Characterization of Human, Ovine and Porcine Mesenchymal Stem Cells from Bone Marrow: Critical In Vitro Comparison with Regard to Humans

For research and clinical use of stem cells, a suitable animal model is necessary. Hence, the aim of this study was to compare human-bone-marrow-derived mesenchymal stem cells (hBMSCs) with those from sheep (oBMSCs) and pigs (pBMSCs). The cells from these three species were examined for their self-renewal potential; proliferation potential; adhesion and migration capacity; adipogenic, osteogenic and chondrogenic differentiation potential; and cell morphology. There was no significant difference between hBMSCs and pBMSCs in terms of self-renewal potential or growth potential. The oBMSCs exhibited a significantly higher doubling time than hBMSCs from passage 7. The migration assay showed significant differences between hBMSCs and pBMSCs and oBMSCs—up to 30 min, hBMSCs were faster than both types and after 60 min faster than pBMSCs. In the adhesion assay, hBMSCs were significantly better than oBMSCs and pBMSCs. When differentiating in the direction of osteogenesis, oBMSCs and pBMSCs have shown a clearer osteogenic potential. In all three species, adipogenesis could only be evaluated qualitatively. The chondrogenic differentiation was successful in hBMSCs and pBMSCs in contrast to oBMSCs. It is also important to note that the cell size of pBMSCs was significantly smaller compared to hBMSCs. Finally, it can be concluded that further comparative studies are needed to draw a clear comparison between hBMSCs and pBMSCs/oBMSCs.


Introduction
As early as the late 1960s, plastic-adherent cells in the bone marrow were discovered, which are known to us today under the disputable name of "mesenchymal stem cells" [1]. Mesenchymal stem cells (MSC) are multipotent, self-renewable cells with the ability to differentiate into various cell types such as chondrocytes, adipocytes and osteoblasts [2]. Consequently, these cells play an important role in many studies of repair and reconstruction of various tissues due to their diverse possibilities [3]. MSCs are not only able to differentiate into a wide variety of specialized cells, but they also have a diverse range of origins. Bone marrow, adipose tissue, umbilical cord, muscle, synovium and placenta are some of the origins from which the cells are isolated [4]. Among those, bone marrow is the most productive and easily accessible source of mesenchymal stem cells. Therefore, BMSCs are most popularly used in clinical research [3].
In order to be able to transfer novel therapies to patients, these must be tested in large animal models in addition to in vitro experiments in the laboratory. Further development of stem cell therapy is also driven by the limitations of current treatment options for various medical problems in different animal species [5]. The choice of the large animal model depends on the purpose and the comparability to humans [6].

Donor Data Recruitment
The human-bone-marrow-derived mesenchymal stem cells (hBMSCs) were obtained from voluntary donors at the Department of Trauma Surgery at Hannover Medical School. After receiving extensive information, the donors consented to bone marrow donation during an elective surgery under anesthesia by iliac crest puncture. The study protocol and process of sample donation complied with the Declaration of Helsinki, and the ethics committee of Hannover Medical School (Votum No. 2562) gave ethical approval. For the present experiment, three donors were randomly selected who met ISCT criteria in previous experiments carried out in the Department of Experimental Trauma Surgery at Hannover Medical School. The porcine-bone-marrow-derived mesenchymal stem cells (pBMSCs) and ovine-bone-marrow-derived mesenchymal stem cells (oBMSCs) were obtained from three animals each euthanized during other animal experiments at the Institute for Laboratory Animal Science (Animal testing application 20/3417) of the Hannover Medical School and as part of a cadaver study at the small animal clinic of the University of Veterinary Medicine Hannover. All donor samples used were collected within 15 min of death and were plastic-adherent. Bone marrow was harvested from the iliac crest in all donors of all species. The human donors were female and aged between 18 and 20 years. The pig and sheep donors were female and approximately three to four years old.

BMSCs Isolation and Cultivation
The extraction and isolation of pBMSC and oBMSC was performed as described before for human BMSC [26]. In brief, a sterile skin incision was made at the iliac crest, followed by insertion of the Jamshidi™ bone marrow puncture needle. The bone marrow was aspirated under maximum suction into the syringes coated with heparin. Samples were stored at 4 • C in a NaCl-heparin mixture for a maximum of 24 h until BMSC isolation.
Afterwards, bone marrow was diluted 1:3 with Phosphate-Buffered Saline (PBS; Dulbecco (#L1825), Biochrom, Berlin, Germany) and separated utilizing a synthetic polysaccharideepichlorohydrin copolymer (Biocoll ® , Biochrom) by centrifugation for 30 min at 500× g without brake. This resulted in the typical gradient phases of plasma, mononuclear cells, Biocoll and erythrocytes. The mononuclear cells, visible as a cloudy white ring, which also contained the BMSCs, were removed, washed again with PBS (Biochrom) and centrifuged with 500× g for 5 min with brake.

Colony-Forming Unit-Fibroblast (CFU-F) Assays and Growth Rate
In order to assess the self-renewal potential of the cells, colony-forming unit-fibroblast assays were performed in passage 4 in this study. For this purpose, BMSCs were seeded as duplicates onto a 6-well plate (9.6 cm 2 ) after expansion at three different concentrations (125, 250 and 500 cells per well) and incubated for 10 days at 37 • C and 5% CO 2 in 3 mL culture medium per well. The cells were fixed with methanol and stained with 1% crystal Life 2023, 13, 718 4 of 16 violet for 30 min. After washing with distilled water, colonies were counted and the number of colonies per 100 cells was calculated [27].
To investigate the proliferation rate, the relative doubling time of the cells in the various cell passages was determined. For this purpose, a defined number of cells was seeded, and when the cells reached 70-90% confluence, they were detached using a 0.05%/0.02% trypsin-EDTA solution (Biochrom). The number of cells at the end of this passage was determined using a Neubauer counting chamber. The following formula was used to calculate doubling time as previously described by Selle et al. [28]: where N 0 = cells seeded, N d = cells counted at the end of the passage and d = days in culture.

Adhesion Assay
The adhesion assay was used to test how many cells adhere to a specific surface (polystyrene, #655180 96 well culture plate, Cellstar) after a defined period of time. For this, a cell suspension with 10,000 cells in 100 µL of culture medium was required per well. In addition, an internal standard with 1000, 2500, 5000, 7500 and 10,000 cells in sixfold determination were applied. Furthermore, a well without cells as a blank control was filled with 100 µL of culture medium. Six wells per donor per time point were each filled with 100 µL of cell suspension (time point t = 0 min). The plate was then incubated at room temperature. After 10, 20, 30 and 60 min, the medium was carefully removed from one experimental series of the 96-well plate and carefully washed with PBS (Biochrom). Then, 100 µL of fresh culture medium was added to the wells. After 60 min (completion of the last time point), 10 µL of WST-1 reagent was quickly pipetted into each well. The plates were then placed in the incubator and incubated at 37 • C and 5% CO 2 . After 90 min, the measurement of absorbance at 450 nm on the Microplate Spectrophotometer (BioTek Instruments, Sursee, Switzerland) was performed and corrected with the absorbance at 630 nm. Afterwards, the blank value was subtracted and the values for each group were averaged.

Migration Assay
This assay was performed to estimate the migration behavior of adherent cells in medium with 1% of serum, here called "deficiency medium". For this purpose, a standardized gap of 500 µm was created in a cell lawn by use of "ibidi inserts" (96 Well Culture Plate, Thermo Fisher Scientific, Schwerte, Germany); it was assumed that the cells would migrate into it to close it. In each compartment of the ibidi chambers, 10,000 cells were seeded in 70 µL of culture medium. The experiment was performed in triplicate and incubation was performed for 24 h at 37 • C and 5% CO 2 . The ibidi inserts were carefully removed afterwards and 3 mL of deficiency medium was added per well. For each scratch, the same four locations were photographed after 12 and 24 h. The cell-free area of the scratch was evaluated using the program ImageJ and calculated as the percentage related to the overgrown area with the Wound Healing Size Tool.

Differentiation
Differentiation in the adipo-, osteo-and chondrogenic directions was performed in passage 4. For adipo-and osteogenesis, 150,000 cells per 9.6 cm 2 (6-well) were seeded and incubated at 37 • C and 5% CO 2 in culture medium. For chondrogenic differentiation, cell pellets were formed in conical tubes of 250,000 cells by centrifugation for 5 min at 200× g and were then incubated at 37 • C and 5% CO 2 in culture medium. The next day, the medium was changed into the respective specific differentiation medium.
On days 0 and 27, the differentiations in osteo-and adipogenesis were stopped. For this, the medium was removed, the wells were washed with 2 mL of PBS (Biochrom) and fixed with 1 mL of 4% formalin for 30 min. Afterwards, the washing was repeated, this time with distilled water.
Chondrogenesis was stopped on day 27 and the chondrogenesis pellets were fixed for histology. Washing and fixation procedures were identical for the 6-well plates. After fixation, the pellet was frozen with Tissue Tek (Tissue Tek OTC Blue, Sakura Finetek, Torrance, CA, USA) embedded, and 5 µm thick cryosections were made (CM 3050S, Leica Biosystems, Wetzlar, Germany).
Calcium ions from osteogenic differentiation that reside in the extracellular mineralized matrix of osteoblasts were stained for 10 min with alizarin red (Roth, 0.5% dissolved in distilled water, pH 4.5). Adipocytes were stained with Oil Red O (Sigma-Aldrich, 5 g/L, dissolved in 60% (w/v) isopropanol) for 25 min. The chondrogenesis pellets were stained with Safranin O for 15 min to analyze glycosaminoglycans in the cartilage. The degree of osteogenic and chondrogenic differentiation was determined by calculating the percentage of stained area relative to total area. For a valid result, three representative images of the respective populations were taken and evaluated.
All analyses were performed blindly without prior knowledge of underlying donor data and with the same settings. For the evaluation of the osteogenesis and adipogenesis, slides of representative images were taken with the light microscope (Olympus CKX41), which were used for all evaluations in this paper, at a magnification of 10 and evaluated using a self-written tool from the Department of Experimental Trauma Surgery of the Hannover Medical School. For the chondrogenesis, the Keyence digital Microscope VHX-7000 was used.

Fluorescence
Cell suspensions of 10,000 and 30,000 cells per mL of culture medium were prepared. For each batch, 3 × 100 µL were seeded into one well of a 96-well plate and incubated for 24 h at 37 • C and 5% CO 2 . The medium from the wells was removed and washed with PBS (Biochrom). Then, 4% formalin was added to the wells and fixed for 20 min at room temperature. Washing was repeated and 0.1% Triton-X-100 (Sigma-Aldrich) in PBS was added for 3 min. The supernatant was removed and washed with PBS. This was followed by adding 100 µL of Phalloidin-iFluor 488 Conjugate (1000X, 2 µL of Stock Solution (1000X) (Biomol, Hamburg, Germany) to 2 mL 1% BSA in PBS (BSA = Albumin bovine Fraction V, pH 7.0, SERVA Electrophoresis, Heidelberg, Germany), which was incubated for 60 min at RT in the dark. After washing with PBS, 100 µL of 2 µM DAPI (Biomol) (4 ,6-Diamidino-2-Phenylindole, Dihydrochloride, cell) was added for 5 min and incubated at room temperature in the dark. Images were evaluated using a mercury vapor lamp on the microscope (Olympus CKX41). DAPI staining becomes optimally visible with an exposure time of 80-140 ms, and Phalloidin-iFluor 488 Conjugate ranges from 1.25 to 2.5 s. The cell size was evaluated using the program ImageJ. To evaluate the results of fluorescence, the length and area of 10 cells were measured and compared using the Measure tool of the program ImageJ-win64.

Statistics
The statistical data analysis in this work was performed with the program GraphPad Prism version 9.3.1. The data analysis of the samples showed a normal distribution. Accordingly, group comparisons were performed with one-way ANOVA with Tukey's post hoc test for correction of multiple comparisons. Grouped data were analyzed via two-way ANOVA with Sidak's post hoc test for correction of multiple comparisons. The graphical representation of the results is shown as scatter dot plots.
Descriptive p-values were determined. A p-value less or equal to 0.05 was considered as a statistically significant difference. In the statistical evaluation of the results, the mean (M) and standard deviation (SD) are given. Biological and technical replicates are denoted with "n" and "N", respectively.

Results
BMSCs from humans, sheep and pigs were compared in terms of their morphology (fluorescence microscopy), stem cell characteristics (CFU-F assay), growth behavior (proliferation capacity) and differentiation ability in the direction of osteogenesis, chondrogenesis and adipogenesis.

Fluorescence Microscopy
As shown in Figure 1, hBMSCs had a shape with thin, long processes. The length of the hBMSCs ranged between 90 and 150 µm (M: 122 µm; SD 1255.23 µm). The oBMSCs were slightly broader in shape and their length ranged from 90 to 170 µm (M = 133 µm; SD: 1022.66 µm). The pBMSCs were small and more round compared to the other species. The length of the pBMSCs ranged between 30 and 70 µm (M = 51 µm; SD: 606.17 µm). The nuclei were of comparable sizes in all three species with a diameter of about 14 µm. The area comparison is shown in Figure 2. The mean area of hBMSCs was 2105.85 µm 2 , that of pBMSCs was 592.82 µm 2 and that of oBMSCs was 2444.94 µm 2 . pBMSC are significantly smaller (p = 0.004, Figure 2). room temperature in the dark. Images were evaluated using a mercury vapor lamp on the microscope (Olympus CKX41). DAPI staining becomes optimally visible with an exposure time of 80-140 ms, and Phalloidin-iFluor 488 Conjugate ranges from 1.25 to 2.5 s. The cell size was evaluated using the program ImageJ. To evaluate the results of fluorescence, the length and area of 10 cells were measured and compared using the Measure tool of the program ImageJ-win64.

Statistics
The statistical data analysis in this work was performed with the program GraphPad Prism version 9.3.1. The data analysis of the samples showed a normal distribution. Accordingly, group comparisons were performed with one-way ANOVA with Tukey's post hoc test for correction of multiple comparisons. Grouped data were analyzed via two-way ANOVA with Sidak's post hoc test for correction of multiple comparisons. The graphical representation of the results is shown as scatter dot plots.
Descriptive p-values were determined. A p-value less or equal to 0.05 was considered as a statistically significant difference. In the statistical evaluation of the results, the mean (M) and standard deviation (SD) are given. Biological and technical replicates are denoted with "n" and "N", respectively.

Results
BMSCs from humans, sheep and pigs were compared in terms of their morphology (fluorescence microscopy), stem cell characteristics (CFU-F assay), growth behavior (proliferation capacity) and differentiation ability in the direction of osteogenesis, chondrogenesis and adipogenesis.

Fluorescence Microscopy
As shown in Figure 1, hBMSCs had a shape with thin, long processes. The length of the hBMSCs ranged between 90 and 150 µm (M: 122 µm; SD 1255.23 µm). The oBMSCs were slightly broader in shape and their length ranged from 90 to 170 µm (M = 133 µm; SD: 1022.66 µm). The pBMSCs were small and more round compared to the other species. The length of the pBMSCs ranged between 30 and 70 µm (M = 51 µm; SD: 606.17 µm). The nuclei were of comparable sizes in all three species with a diameter of about 14 µm. The area comparison is shown in Figure 2. The mean area of hBMSCs was 2105.85 µm 2 , that of pBMSCs was 592.82 µm 2 and that of oBMSCs was 2444.94 µm 2 . pBMSC are significantly smaller (p = 0.004, Figure 2).

Colony-Forming Unit-Fibroblast Assays and Growth Rate
Evaluation of the CFU assay in Figure 3 shows that pBMSCs with a mean of 7.07% had a comparable renewal potential to hBMSCs with 6.27% (p = 0.84). With 1.73%, oBMSC could only form a third of the colonies compared to hBMSC (p = 0.01).
The doubling time in days was comparable in all species in passages 3 to 6 (p ≥ 0.05), whereby oBMSCs showed a wide variance in passages 4-7 ( Figure 4). In passage 7, oBMSCs required a significantly higher doubling time than hBMSCs (p = 0.01) (Figure 4).

Colony-Forming Unit-Fibroblast Assays and Growth Rate
Evaluation of the CFU assay in Figure 3 shows that pBMSCs with a mean of 7.07 had a comparable renewal potential to hBMSCs with 6.27% (p = 0.84). With 1.73%, oBMS could only form a third of the colonies compared to hBMSC (p = 0.01).

Colony-Forming Unit-Fibroblast Assays and Growth Rate
Evaluation of the CFU assay in Figure 3 shows that pBMSCs with a mean of 7.07 had a comparable renewal potential to hBMSCs with 6.27% (p = 0.84). With 1.73%, oBM could only form a third of the colonies compared to hBMSC (p = 0.01). The doubling time in days was comparable in all species in passages 3 to 6 (p ≥ 0.0 whereby oBMSCs showed a wide variance in passages 4-7 ( Figure 4). In passage 7, oB SCs required a significantly higher doubling time than hBMSCs (p = 0.01) (Figure 4).

Adhesion Assay
The adhesion capacity of all three species at four different time points is shown in Figure 5. After 10 min, hBMSCs with 20.9% (SD 3.58%) were significantly different from both pBMSCs with 1.21% (SD 2.1%) (p = 0.006) and oBMSCs with 2.1% (SD 3.14%) (p = 0.006). In general, hBMSCs are found to adhere faster than oBMSCs and pBMSCs.

Adhesion Assay
The adhesion capacity of all three species at four different time points is shown in Figure 5. After 10 min, hBMSCs with 20.9% (SD 3.58%) were significantly different from both pBMSCs with 1.21% (SD 2.1%) (p = 0.006) and oBMSCs with 2.1% (SD 3.14%) (p = 0.006). In general, hBMSCs are found to adhere faster than oBMSCs and pBMSCs.
After one hour, the adherence of the hBMSCs was comparable to the 30 min time point. The adherence of oBMSCs reached 31.86% (SD 21.1%) after 1 h, whereas the adhesion of pBMSCs remained low with 4.17% (SD 3.85%) (p = 0.04) compared to hBMSCs. After 24 h, the BMSCs from all three species were adherent.
After 24 h, the migration capacity of both oBMSCs with 44.8% (SD 16%) and pBMSCs with 50.9% (SD 6.6%) increased compared to the time point 12 h ( Figure 6). However, it was still significantly lower compared to hBMSC with 90.2% (SD 11.5%). For oBMSCs and pBMSCs, a significant reduction in the migration capacity with p ≤ 0.001 was measurable compared to hBMSCs. <2% (SD 0.95%). There was once again a significant difference between pBMSCs (p = 0.004) and the oBMSCs (p = 0.01) compared to hBMSCs.
After one hour, the adherence of the hBMSCs was comparable to the 30 min time point. The adherence of oBMSCs reached 31.86% (SD 21.1%) after 1 h, whereas the adhesion of pBMSCs remained low with 4.17% (SD 3.85%) (p = 0.04) compared to hBMSCs. After 24 h, the BMSCs from all three species were adherent.
After 24 h, the migration capacity of both oBMSCs with 44.8% (SD 16%) and pBMSCs with 50.9% (SD 6.6%) increased compared to the time point 12 h ( Figure 6). However, it was still significantly lower compared to hBMSC with 90.2% (SD 11.5%). For oBMSCs and pBMSCs, a significant reduction in the migration capacity with p ≤ 0.001 was measurable compared to hBMSCs. Figure 6. Migration assay. Degree of migration (%) in oBMSCs and pBMSCs compared to hBMSCs after 12 and 24 h. Significant differences between the two animal species and hBMSCs were measurable after both time points, where hBMSCs migrated the fastest. n = 3, N = 3 ** p < 0.002, *** p < 0.001.

Osteogenic Differentiation
Representative pictures of alizarin red staining showing mineral depositions of calcium are shown in Figure 7A on day 27 of differentiation of hBMSCs, oBMSCs and pBM-SCs. All donors of each species were able to induce osteogenic differentiation (Figure 7). On day 27, 75.6% (SD 21.7%) of the area of hBMSCs, 92.5% (SD 3.7%) of the area of oBM-SCs and 97.9% (SD 4.3%) of the area of pBMSCs were positively stained with alizarin red. These results indicated a better osteogenic differentiation capacity of oBMSCs (p = 0.03) ✱✱ ✱✱✱ ✱✱✱ ✱✱✱ Figure 6. Migration assay. Degree of migration (%) in oBMSCs and pBMSCs compared to hBMSCs after 12 and 24 h. Significant differences between the two animal species and hBMSCs were measurable after both time points, where hBMSCs migrated the fastest. n = 3, N = 3 ** p < 0.002, *** p < 0.001.

Osteogenic Differentiation
Representative pictures of alizarin red staining showing mineral depositions of calcium are shown in Figure 7A on day 27 of differentiation of hBMSCs, oBMSCs and pBMSCs. All donors of each species were able to induce osteogenic differentiation (Figure 7). On day 27, 75.6% (SD 21.7%) of the area of hBMSCs, 92.5% (SD 3.7%) of the area of oBMSCs and 97.9% (SD 4.3%) of the area of pBMSCs were positively stained with alizarin red. These results indicated a better osteogenic differentiation capacity of oBMSCs (p = 0.03) and pBMSCs (p = 0.006) compared to hBMSCs. As expected, the non-induced samples showed no signs of osteogenic cell differentiation.  Figure 8 shows the differentiation properties of the stem cells of the compared species towards adipogenesis. Each species was visibly suitable for adipogenic differentiation, but due to technical difficulties in washing off the cells, only a qualitative statement was possible. As expected, the non-induced samples showed no signs of adipogenic cell differentiation. Figure 8 shows the differentiation properties of the stem cells of the compared species towards adipogenesis. Each species was visibly suitable for adipogenic differentiation, but due to technical difficulties in washing off the cells, only a qualitative statement was possible. As expected, the non-induced samples showed no signs of adipogenic cell differentiation.

Chondrogenic Differentiation
Representative pictures of chondrogenic differentiation after 27 days are shown in Figure 9A. Quantitative evaluation revealed a chondrogenesis level with 16.54% in hBM-SCs (SD = 3.95%) and 23.16% in pBMSCs (SD = 13.4%) on day 27. Only 0.18% were stained with Safranin O in the oBMSCs (SD = 0.2%). These results indicate no significant chondrogenic differentiation between hBMSCs compared to pBMSCs and oBMSCs. Only hBMSCs and pBMSCs were able to perform a chondrogenic differentiation in pellet culture in all three donors. In sheep, no differences were observed between the pellets incubated in control medium or chondrogenic medium. As expected, the non-induced samples showed no signs of chondrogenic cell differentiation.

Chondrogenic Differentiation
Representative pictures of chondrogenic differentiation after 27 days are shown in Figure 9A. Quantitative evaluation revealed a chondrogenesis level with 16.54% in hBMSCs (SD = 3.95%) and 23.16% in pBMSCs (SD = 13.4%) on day 27. Only 0.18% were stained with Safranin O in the oBMSCs (SD = 0.2%). These results indicate no significant chondrogenic differentiation between hBMSCs compared to pBMSCs and oBMSCs. Only hBMSCs and pBMSCs were able to perform a chondrogenic differentiation in pellet culture in all three donors. In sheep, no differences were observed between the pellets incubated in control medium or chondrogenic medium. As expected, the non-induced samples showed no signs of chondrogenic cell differentiation.

Discussion
Since cell-based methods are being used more frequently in innovative medicine aim of this study was to determine whether porcine-or ovine-bone marrow-derived enchymal stem cells, which were isolated and cultured with the established hBMSC tocol, are more similar to human-bone-marrow-derived mesenchymal stem cells in o to select an appropriate animal model for preclinical research.
The results showed that ovine BMSCs have a similar cellular appearance to hBM with adequate osteogenic and adipogenic differentiation potential, but an impaired c drogenic differentiation potential. The time to adhere to the cell culture plate and the gration time were prolonged for oBMSCs compared to hBMSCs. Porcine BMSCs ha

Discussion
Since cell-based methods are being used more frequently in innovative medicine, the aim of this study was to determine whether porcine-or ovine-bone marrow-derived mesenchymal stem cells, which were isolated and cultured with the established hBMSC protocol, are more similar to human-bone-marrow-derived mesenchymal stem cells in order to select an appropriate animal model for preclinical research.
The results showed that ovine BMSCs have a similar cellular appearance to hBMSCs with adequate osteogenic and adipogenic differentiation potential, but an impaired chondrogenic differentiation potential. The time to adhere to the cell culture plate and the migration time were prolonged for oBMSCs compared to hBMSCs. Porcine BMSCs have a different cellular appearance with a smaller and more round cell body, but suitable osteogenic and chondrogenic differentiation capacity. The adhesion capacity is slower than oBMSCs and slower compared to hBMSCs, whereas the migration capacity is faster than oBMSCs but slower compared to hBMSCs. Apart from this study, there is no experimental setup with all three species under the same culture conditions for direct comparison [18,20,29]. Consistent with our study, Schweizer et al. showed that cell sizes in fluorescence staining between pBMSCs and hBMSCs were significantly different and hBMSCs had a cell body almost twice as large [26]. Furthermore, Noort et al. showed that there was no difference in growth potential between hMSCs and pMSCs over a 3-week period [18]. Cells of both species were cultured in a different basal medium (medium 199) with dextrose as the carbohydrate source, to which an endothelial cell growth factor and heparin were added.
In our study, the carbohydrate source was glucose, and in addition, FGF2 was used as a growth factor and no heparin was added [18]. Despite differences in the medium, the results of our study were confirmed regarding the pBMSCs. Between hBMSCs and oBMSCs, a significant difference was only visible from passage 7. However, this finding should be interpreted with caution due to the high variability of the individual data from this passage. In order to be able to make an exact statement on this point, repetitions in late passages with several donors are necessary. Rentsch et al. showed that the doubling time of sheep MSCs is 1.2-fold higher than hBMSCs [30]. A comparable medium was used as in the present work and the doubling time was about 50 h calculated on average over five passages. In contrast, we could only show in passage 4 that the mean of the doubling time of the sheep was higher than that of the human.
Cell migration is an important function that plays a major role in tissue repair processes [31]. In this work, migration assay was used to compare the stem cells of different species in terms of migration with the same environmental factors. In our study, a less significant difference was found between the animal species compared to hBMSCs. This could be related to the results of the adhesion assay, as hBMSCs also performed significantly better than oBMSCs and pBMSCs in the migration assay. However, since the adhesion and migration assays were performed under conditions designed for the cultivation of human BMSC, the results may be different with customized media tailored to the needs of oBMSCs or pBMSCs. Osteogenic differentiation was significantly increased in oBMSCs and pBMSCs compared to hBMSCs in our study. These results are in line with the publication of Haddouti et al., who demonstrated a strong osteogenic potential of oBMSCs as early as 21 days, which showed a slower mineralization process than hBMSCs, under conditions comparable to our osteogenesis protocol and using the same staining with alizarin red [6]. The literature also includes studies that have tested the osteogenic potential on so-called 3D scaffolds. However, these in vivo experiments have shown that oBMSCs not only differentiate osteogenically on more materials than hBMSCs, but also form a higher percentage of bone than hBMSCs [29]. Noort et al. examined osteogenic differentiation of pBMSCs under three different protocols that are for humans, in which differentiation was successful in most donors [18].
After completion of adipogenic differentiation and staining, hBMSCs had more stained fat vacuoles compared with oBMSCs and pBMSCs in the current study. Especially for pBMSCs, this might be due to the fact that fat vacuoles are secreted during differentiation, and consequently, vacuoles are no longer fixed intracellularly. Thus, wash-off of the stained vacuoles could have happened during the staining and washing process. This observation is in agreement with a study performed with pBMSCs [18]. The reason for this could additionally be excessive lipid formation leading to many unfixed vacuoles. In this context, Noort et al. showed that pMSCs formed fat much faster than hMSCs [18]. For oBMSCs, there are only publications that provide a successful adipogenic differentiation but do not report about the morphology of the fat vacuoles. Confirmation of the success of adipogenesis in sheep was additionally provided by the study of McCarty et al. Again, positive results were obtained after four weeks in induced medium intended for human stem cells. However, the number of stained fat vacuoles was also low, which is consistent with our study [32]. Regarding the chondrogenic differentiation, our study showed that pBMSCs are comparable to hBMSCs. This is in line with the findings of Noort et al., supporting the findings that pMSCs are able to differentiate in the chondrogenic direction. However, chondrogenic differentiation in oBMSCs was not successful in our study. In turn, Haddouti et al. showed that oMSCs in pellet culture can perform chondrogenesis after 21 days. They cultured them on agarose gel and stained with Alcian Blue, and the medium was comparable to the one used in our study [6]. In addition, Zscharnack et al. showed that chondrogenic differentiation of oMSCs is enhanced under low oxygen conditions (5% O 2 ) [33]. Furthermore, mechanical stimulation showed the improvement of chondrogenesis potential [34].

Limitations
As this study deals with donated hBMSCs and BMSCs from pigs and sheep which were sacrificed due to other reasons, some limitations must be considered. We used BMSCs from healthy, middle-aged patients in this study, which cannot reflect the complex human situation in a clinical setting with different comorbidities. Apart from that, the age and sex of all human and other BMSC donors were not taken into consideration. In addition, the conditions chosen for BMSC isolation and cultivation were optimally designed for hBMSCs. There is a lack of uniform protocols for working with oBMSCs and pBMSCs that are optimal for the cells and can be applied uniformly across laboratories. Further, the site

Conclusions
The results on the properties of stem cells from sheep and pigs in terms of in vitro self-renewal, growth, adhesion and migration potential, as well as differentiability and cell size, showed various differences compared to hBMSCs.
Based on cell morphology, oBMSCs are more comparable to hBMSCs, while pBMSCs are more comparable to hBMSCs based on successful chondrogenic differentiation and their self-renewal potential. Under the standardized culture conditions defined here, various differences between species are thus present, so caution should be exercised in in vitro studies when directly transferring oBMSCs and pBMSCs to humans. However, no clear recommendation can be made; thus, further comparative studies are necessary. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.