Monitoring of transplanted human Mesenchymal Stem Cells from Wharton’s Jelly in xenogeneic systems in vivo

Mesenchymal stem cells (MSCs) are ideal candidates for cell-based therapy aimed at tissue repair and immunomodulation. Aim. To study the survival of transplanted human MSCs from umbilical cord Wharton’s Jelly (hWJ-MSCs) in the animal model of experimental osteoarthritis (OA) in rats after injecting cells into a knee joint and to explore the effect of collagen scaffold on the cell survival in vivo. Methods. MSC isolation and cultivation in vitro. Immunological phenotyping of propagated hWJ-MSCs was performed by fl ow cytometry. The retention of transplanted cells was studied by the PCR revealing of human specifi c sequences in genomic DNA extracted from animal tissues. Results. hWJ-MSCs, both individual and grown on scaffold, were used and it was shown by PCR that human alpha-satellite DNA was detected on the fi rst day in the immunocompetent OA animals inside the injured knee joint. In the collagen matrix (in the model of subcutaneous implantation) human alpha-satellite DNA was detected on the 5th day but was not detected on the 12th day. Conclusions. According to the PCR results, hWJ-MSCs survived in the OA animal model for a short period. Collagenic scaffold increased the residence time of donor cells in the recipients. hWJ-MSCs may be considered as a perspective cell source for the treatment of OA in human.


Introduction
MSCs have been widely investigated over the years due to a combination of unique features such as their availability, ease of cultivation, multiplication ex vivo, and positive therapeutic effects in animal models of various human diseases.A wide application of cell therapy with MSCs in practice depends on complete understanding of mechanisms of their action.MSCs fi rst attracted attention due to their ability to differentiate into different directions [1,2].At present, more and more researchers suggest that the regenerative potential of MSCs is associated with pa-racrine actions that coordinate the complex processes of restoration of damaged tissues and organs [3][4][5][6][7].MSCs have been shown to secrete numerous growth factors like the transforming growth factor β, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, vascular endothelial growth factor, cytokines (interleukin 6, 10) and chemokines, prostaglandins, etc. [8][9][10].
In vitro MSC characteristics usually suggest their in vivo potency.However, the in vitro predictions do not always correlate with the in vivo results.Suc cessful application of MSC-therapy to humans requires the characterization through in vivo animal models, and xenogeneic transplantation is needed for the pilot study of therapeutic features of human MSCs.Although MSCs are immunosuppressive and immunoprivileged and can be tolerated after allotransplantation [11,12], the cells cannot overcome the immunological barrier in a xenogeneic environment [13].However, the MSC therapeutic effi ciency was confi rmed in several different cross-species models [14].Numerous studies have demonstrated that the transplanted xenogeneic MSCs are able to avoid the immune surveillance initially (e.g., during one week after transplantation), but are fi nally rejected even with immunosuppression [15].Despite some limitations of xenogeneic animal models in the study of human MSCs, the animal model assays improve our understanding of the mechanisms underlying the therapeutic effects.
MSCs have a signifi cant potential for the treatment of OA, that is why several MSC-therapies are intensively developed for the OA-associated cartilage defects treatment [16,17].However, many questions remain unanswered such as the required cellular dose, the best source of MSCs, the fate of transplanted cells, etc.] [17].hWJ-MSCs are considered to be much more proliferative, immunosuppressive, and even therapeutically active than other MSCs, and thus, more prospective for the OA treatment [18].The MSCs survival after their transplantation is crucial to the therapeutic effi cacy but there is little information about the fate of transplanted cells.In this context, the aim of the present short-term study was to examine the survival of hWJ-MSCs in immunocompetent OA rats in an early period following the transplantation suggesting that at this stage the immune system contribution to the elimination of cells would not be essential.The survival of collagen scaffold immobilized hWJ-MSCs was also assessed in vivo.

Experimental Design
For the overall experimental design, one source of cells has been assayed in vivo in two site-directed implantation models for examining their retention.hWJ-MSCs were used after the second passage in culture.For Experiment 1 (local injection), culture expanded hWJ-MSCs were injected into a knee joint.For Experiment 2, the cells were seeded in 3-D scaffolds and implanted subcutaneously.All the experiments were performed without immunosuppression.To track the fate of the donor hMSCs, the tissue samples were collected at different time points for PCR analysis.

Laboratory animals
Wistar rats (males with we ight 180-200 g) and mouse line BALB/c were used as experimental animals.The animals were kept under standard conditions.All manipulations with animals were carried out in accordance with the requirements of Guide for the Care and Use of laboratory Animals [19] and the law of Ukraine N 3447-IV of 21.02.2006«The protection of animals from cruel behavior» [20].

Isolation and culture of hWJ-MSCs
The human umbilical cord samples were collected after informed consent of puerpera and processed by the method described earlier [21].To confi rm a proper surface mar kers' expression (CD73, CD90, CD105 and CD34), the ex vivo propagated cells were analysed by fl ow cytometry at passage 2 [22].FACSanalysis of the MSC markers expression (MAbs CD105, CD90, CD73 -BD, USA) was performed with BD FACSAria cell sorter (Becton Dickinson Biosciences, San Jose, CA, USA) and BD FACSDiva software (v 6.1.2).

Obtaining the experimental model of osteoarthritis with I-acetic acid in rats
The animal models were established by means of intra-articular injection of iodine-acetic acid.Rats were injected with 0.1 ml of iodine-acetic acid (3 mg of iodine-acetic acid in 50 μl of 0.9 % NaCl) and monitored for signs of arthritis onset based on paw swelling and clinical scores [23].Animals with osteoarthritic signs (score 2, pronounced edema) were enrolled in the study (n = 19).On day 7, osteoarthritic and intact (n = 10) animals were injected with 1.5  10 6 cells in 100 μl of PBS.Additionally, two animals (sham-operated males) received injections of an equivalent volume of PBS.

Fabrication of collagen scaffolds and cell seeding.
Type I collagen was extracted from bovine tendons using the pepsin-based acidic extraction method described in [24], lyophilized, and stored at -20 °C for further application.Porous collagen scaffolds were prepared by freeze-drying the collagen type I solution (10 mg/ ml) in 0.1 M acetic acid as described earlier [25].The scaffolds were UV-treated, swollen overnight in 0.1 M HEPES pH 7.4, and then equilibrated in cell cultivation medium for 24 hours at 37 °C and 5 % CO 2 .Approximately 1 × 10 6 -1.5 × 10 6 hWJ-MSCs were seeded per 1 cm 2 of scaffold.The cells were cultivated on scaffold for 24 hours at 37 °C and 5 % CO 2 prior to implantation.After implantation for different time points, the scaffolds were removed.

Isolation of DNA
Genomic DNA was extracted from synovial fl uid using the salt out method (according to Grinberg,1989) [26].DNA from synovial tissues (swabs) and scaffolds was isolated as previously described by the method of Bruijn et al. [27].The DNA concentration and purity were determined by optical density using the Thermo Scientifi c NanoDrop 2000 UV-Vis spectrophotometer.

Polymerase chain reaction
To detect transplanted human cells in rats and mice, the polymerase chain reaction analysis was performed using primers to amplify human-specifi c 850bp fragment of the alpha-satellite DNA on human chromosome 17 [28]; polymerase chain reaction was performed as described in the work of Becker et al. [28].Each reaction contained the same amount of genomic DNA template (100 ng in 20 μl of reaction mixture).DNA from cultured hWJ-MSCs served as a positive control and DNA from an untreated animal was used as a negative control.

Results and Discussion
hWJ-MSCs were isolated from Wharton's Jelly, propagated in culture, harvested at the second passage and confi rmed to be MSCs according to the International Society for Cellular Therapy's minimal criteria for defi ning MSCs.In accordance with the FACS analysis hWJ-MSCs were positive for CD73, CD90, and CD105 antigens more than 90 % and were negative for CD34 antigen (< 2 %).The cell viability was confi rmed with trypan blue dye exclusion (> 95 %).The retention of hWJ-MSCs in the animal models was detected through the presence of the human-specifi c alpha-satellite DNA in the analysed samples.A sensitive PCR assay was performed for detection of the human cells in animal tissues.The method has a high sensitivity due to the highly repetitive alpha-satellite sequence.PCR primers we- re specifi c for the hMSC donor DNA with the sensitivity of 1/10 5 -1/10 6 , in serial dilutions.The electrophoregram of alpha-human DNA PCR product is shown in Fig. 1.
To study the survival of hWJ-MSCs in OA animal, the experimental rats were injected with cells through intra-articular route.The animals were sacrifi ced according to the standard protocols for euthanasia after 0.5, 2, 24 hours and 7 days of transplantation.The number of animals selected for PCR analysis was minimized to two at each harvest time point (Table 1).The joints were opened, the synovial washouts and tissues of hWJ-MSCs injected (injured and healthy) and control joints were used to isolate DNA, which was used as a template for PCR (Fig. 2).
As seen from the results of PCR, the human DNA was detected in joint cavity, at least during the fi rst day.At the 7 th day of transplantation, the alpha-satellite DNA in amplifi cation reaction was not detected.Early cell loss was observed in both OA-induced and intact animal groups.The exact date of human DNA clearance remains unclear because the samples were harvested on days 1 and 7 after in vivo hWJ-MSCs delivery.
It is known that biocompatible 3-D scaffolds can prolong the the transplanted cells survival in the injury site.In order to examine whether the collagen scaffolds improve the hWJ-MSCs survival in the xenogeneic setting, the same hWJ-MSCs were seeded to scaffolds and subcutaneously implanted into immunocompetent mice without appropriate immune suppression.The collagen matrices were recovered from the implantation sites with different time points at 1, 5 and 12 days for the isolation of genomic DNA.The results of PCR for the presence of human DNA in the grafts in mice are shown in Fig. 3.
The PCR assay demonstrated the complete loss of human DNA in the samples at the 12 th day.This suggests that hWJ-MSCs survived in the BALB/c mice at least for the fi rst week of post-transplantation.
Using a species-specifi c PCR analysis of the human genomic DNA we have revealed short-time survival of the transplanted cells in two different animal models in vivo.It is known that a local administration of cells promotes a direct physical contact of  donor and recipient cells which leads to a more effi cient exchange of the molecular signals that coordinate regenerative processes.We have observed a sudden cell loss during the fi rst days following the local hWJ-MSCs transplantation in the applied experimental model of OA.The early cell loss during intra-articular injection can occur for a variety of reasons.For example, the infl ux of monocytes/ macrophages into the injured knee joint provides unfavourable conditions for the exogenous stem cell survival, confi rmed by the absence of human DNA in the samples.On the other hand, hypoxia and serum deprivation within the fi rst 24 hours after transplantation contribute to the cell death.Additionally, a sharp decrease in detectability of the human DNA in animal tissues may be associated with the PCR limitation.All mentioned above reasons can be relevant.In contrast, the hWJ-MSCs immobilised in a scaffold avoided an immediate contact with the host immune cells in the early post-transplantation period as they were partially protected and thus did not undergo a direct immune attack by the host within the fi rst week after transplantation.Moreover, a scaffold partially mimics in vivo conditions that prolongs the cell survival.There was an appreciable difference in the survival time of the hWJ-MSCs immobilized on a scaffold and transplanted as a three-dimensional structure compared to those transplanted as the cell suspension.A tendency to early death of the transplanted human MSCs in immunocompetent animals is shown in recent studies [30,31].Toupet et al. reported that survival and biodistribution of xenogeneic MSCs are not affected by the degree of infl ammation in arthritis [31].On the other hand several studies indicated that even such short-term viability of transplanted cells (one day) can be suffi cient to initiate the regeneration of damaged tissues [15,32,33].Therefore the therapeutic potential and functional activity of human MSCs may initially be tested in such animal models.Our data suggest that the optimization of experimental protocols for the hWJ-MSCs isolation, cell preconditioning and cell formulation are required to improve the cell survival after transplantation.

Conclusions
hWJ-MSCs were applied in the OA animal model to assess a potential of these cells for treatment of OA.According to the PCR results, hWJ-MSCs survived in the OA animals for a short period.The collagen scaffold increased the cell residence time in recipients.hWJ-MSCs may be considered as a perspective cell source for the treatment of OA in humans.