of the isolated cells. After 21 days of induction with the corresponding reagents, cells were stained with Oil Red O or Alizarin Red S to visualize adipogenesis or osteogenesis, respectively. After adipo-induction, small red-stained oil drops were clearly observed in the cytoplasm (Figure 1C, c), and after osteo-induction, cells showed obvious red-stained calcium nodes (Figure 1C, f). As controls, fresh-cultured cells and 21-day cultured cells without induction were processed and stained in parallel. Fresh-cultured cells were not stained by either dye (Figure 1C, a and d), and 21-day cultured cells without induction showed only slight staining (Figure 1C, b and e).

      All of the characteristics regarding morphology, surface marker expression pattern, and multipotent differentiation potential met the minimal criteria for defining MSCs recommended by the ISCT. Thus, these results confirmed that the cells isolated from WJ were MSCs.

    • In permissive cells, the HCMV genome is temporally expressed. The IE genes are transcribed after virus entry and rely mainly on host cell cycle progression factors for expression. Prior to viral DNA synthesis, the early genes are expressed with the aid of IE gene products. Finally, L genes are transcribed after the initiation of viral DNA replication, with substantial release of virions. The morphology of infected cells gradually changes during the progression of virus infection, including vesicle formation, cell enlargement, and cell lysis (Lecointe et al., 1999). To reveal the permissiveness of MSCs for HCMV infection, the isolated MSCs were infected with HCMV at an MOI of 3, and the cells were observed at different time points post-infection. Upon HCMV infection, MSCs displayed visible morphological changes beginning at 24 hours post-infection (hpi) and exhibited an increasingly rounded-up shape from 48 hpi to 96 hpi (Figure 2). These results indicated the potential permissiveness of MSCs for HCMV infection.

      Figure 2.  HCMV infection of MSCs. MSCs were infected with HCMV at an MOI of 3. Cytopathogenic effects of HCMV infection on MSCs were observed through an inverted microscope. Morphology of the cells at the indicated time points after HCMV or mock infection. Magnification, ×100.

      The viral life cycle includes viral entry, viral replication, protein expression, and viral progeny release. To further characterize the HCMV infection properties in MSCs, viral entry, replication initiation, and gene expression were examined using IFA and western blotting, and viral release was determined by PFU counting.

    • After viral entry into permissive cells, some tegument proteins (such as pp65 and pp71) are also delivered into the cells and further transported into the nucleus. Thus, pp65 was chosen as an indicator to determine whether HCMV entry occurred in the first 24 hpi. As shown in Figure 3, pp65-positive cells were present as early as 4 hpi (Figure 3A), and the positive rate was 94.33 ± 8.02 % at 4 hpi and reached 100% after 8 hpi (Figure 3B).

      Figure 3.  HCMV entry in MSCs. MSCs were infected with HCMV at an MOI of 3, and viral tegument protein pp65, serving as a virus entry indicator, was measured by IFA at the indicated time points (upper, scale bar, 10 μm). The IFA images were used to calculate the percentage of pp65+ cells, and the results are presented as the mean ± SD of five independent experiments.

    • Entering the host cell is just the first step of the HCMV infection process, and it does not necessarily lead to the initiation of viral replication. Because the critical trans-activating protein immediate early protein I (IE1) is one of the first strongly expressed proteins during HCMV replication, IE1 synthesis was measured as an indicator of viral replication initiation (Landolfo et al., 2003). As early as 4 hpi, IE1 was clearly observed in the nuclei of MSCs (Figure 4A), indicating that HCMV replication was initiated in these cells. The amount of IE1 continued to accumulate in the cells (Figure 4A), and the ratio of IE1-positive MSCs increased from 5.44 ± 1.53% at 4 hpi to 45.03 ± 4.89% at 36 hpi (Figure 4B).

      Figure 4.  Initiation of HCMV replication in MSCs. MSCs were infected with HCMV at an MOI of 3, and IE1 expression, as an indicator of viral replication initiation, was examined by IFA at the indicated time points (upper, scale bar, 10 μm). IE1+ cells were counted according to the IFA data, and the frequency of IE1+ cells is presented as the mean ± SD from five independent experiments (lower).

    • The viral UL44 HCMV DNA polymerase processivity factor is a component of the viral replication center and is necessary for effective viral DNA replication. Thus, UL44 was measured by IFA to assess viral genome replication. Cells containing UL44 were further categorized according to the subcellular location as UL44+ cells and UL44 foci+ cells. The designation of UL44+ indicated that positive signals existed in the cells. The designation of UL44 foci+ indicated that positive signals existed in the nuclei, formed foci, and then gathered gradually starting at 24 hpi, which represented the hallmark of HCMV genome replication (Luo et al., 2010). In Figure 5A, UL44+ cells and UL44 foci+ cells are indicated by arrows and triangles, respectively. At 12 hpi, the UL44+ signals exhibited diffuse staining in MSCs. After 24 hpi, some cells had areas of accumulated signals underlying a diffuse pattern and were designated UL44 foci+ cells, indicating viral replication sites. In addition, as previously demonstrated by a number of groups (Penfold and Mocarski, 1997; Luo et al., 2008), the accumulation of UL44 signal increased after 36 hpi, mainly forming bipolar foci at 36 hpi and large nuclear domains at 48 hpi. To evaluate the replication efficiency of HCMV in MSCs, the UL44 foci+/IE1+ values were calculated at different time points. The results showed that the value UL44 foci+/IE1+ values sharply increased after 36 hpi, from 20.87 ± 3.32 % at 24 hpi to 76.22 ± 4.08% at 36 hpi and to 92.04 ± 8.72% at 96 hpi (Figure 5B), indicating a high level of viral genome replication after 24 hpi in MSCs. Together, the results indicate that efficient replication of the viral genome began shortly after viral entry into MSCs.

      Figure 5.  The efficiency of HCMV genome replication in MSCs. (A) UL44 and IE1 were double stained in HCMV-infected MSCs, and the UL44 foci+/IE1+ value served as an indicator of the efficiency of viral genome replication (upper, scale bar, 10 μm). Cells expressing UL44 are labeled as UL44+ cells (arrows), and cells with aggregating UL44 are labeled as UL44 foci+ (triangles). (B) The frequencies of UL44+ foci/IE1+ cells were calculated to evaluate the efficiency of viral genome replication.

    • After the initiation of viral replication by IE gene products, the viral E and L gene products are synthesized in a time-dependent manner. During the time course of viral infection, the major immediate early proteins IE1 and IE2 could be detected at 4 hpi. The early proteins UL44 and pp65 with different subtypes were present at 48 hpi. The representative late stage protein gB was also detected at 48 hpi (Figure 6A).

      Figure 6.  Viral protein expression and viral particle shedding. (A) Representative viral protein expression in HCMV-infected MSCs. Cells were collected at the indicated time points, and western blotting was performed to examine the expression of viral proteins. β-actin served as a control for cell amount normalization. (B) MSCs were infected with HCMV (Towne strain) at an MOI of 3. Supernatants were collected at the indicated time points for virus shedding titration.

    • After viral replication and viral protein expression, viral progeny products are assembled and released. To further analyze viral progeny, supernatants of HCMV-infected MSCs were collected at designated times to determine viral titer by PFU counting. As shown in Figure 6B, the viral titer in MSCs presented a typical logarithmic growth pattern and progressed into a slow growth state (Figure 6B). These results further indicated that MSCs are fully permissive for HCMV infection.

      Thus, the following results indicate that MSCs are fully permissive for HCMV infection: viral entry into MSCs as early as 4 hpi, initiation of viral replication at 4 hpi, and release of viral progeny products.

    • MSCs have great therapeutic potential due to their capacity for self-renewal and multi-potency, and they are capable of differentiating into bone, cartilage, fat, tendon, muscle, and marrow stroma (Pittenger et al., 1999). HCMV is a ubiquitous pathogen that can infect most organs, including the brain, lungs, heart, salivary gland, liver, kidney, and bone marrow. MSCs are involved in multi-organ formation, but whether MSCs play a role in the pathogenesis of congenital HCMV infection remains unclear. In this study, we determined the susceptibility of MSCs for HCMV infection and confirmed that MSCs are fully permissive for HCMV infection. These results represent new information that will aid in determining the pathogenesis mechanism of HCMV infection.

      A large number of reports have shown that MSCs can be derived from various tissues, including bone marrow (BM), periosteum, adipose tissue, skeletal muscle, deciduous teeth, fetal pancreas, lung, liver, cord blood, and umbilical cord tissues (Zuk et al., 2001; Lu et al., 2006; Wei et al., 2013; Rylova et al., 2015). Previously, BM was the major source of MSCs for use in cell therapy. However, the aspiration of BM involves invasive procedures, and the frequency and differentiation potential of BM-MSCs decrease significantly with age. Cord blood and umbilical cord tissue might be ideal sources of MSCs because of their advantages of having very similar cell characteristics, being widely available, requiring a non-invasive collection procedure, and being associated with relatively low ethical risks. Regarding the human umbilical cord, MSCs can be found in the umbilical cord lining, sub-endothelial layer, perivascular zone, and WJ (Watson et al., 2015). Among the regions of the umbilical cord, WJ is the best source of cells because MSCs in WJ are maintained in an early embryologic phase and therefore have retained some of their primitive stemness properties (Watson et al., 2015). Therefore, we chose WJ as the MSC source in this study. Following the criteria for the identification of MSCs proposed by the ISCT, we examined the plastic adherence, surface markers, and differentiation properties of the isolated MSCs. The results indicated that the cells isolated from WJ were MSCs because they met all criteria.

      The results of the cell susceptibility analysis showed that MSCs are fully permissive for HCMV infection, as indicated by viral pp65 protein entry into cells immediately after HCMV infection, viral IE protein expression, viral replication, viral E and L protein expression, and viral particle release. MSCs are found in almost all tissues and are a subset of non-hematopoietic stem cells that originate from the mesoderm during embryonic development. MSCs can differentiate into not only mesoderm lineages but also ectodermic and endodermic cells (Bruder et al., 1994; Wei et al., 2013). Thus, if MSCs were infected with HCMV in an embryogenesis stage, the transduction of a series of signals might be affected by the infection and lead to abnormal morphogenesis and organ functions, further exacerbating the disease in HCMV-infected neonates.

      Because of their multi-lineage differentiation potential and immunomodulatory functions, MSCs have promise for use in the treatment of numerous diseases. A large number of studies have assessed the therapeutic efficacy of MSCs on disease progression in experimental animal models and human clinical trials and found that MSC administration can promote tissue repair by secreting soluble factors that alter the tissue microenvironment or inducing cells to transdifferentiate into epithelial cells and similar lineages (Phinney and Prockop, 2007). However, HCMV infection might influence the effective modulation or transdifferentiation of MSCs and potentially lead to the disordered repair of tissues. In addition, infection of MSCs with HCMV possibly leads to complications such as pneumonia, myocarditis, retinitis, and gastroin-testinal ulceration. HCMV infection also leads to disease in various tissues, including the brain, lung, liver, and gastrointestinal tract (Landolfo et al., 2003).

      MSCs are located within the connective tissue compartments of many organs and organ systems and thus have a widespread distribution throughout the body (Young et al., 1995). In addition, the therapeutic mechanisms of MSCs include many cytokines, transcriptional factors, and signaling pathways (Mrugala et al., 2009; Le Blanc and Mougiakakos, 2012; Dudakovic et al., 2015; Laranjeira et al., 2015; Sela et al., 2015; Tan et al., 2015). Among the various factors involved, HCMV infection can activate transforming growth factor-β in renal tubular epithelial cells (Shimamura et al., 2010) and osteosarcoma cell lines (Kwon et al., 2004), down-regulate epidermal growth factor receptor (EGFR) in human fetal lung fibroblasts (Beutler et al., 2003), induce platelet-derived growth factor receptor (PDGFR) expression in human vascular smooth muscle cells (Reinhardt et al., 2005a), and dysregulate PDGFR, EGFR, Wnt/β-catenin, Notch, and NF-κB signaling pathways (Cinatl et al., 2001; Fairley et al., 2002; Reinhardt et al., 2005b; Bentz and Yurochko, 2008; Isern et al., 2011; Angelova et al., 2012; El-Shinawi et al., 2013; Kapoor et al., 2013; Mathers et al., 2014; Li et al., 2015). The exact nature of the relationship between MSCs and the pathogenesis of HCMV infection remains to be determined.

      In conclusion, MSCs are fully permissive for HCMV infection, as indicated by viral entry into MSCs as early as 4 hpi, replication initiation, viral protein expression, and viral progeny product release. The results of this study provide a new perspective on the pathogenesis of HCMV infection.

    • The work was supported by the National Science Foundation of China (81071350, 81271850, and 31170155) and the National Program on Key Basic Research Project (973 program 2011CB504804 and 2012CB519003).

    • There were no conflicts of interest in the study. Additional informed consent was obtained from all patients for which identifying information is included in this article.

    • GHQ conceived and performed the experiments. MHL and SC designed and oversaw the study. FZ contributed to the western blot and IFA analyses.

    Figure (6)  Reference (52) Relative (20)

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