Lapine CD133 + CD34 + endothelial progenitor cells for 1 musculoskeletal research in preclinical animal trials

sorted using CD34 + CD133 and CD34 + VEGFR-2 + Further, with analysis, were found to be one third (3%) as compared to human CD34 + CD133 + EPCs (12%). These CD34 + CD133 + rEPCs/hEPCs were double-positive for 63 acLDL uptake, ULEX binding, CD34, CD309, and CD31; whereas negative for CD133, CD14 64 and CD45. Also, EPCs from both species demonstrated functional characterization.

To our knowledge, this is the first study to characterize rabbit EPCs in the full spectrum. 100 Further, we hypothesized that the rEPCs were similar to hEPCs regarding the proliferative 101 potential, functional characterization, and the expression of surface markers. Hence, rEPCs 102 could provide an excellent research model for translational research in medicine. Additionally, 103 an in-depth understanding of the origin of EPC will be beneficial to researchers and the 104 Subsequently, the buffy coat containing rabbit MNCs at the interface was carefully withdrawn. 132 On the other hand, Ficoll-Paque Premium (GE Healthcare Biosciences, PA, USA) was used 133 according to the manufacturer's instructions to isolate MNCs from human blood. Briefly, the 134 blood was first diluted with PBS (1:1 ratio). The diluted blood (4 ml) was then carefully layered 135 over Ficoll-Paque Premium (3 ml), followed by centrifugation at 400 × g for 40 mins. These 136 MNCs (buffy coat) at the interface were then carefully withdrawn and processed further. 137

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The MNCs from the rabbit and human PB were washed twice with PBS at 100×g to remove 139 separating media and platelets. These MNCs were then plated at three different densities of 140 0.5´10 6 cells, 1´10 6 cells/cm 2 and 5´10 6 cells/cm 2 (n=3) in the custom coated six-well plates 141 (2.5 µg/cm 2, human plasma fibronectin, Gibco; ThermoFisher Scientific, MA, USA) [1] in the 142 medium X [EGM-2 or EGM-2MV; Lonza, Basel, Switzerland)]. The MNCs were cultured 143 according to the depletion technique or the re-plating technique (Hills technique) in either 144 EGM-2 or EGM-2MV supplemented with an additional 8%, and 5% fetal bovine serum (FBS) 145 respectively. acLDL uptake and lectin binding were used to test the efficacy of the protocols 146 or the type of medium in EPCs yield. After the first medium change, the cells were carefully 147 monitored under a microscope every day for 14 days. 148

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After testing appropriate culture conditions, second and subsequent media was changed 150 accordingly. At 70-80 % confluency, EPCs were passaged to obtain a sufficient number for 9 double sorting (CD34 + CD133 + or CD34 + VEGFR-2 + ) with FACS. The protocol was described 152 later in this section, along with the analysis of surface markers. 153 Culturing and expansion of sorted CD34 + CD133 + or CD34 + VEGFR-2 + EPCs 154 Both sorted CD34 + CD133 + or CD34 + VEGFR-2 + EPCs were cultured, expanded, and evaluated 155 for their characteristic morphology by microscopic examination. The EPCs which maintained 156 their morphology over subsequent passaging were used for analysis and comparison. 157

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The proliferative potential of EPCs was determined by the CCK-8 assay (Sigma-Aldrich). In 159 brief, rCD34 + CD133 + EPCs and hCD34 + CD133 + EPCs (r/h CD34 + CD133 + EPCs) at P5 passage 160 were seeded in 96-well plates at a density of 1000 cells/well and cultured in medium X at 37°C 161 in a humidified atmosphere containing 5% CO 2 . The Optical density (OD) was measured every 162 two days for two weeks. On the day of evaluation, medium X was replaced by a phenol red-163 free culture medium containing 5% FBS, 1% P/S (working medium), and 10μL of CCK-8. The 164 plates were measured for absorbance at 460 nm (OD) by a microplate reader after incubating 165 for 2 hrs at 37°C. The experiment was performed in triplicate, with each set having three wells. After obtaining the homogeneous CD34 + CD133 + /CD34 + VEGFR-2 + EPCs by FACs, the 173 percentage of cell surface markers, such as CD34, CD133, CD31, VEGFR-2, CD45, and CD14 10 were evaluated by expanding the respective cell population. 1.5-3.0×10 6 cells and 0.5-1.0×10 6 175 cells were used for sorting and analysis, respectively. The cells were simultaneously blocked 176 (0.1% BSA) and incubated with the antibody (Supplementary Table 1

246
Rabbit and human blood sampling 247 Both the rabbit and human blood were successfully withdrawn. However, extracting rabbit 248 blood was technically challenging and required extended time as compared to removing human 249 blood (Fig. 1.A). After centrifugation, the buffy coat of MNCs (Fig. 1.B.1) was carefully 250 pipetted and transferred into sterile tubes (Fig. 1.B.2). However, while withdrawing the MNCs, 251 if any blood contamination occurred, the pipettes were discarded immediately. 252

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We found that MNCs at 1.0´10 6 cells/cm 2 yielded optimal results. No ECs were obtained at 254 0.5´10 6 cells, whereas at 5.0´10 6 cells/cm 2 , cell clumping, and cell death occurred (Fig. 2.A). 255 In our preliminary experiments, the Hills technique was found to be ineffective and was only 256 successful in 20% of cases (1/5 blood samples) tested, whereas the depletion technique yielded 257 EPCs in 40% of cases (2/5 blood samples). Both EGM-2 and EGM-2MV gave positive results, 258 but EPCs in EGM-2MV emerged earlier and greater in number as observed microscopically 259 ( Fig. 1.B). acLDL uptake and lectin binding confirmed the emergence of EPCs (Fig. 3). In the 260 depletion technique, the first medium was changed on the fourth day for both rabbit and human 261 MNC's culture. From day 5 to day 7, 28% (7/25) of rabbit blood samples (rBS) and 40% (4/10) 262 of human blood samples (hBS) yielded cobblestone EPCs in three different patterns; blood 263 island-like cell clusters, biphasic EPCs, and de novo EPCs (Fig. 3). Spindle-shaped cells (Fig.  264 3A) occurred in the rest of the cases (referred to as eEPCs) and died within 1-2 weeks, whereas 265 blood island-like cell clusters (Fig. 3B) and biphasic EPCs (Fig. 3C) contained a mixture of 266 eEPCs and lEPCs (ECFCs). De novo EPCs (Fig. 3D) were essentially the lEPCs (ECFCs) only. 267

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The growth curve (Fig. 5C, Table 2) revealed a statistically significant difference in overall 280 time points (P<0.05) as wells as between time points (P<0.05) and groups (P<0.05). 281 Significant growth was observed from day 2 to day 8 (P<0.05). PDT for rEPCS was 21.18 hrs, 282 whereas, for hEPCs, it was 20.01 hrs. Overall, both rEPCs and hEPCs revealed a similar growth 283 pattern. 284

eNOS assay 304
A significantly increased NO production in both r/h CD34 + CD133 + EPCs was observed with 305 0.5µM of b NADPH. Further, at different concentrations of b NADPH, it was found that the 306 rEPCs released higher but statistically insignificant (P>0.05) NO as compared to hEPCs (Fig.  307 9B, Table 3). 308 16

309
As blood is the source of EPCs, the foremost question was how much blood could be withdrawn 310 from an animal/human? A rule of thumb is the 10 percent-10 percent rule. Accordingly, safe 311 sampling volume is 10 percent of the total blood volume, which is estimated to be 10 percent 312 of the animal's body weight. In other words, safe sampling volume is 1% of total body weight 313 [27]. Therefore, 30 ml of blood was withdrawn from the rabbit of approximately 3 kg, whereas 314 in humans, a standard 30 ml of blood was collected. It was also observed that the greater the 315 amount of blood, the higher were the chances of isolating EPCs because only 0.05-0.2 316 lEPCs/ml are present in the blood [33]. Various studies had also collected a similar amount of 317 blood (30-100 ml) for isolating EPCs [9,13,34,35]. 318 If lEPCs are difficult to isolate, the next question was, how many cells should be seeded/cm 2 319 of the culture ware? There were two essential considerations; firstly, efficient MNC recovery, 320 and secondly, appropriate seeding density. MNCs were isolated by various techniques, such 321 as; magnetic bead separation, FACS, and density gradient centrifugation. However, density 322 gradient centrifugation with Ficoll-Paque is the most widely used technique. For isolation of 323 hMNCs, we adhered to Ficoll-Paque Premium. However, for isolation rMNCs, we used a dual 324 Histopaque technique because the density of rabbit MNCs is higher as compared to humans, 325 and that could give the highest and purest mononuclear cell recovery [29]. As the density of 326 solution increases, the osmotic shock is reduced in Histopaque as compared to Ficoll-Paque, 327 thereby reducing cell death and increasing cell recovery [29]. 328 329 We also found that only 1.0´10 6 MNCs/cm 2 yielded EPCs. In another study, 1.5´10 6 cells/cm 2 330 was used as the initial seeding density. However, researchers did not test the minimum cell 331 concentration required for EPCs isolation [34]. Another important consideration is whether the 332 coating of culture ware can improve the efficiency of EPCs isolation? We used custom made 333 fibronectin-coated dishes instead of collagen because morpho-differentiation and proliferation 334 of EPCs were found to be significantly higher in fibronectin-coated dishes/flasks as compared 335 to the collagen-coated dishes [1]. 336 Culture media is decisive for defining cell characteristics. In literature, there is a discrepancy 337 in the type of media used for the isolation of EPCs from PBMNCs [9, 35-37]. In our study, we 338 found that EPCs emerged on day six in EGM-2MV as compared to day 9 in EGM-2. Further, 339 uptake of acLDL and UEA-1 uptake was higher in EGM-2MV as compared to EGM-2, 340 indicating that they were more metabolically active in EGM-2MV than EGM-2. Our results 341 were in correspondence with another study in which EGM-2MV increased colony count, cell 342 differentiation, adhesion, tubulogenic potential, and NO production of EPCs cells, particularly 343 lEPCs [38]. In this study, we were able to successfully isolate CD34 + CD133 + from the heterogenous EPC 355 population and expand EPCs. However, when the expanded r/hCD34 + CD133 + EPCs 356 18 populations were further analyzed, CD133 was found to absent from both cell populations. The 357 results confirmed that CD133 is an early marker, and when sorted CD34 + CD133 + were 358 expanded, they transformed into mature cells and lost their CD133 expression. The results were 359 supported by another study where the authors found that EPCs differentiated into mature cells 360 having cobblestone appearance (lEPCs), which were CD34 + VEGRF-2 + but CD133 -[17]. In 361 another study, researchers were able to generate endothelial cells (lEPCs) from CD133 + cells 362 [18]. In sharp contrast, some researchers were neither able to detect VEGFR-2 transcripts in 363 CD133 + cells nor generate lEPCs from CD133 + cells. They hypothesized that CD34 + VEGFR-364 2 + CD133cells within the heterogenous CD34 + fraction might be the origin of the lEPCs [16]. 365 The tubulogenic assay is a specific assay for endothelial cells. In the present study, 366 characteristic tubules were formed on Matrigel. eEPCs failed to create a tube-like structure, 367 whereas lEPCs did, indicating an inferior endothelial function of eEPCs in vitro. lEPCs 368 expressed FLK-1 which might be responsible for VEGF mediated tube formation [9]. In the 369 current study, both rCD34 + CD133 + EPCs and hCD34 + CD133 + EPCs did form a tubular 370 network, as seen on microscopic examination, but the junctional area, tubule thickness, and 371 length of tubules were larger in rEPCs as compared to hEPCs. It might explain why rabbits are 372 called as potential healers as compared to humans. 373 The selectivity of endothelial cells can also be demonstrated by the ability to bind with DiI 374 acLDL [30] and UEA-1 uptake [31]. The distinct advantages of Dil-ac-LDL assay include its 375 reproducibility, uniform labeling without any permeabilization or fixing, non-sensitivity to 376 trypsinization, and no effect on the growth rate of endothelial cells. On the other hand, lectin 377 binds specifically to L-fructose residues on endothelial cells [41]. Both types of EPCs bind 378 with acLDL and uptake UEA-1, but the level of binding and uptake in eEPCs was less as 379 compared to lEPCs suggesting higher metabolic activity in lEPCs [9]. Our results revealed the 380 19 same that the uptake and binding intensity in the primary culture of EPCs were significantly 381 less than that in homogenous and sorted CD34 + CD133 + EPCs. 382 The detection of the release of NO is another functional assay to characterize EPCs. NO is 383 generated in vivo through the conversion of L-arginine to L-citrulline by NO synthase (NOS). 384 It mediates many physiological and pathophysiological processes in the human body [42]. 385 DAF-2DA is a highly sensitive, specific, and a membrane-permeable fluorescent probe where 386 DAF-2 reacts rapidly and irreversibly in solution with NO and NO-derived reactive species in 387 a concentration-dependent manner to produce the highly fluorescent product 388 triazolofluorescein (DAF-2T) [32]. We measured the RFI of CD34 + CD133 + EPCs by using b 389 NADPH as it is one of the co-factors in the pathways of NO production [43]. We found no 390 significant difference in the production of NO in either human or rabbit EPCs. 391 The other exciting finding in the present study, apart from the similarities between rEPCs and 392 hEPCs, was the emergence of two distinct colonies of EPCs. Classically, EPCs were suggested 393 to grow from blood-island cell-like clusters that have round cells in the center and spindle shape 394 cells in the periphery. The spindle-shaped cells are traditionally classified as eEPCs because 395 they appear earlier in culture, whereas lEPCs, emerged later in the culture. We found two other 396 patterns apart from blood-island like structures, biphasic EPCs, and de novo late EPCs. In 397 "biphasic EPCs", lEPCs did not need to emerge after eEPCs. In fact, both eEPCs and lEPCs 398 were simultaneously growing together, and because early EPCs have a short life span, they 399 eventually die, allowing lEPCs to proliferate and increase in number. The corresponding 400 presence of both eEPCs and lEPCs in our study, proved previous studies stipulating lEPCs 401 arise from cells other than eEPCs [9, 15, 35]. On the other hand, "de novo lEPCs" are termed 402 so because these lEPCs emerge without any significant eEPCs in a culture. The said pattern is 403 reminiscent of endothelial colony-forming cells (ECFCs) described in various studies [13, 33, 404 44]. Therefore, the appearance of biphasic EPCs and de novo lEPCs suggested that some lEPCs 405  representative images taken on day 6 1) 0.5´10 6 cells/cm 2 2) 1.0´10 6 cells/cm 2 3) 5.0´10 6 631 cells/cm 2 (n=3, 10X, Pixel size: 0.9 µm, Scale bar: 100 μm) B. Influence of culture medium. 632 Emergence of rEPCs on day 6 after culturing 1.0´10 6 cells/cm 2 in different mediums. 633 Representative images (n=3, 10X, Pixel size: 0.9 µm, Scale bar: 100 μm) in 1) EGM-2 medium 634 2) EGM-2MV medium. 635 tubule length, whereas, mean (μm 2 ±SD) junctional area were analyzed in eight randomly 667 selected areas at 5 hrs as well as 10 hrs. The mean surface area of rEPCs was significantly 668 higher than the mean surface area of hEPCs (P<0.05) at both 5 hrs and 10 hrs. rCD34 + CD133 + 669 35 had a significantly thicker junctional area, tubule thickness, and a longer tubule length (P<0.05) 670 as compared to hCD34 + CD133 + EPCs. 671