Characterizing the SLC39A14 zinc transporter and collagen products of the chondrocytes from human umbilical cord-derived mesenchymal stromal cell

Open access: www.balimedicaljournal.org and ojs.unud.ac.id/index.php/b j Characterizing the SLC39A14 zinc transporter and collagen products of the chondrocytes from human umbilical cord-derived mesenchymal stromal cell Ketut Dewi Kumara Wati1,2*, Endah Dianty Pratiwix3, Yanni Dirgantara3, Cynthia Retna Sartika3, Meita Dhamayanti1,4, Budi Setiabudiawa1,4, Ida Parwati1,5 Background: The SLC39A14 zinc transporter showed a role in the growth of the mouse model. There was no report regarding SLC39A14 protein expression in human chondrocyte, which might prevent further identification of SLC39A14’s function in human growth. This study aims to describe the SLC39A14 protein expression in chondrocyte using human umbilical cord (UC)-derived mesenchymal stromal cell (MSC) isolation and differentiation. Methods: A cross sectional study was conducted at the Faculty of Medicine, Universitas Padjajaran Bandung, Indonesia, in 2019. The UC was derived after the respondents signed informed consents were documented. Specimens transferred, UC tissue digestion, cell isolation, cultures, and passages were done under the laboratory standard protocols. The immuno-phenotyping and differentiation toward chondrocyte procedure were done after third passage. The SLC39A14 expression of the chondrocytes was observed under immunofluorescence microscopy after staining using polyclonal anti SLC39A14 antibody. The collagen 2A and 10A production was measured using the medium supernatant and analyzed using GraphPad Prism 8 edition. Results: The adherent fibroblast-like cells appeared on day 5th, which continued to proliferate and showed MSC characteristic with positive markers for CD 73, CD90 and CD105, but absent for negative lineage markers. Upon differentiation, cells positive for Alcian blue staining denoted the chondrocyte which shown capacity for further proliferation on Methyl Tetra Toluene assay. Zinc transporter SLC39A14 was expressed on cell membrane. The Collagen 2A produced at a mean level 0.097 ng/ml (SE 0.011;95%CI 0.073-0.12) and Collagen 10A at mean level 31.078 ng/ml (SE 3.792;95%CI 22.870-39.287). Conclusion: Zinc transporter SLC39A14 was expressed in monolayer human chondrocyte from UC-derived MSC. The chondrocyte shown capacity for proliferation and collagen-producing enables future utility to identify the role of SLC39A14 in chondrocyte pathology.


INTRODUCTION
The umbilical cord (UC) has been known as a source for mesenchymal stromal cell (MSC), which can be directed into a differentiation toward chondrocyte, osteocyte, and adipocyte cells. 1 Friendenstein was the first who described the supportive hematopoietic cells of bone marrow as MSCs. He exhibited that MSCs could differentiate to bone in vitro and a subset of the cells had a high proliferative potential (CFU-F) when plated at low density in tissue culture. 2, 3 Based on in vitro study, MSCs is the spindle-shaped plastic-adherent cells isolated from bone marrow, adipose, and other tissue sources, with multipotent differentiation capacity. 2, 3 Thus, the International Society for Cellular Therapy (ISCT) has promoted that these spindle-shaped, plastic-adherent cells be termed, "mesenchymal stromal cells" of MSCs. 4 Many standard procedures have been reported about this process to enable replication. 5, 6 Based on the multipotent properties of MSCs, it has been known that MSCs can differentiate into chondrocyte cells through a particular growth factor. 7, 8 A recent report in animal study found that disturbance in zinc (Zn) influx in the growth platechondrocytes explained the mechanism of growth failure. This study contributed to a new paradigm primarily related to the SLC39A14 zinc transporter while also highlighted Zn as a ubiquitous metal in human body. 9-13 Zn is an important trace element that is involved in various cellular events by regulating the structural and catalytic functions of transcription factors or enzymes. 14 Zn homeostasis ORIGINAL ARTICLE is tightly controlled by two types of Zn transporter such as SLC39/ZIP importers and SLC30/ZnT exporters which is well known participate in various physiological events. 15 Abnormal Zn homeostasis is related to vertebrate growth retardation as well as lead to dwarfism with reductions in the growthplate width, which is correlated with a decreased of cellular Zn content. 16 The molecules responsible for Zn homeostasis in growth failure have been elusive, and data regarding how Zn affects the intracellular signaling that regulates growth-related endocrine processes still limited. However, a previous study has mentioned that SLC39A14 molecule plays a pivotal role in chondrocyte differentiation through Zn metabolism. 9 SLC39A14 transporter is knowns as a SLC39/ ZIP family member, transports the Zn ion into cells in an in-vitro culture system. 17 A study conducted by Hojyo notified that the PTHr-PKA-CREB signaling pathway of the chondrocyte proliferation phase would not be able to occur when zinc was deficient, leading to growth failure. 9 Using an insitu hybridization staining procedure, Hojyo and the coworkers identified SLC39A14 molecule in the mouse growth plate-chondrocyte. 9 To date, there was no report regarding SLC39A14 zinc transporter identification in human chondrocyte.
We sought to extend the finding, by identification of the zinc transporter SLC39A14 expression in the human chondrocyte from UC derived MSC. We expect to expand the utilization of the MSC for the chondrocyte differentiation intended for future research in human disease mechanism. Based on those mentioned above, this study aims to characterize the SLC39A14 zinc transporter and collagen products of the chondrocytes from human umbilical cord-derived mesenchymal stromal cell.

Preparation of material and methods
The UC derived from Caesarean-delivery, term and non-complicated pregnancy, after exclusion for metabolic and infectious diseases, while a baby born vigorously, with no significant congenital anomaly. The UC derived only after a signed informed consent was documented.
All laboratory works were delivered in a sterilized hood started with artery and venous vessels removal to collect the Wharton's jelly and the remaining tissue. The Collagenase type 1 (Gibco-Nordmark-USA) 3mg/ml was used for enzymatic digestion then incubated for one hour in 5% CO2, 37 o C to get the supernatant for the Monolayer cultures with 10 mL DMEM abundant glucose (DMEM enriched with 4.5g/l glucose and L glutamine, 5% human plasma lysate, 2 mM glutamine, also penicillin 100 u/ml and streptomycin 100 μg/ml) and MesenPRO RS TM medium. 18

MSC culture and harvesting
Cultures were done in a humidified incubator (Thermo Scientific Forma Series 3 Water Jacketed-CO2 incubator-USA) with 5% CO2 and 37 0 C temperature environment. The medium was changed in the first 24 hours to remove debris, then every three days afterward, to observe the appearance of separated cells. 18 At the 70-80% confluence, cell was passaged and subcultured until the third passage (P3).
Cells were harvested after P3, followed by cell counting using Trypan blue (Sigma Aldrich-USA). The live-cell was identified from the transparent and shined appearance, while the dead cell would have a broken membrane, so it absorbed the stain, resulting in blue color under a microscope. A number of live cells were calculated according to the formula: live cells in 4 areas/ 4 x 10 dilution x 10 4 (a constant determined by the manufacturer). Cell viability was calculated using following formula: live cells/total cell x 100%.

MSC differentiation into chondrocyte
For differentiation, the MSC was washed then trypzinized using TrypLE TM Express. After incubation, the cells were rewashed with PBS and resuspended with 0.5 mL Stempro® Chondrogenesis Differentiation Kit™ (Thermo Fisher-life Technologies-USA), in a six-well plate to observe growth and 96 wells plate to optimize cell number in the well for subsequent research procedure. 20 We applied 3200cell/100µL and changed the medium every seven days to avoid damaging the cells, then replace into a basal medium as rapid proliferation was noted. After 21 days, cultures were fixed and stained with 1% Alcian-Blue to identify chondrocytes under a phase-contrast inverted microscope.

Methyl Tetra Toluene assay and Immunofluorescence Staining
We used a 14 days age chondrocyte to carry out Methyl Tetra Toluene (MTT) assay per manufacturer protocol (Thermo Fisher Scientific -USA), Incubation was done at 37°C for 4 hours in the dark, followed by the stop solution (SDS), and read the absorbance at 570 nm. 21 After MTT assay was carried out, we performed the SLC39A14 immunofluorescence staining ( Collagen 2A-10A measurement and data analysis Both Collagen 2A and 10A measurement procedure were based on Sandwich ELISA, using culture medium supernatant according to manufacturer protocol. (LSBio-Seattle, USA). Optical density (OD) value for each well was determined using a microplate reader at 450 nm. 24,25 Cell morphology was observed with 100x magnificent under an inverted microscope (Observer Z1-Zeiss-Germany), and the image was taken using Axio Visio 4.8 software. A 10 x magnification picture was taken using a hand-phone camera (iPhone 7) for immunofluorescence image. For publication preparation, All images from immunofluorescence procedures were equally edited on Photoshop 8.0 software by mounting 3 layers without any further color changes and no additional editing was done. Data was analyzed and presented on GraphPad Prism 8 edition

RESULTS
Our study found that the stellate cells appeared on 5 th day and continue to confluence, when subculture was done, acquiring as much as 1.2175 to 6.65x 10 6 and viability 98.88 to 99.18% MSC, which was confirmed in Figure 1. The immunophenotyping evaluation also showed a positive result for CD73, CD90, and CD105, but negative results for hematopoietic lineage and HLA-DR confirming for mesenchymal stromal cells based on ISCT research standards (Figure 1).
Incubation of the MSC in the chondrocyte differentiation medium produced cells with positive staining in 1% Alcian blue (Figure 2). The positive staining confirms the proteoglycan which arises from chondrocyte cells (Figure 2). The initial cell number of 3200 cells per 100 mL yielded a high proliferation on day 10. Hence, we quit using the differentiation medium and used a maintenance medium instead.
A culture on a-24 wells-plate was done to identify SLC39A14 membrane expression shown positive staining on immunofluorescence

DISCUSSION
The human umbilical cord has raised more attention in recent years as a source for the MSC. 1-3 The ISCT defined minimum criteria for the MSC based on the ability to adhere to plastic dish, positivity on CD73, CD90 and CD105 and negative for CD34, CD45, CD11b, CD19, and HLA-DR, as well as ability for differentiation. This study samples showed 95.7% to 97% positivity for the MSC surface antigen and 3.5% for negative lineage, and ability to differentiate into the chondrocyte. This result determined the isolation of umbilical tissue stromal cells were the MSC.
The chondrocyte which resides typically within a lacuna and embedded in the proteoglycans matrix in the growth plate also shown similar inhabitation while in the monolayer culture in our study. 26 The confirmatory 1% Alcian blue staining even shown the proteoglycans enhancement, including matrix fibril accentuated from the chondrocytes. Our study also shown similar morphology to the study by Oppenheimer et al. 27 The zinc transporter 14 or SLC39A14 protein was a member of the solute carrier family. This carrier was used together with iron, hence getting the name zinc/iron transporter (ZIP), although having more functional role in different cells, such as for manganese influx. 11,12, 28 The disturbance of SLC39A14 prevented zinc influx in the chondrocyte, hence, interfered different pathways resulting in acceleration of hypertrophic phase as documented in various studies. 30, 31 The zinc transporter SLC39A14 was recognized in the hypophysis, liver and growth plate chondrocyte by Hojyo and co-worker in mouse model using insitu hybridization. 9 We used primary culture cell from human UC and provided evidence to identify the SLC39A14 in human chondrocyte using an immunocytochemistry staining. 29 We could not able to find any literature mentioned identification of the SLC39A14 in human chondrocyte previously, hence this study presumably the first. To visualize the immunofluorescence antigen-antibody complex better, we used fluorophore-conjugated antibodies (Alexa Fluor). The staining procedure allowed us to identify membranous location of the SLC39A14 protein on the chondrocyte while the nucleus was identified separately on a counterstaining method with DAPI. This localization would support future research regarding zinc role in the chondrocyte as a continuum for the similar studies in animal. 30, 31 While on monolayer culture, our study showed the chondrocyte produced the collagen, such as collagen type 2A (col2a1) which is typical for the proliferation zone or collagen type 10A (col10A1) in the hypertrophic zone. 26 These products could  microscopy ( Figure 3). According to the immune staining, it was found a positive result (green color) of SLC39A14 receptor using Anti SLC39A14 alexafluor green filter in 40 times magnification. DAPI-blue filter used in this study also emphasized the positive outcomes for nucleus and confirmed the localization of SLC39A14 zinc transporter on the membrane (Figure 3). Our 12 independent samples showed mean Collagen 2A concentration was at 0.097 ng/ml (SE 0.011; 95%CI 0.073-0.12) and mean Collagen 10A level was 31.078 ng/ml (SE 3.792;95%CI 22.870-39.287), as shown in Figure 4. These results indicate that based on the average value, the Collagen 10A level was higher compared with Collagen 2A concentration after 7 days on the differentiation medium and 3 days on maintenance medium ( Figure 4). be used as markers to identify which phase of the differentiation was occurring. These collagen 2A1 and collagen 10A1 were in accordance with Hojyo result also to the study by Yuan et al., using humancartilage-chondrocyte. 9,32 A study conducted by Yuan et al. found that the human UC as a source gave advantage from low risk of environmental exposure or disease compared to adult cartilage. 32

CONCLUSION
This study showed the identification of SLC39A14 zinc transporter in chondrocyte from human UC derived-MSC differentiation and the collagen product. This attempt provided an alternative option for a self-engineered-chondrocyte in cell research.

ETHICAL CONSIDERATION
This study was a part of research: Role of HIV-1 Tat protein to the chondrocyte SLC39A14-zinc transporter and zinc utility as the mechanism of stunting in children with HIV infection.

CONFLICT OF INTEREST
The author reports no conflicts of interest in this work.

FUNDING
The study was funded by the Faculty of Medicine, Universitas Udayana, Bali, Indonesia