2 Gene expression profiling of canine adipose-derived stem...

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Gene expression profiling of canine adipose-derived stem cell preparations: 4 5 Effects of tissue preparation and photo-activation 6 7 8

9 10 11 12 13 Donald A. Cohen, PhD 14 15 16 17 18 19 University of Kentucky, College of Medicine 20

21 Dept. of Microbiology, Immunology and Molecular Genetics 22 23 800 Rose Street, Room MS419 24 25 Lexington, KY 40536-0298 26 27 28 29 30 31 32 33

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35 Running title: canine adipose SVF microarray 36

37 38

39 Corresponding author: 40

41 Donald A. Cohen, Ph.D. 42

43 University of Kentucky, College of Medicine 44

45 Dept. of Microbiology, Immunology and Molecular Genetics 46

800 Rose Street, Room MS419

48 Lexington, KY 40536-0298

50 Tel: 859-323-5131

52 Fax: 859-257-8994

53 54

Email: [email protected] 55

mailto:[email protected]

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Abstract 4 5

6 Adipose stem cells (ASCs) are a valuable tool in veterinary medicine for 7

8 treatment of acute tissue injuries and osteoarthritis. The source of ASCs, the stromal 9 10

vascular fraction (SVF), is heterogeneous and includes both mesenchymal stem cells

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13 (MSCs) and non-MSCs. The role that non-MSCs play in stem cell therapy is unclear, 14 15 but non-MSCs can secrete a variety of growth factors and cytokines which could 16 17

18 contribute to effective ASC therapy. Little is known about the factors released by non- 19

20 MSCs and how adipose tissue processing affects the spectrum of factors produced by 21 22 ASC preparations. Microarray studies were performed to identify expression patterns 23 24

25 by cryopreserved ASC preparations that were prepared by three different veterinary 26

27 stem cell companies from the same source of canine adipose tissue. Results indicate 28 29

that tissue processing procedures affected recovery of viable cells from cryopreserved 30 31

32 ASC preparations, overall gene expression by ASC preparations, and expression of 33

34 genes which are important for effective tissue regeneration. Additional studies 35 36

investigated whether treatment of ASC preparations with platelet-rich plasma (PRP) and

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39 photo-activation would modify gene expression in a manner that would increase ASC 40 41 regenerative potential. Treatment with PRP and photo-activation led to increased 42 43

44 expression of numerous genes, many of which can promote tissue regeneration by 45

46 ASCs. These studies indicate that adipose-derived non-MSCs contribute important 47 48 cytokines and growth factors in ASC preparations which likely enhance regenerative 49 50

51 capacity of MSCs. Moreover, cell viability and the spectrum of factors produced in ASC 52

53 preparations are dependent on the procedures used to prepare ASCs from adipose 54 55

tissue samples.

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6 Keywords: adipose, mesenchymal, stem cell, microarray, flow cytometry

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differentiation during ASC therapy remains unclear, but it is known that each of these

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Introduction 4 5

6 Adipose-derived stem cells (ASCs) are becoming a valuable therapeutic tool in

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9 veterinary regenerative medicine. The use of ASC therapy in private veterinary practice 10 11 is increasing rapidly for treatment of a number of acute conditions, such as injuries to 12 13

tendons, ligaments, bone or cartilage and for chronic conditions, such as osteoarthritis.

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16 ASCs have been isolated from and have shown therapeutic benefit in a number of 17 18 species, including canis, and equus [1-4]. ASCs are often referred to as mesenchymal 19 20

21 stem cells (MSCs), due to their mesenchymal tissue origin during embryogenesis and 22

23 their developmental potential [5]. MSCs have been isolated from a number of different 24

25 tissue sources within the body, such as bone marrow, blood, adipose tissue, muscle 26 27

28 and cartilage [6-9]. While many basic and clinical studies were performed initially using 29 30 bone marrow-derived MSCs, the fact that MSCs are present in adipose tissues at 100 – 31 32

1000 times greater concentrations than in bone marrow has led to greater interest in

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35 ASCs for regenerative stem cell therapy. The use of ASCs has made veterinary stem 36 37 cell therapy much more cost effective and has catapulted ASC therapy from the 38 39

research lab into private veterinary practice. ASCs obtained from the stromal vascular

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42 fraction (SVF) of adipose tissue possess multi-lineage differentiation capacity which 43 44 allows them to develop into a variety of cell types, including chondrocytes, osteoblasts, 45 46

47 myocytes, endothelial cells and others [5, 10-19]. However, SVF of adipose tissue 48

49 contains a heterogeneous mixture of cells, including not only MSCs, but also variable 50 51

numbers of endothelial cells, smooth muscle cells, fibroblasts, preadipocytes and 52 53

54 immune cells [20, 21]. The role that these additional SVF-associated cells play in ASC

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ASC lineage differentiation. The current study was designed to identify growth factor

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cell types can secrete a variety of growth factors and cytokines which could contribute 4 5

6 to the effectiveness of ASC cell preparations at an injured tissue site [22-25]. 7 8

9 Several companies have entered the arena of regenerative stem cell therapy and 10 11 are offering enriched ASCs and ASC preparation kits for veterinary clinical practice. 12 13

The quality of ASC preparations (ASC-P) is very important in order to achieve the

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16 expected clinical outcome of tissue regeneration. That quality depends not only on the 17 18 number of ASCs present in the cell preparation, but also on the potential of the ASCs to 19 20

21 survive, proliferate and differentiate at the tissue site where they are administered. 22

23 While there are many basic scientific issues that still must be addressed to fully develop 24

25 the clinical potential of ASC therapy, it is certain from many studies that differentiation of 26 27

28 ASCs into distinct tissue types requires unique sets of growth factors, cytokines and 29 30 other soluble factors. Studies on induction of culture-expanded adipose MSCs in vitro 31 32

into osteoblasts, chondrocytes, etc. have shown that many of these critical factors must

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35 be supplied from exogenous sources for MSC lineage differentiation to occur effectively. 36 37 Given the potential importance of paracrine factors produced by stromal vascular cells 38 39

in the viability, proliferation and differentiation of ASCs, the therapeutic quality of SVF

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42 preparations will depend not only on the number of ASCs in the preparation, but also on 43 44 the number of non-ASCs which are actively producing factors which may promote MSC 45 46

47 regenerative functions in a paracrine fashion. While use of uncultured ASC 48

49 preparations from SVF has shown clinical benefit and holds great promise for 50 51

regenerative tissue therapy, little is known about what factors are released by SVF- 52 53

54 associated cells in ASC cell preparations and whether these factors are important for

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gene profiling analysis was performed.

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and cytokine genes expressed by ASC cell preparations that were prepared by three 4 5

6 different veterinary adipose stem cell companies from the same identical source of 7

8 canine adipose tissue. A second study was performed to determine if treatment of 9 10

canine SVF with photo-activated platelet-rich plasma (PRP) would modify SVF gene

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13 expression patterns in a manner that would increase the regenerative potential of 14

15 canine ASCs in SVF preparations. 16 17

18 Materials and Methods 19 20 21 Preparation of Canine Stromal Vascular Fractions 22 23

24 For the study comparing gene expression profiles in different SVF preparations 25 26

27 from an identical source of adipose tissue, approximately 60 grams of adipose tissue 28 29 was surgically extracted from a 12 year old neutered male Corgi by a private 30 31

veterinarian in the central Kentucky area. Three equal samples of approximately 20

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34 grams of adipose tissue were shipped on ice to 3 different companies which specialize 35 36 in adipose-derived veterinary stem cell therapy: Company M; Company V and 37 38

Company S. All 3 ASC preparations were cryopreserved at their respective facilities.

40 41 Approximately one month later, cryopreserved samples were retrieved from all 42 43 companies and transported frozen to the University of Kentucky for gene profiling and 44 45

viability analysis. All samples were rapidly thawed in a 370C water bath and then

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48 maintained on ice. Cell viability and concentration were determined in a blinded fashion 49 50

using standard Trypan blue hemocytometer procedures. Total RNA was immediately 51 52

53 purified from each cell preparation using Qiagen RNA mini-prep kits (Qiagen Inc, 54

55 Valencia, CA) according to manufacturer instructions. RNA was stored at -800C until

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antibody as suggested by the manufacturer. A minimum of 100,000 washed cells were

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For the study to evaluate the contribution of photo-activated PRP on SVF gene 4 5

6 expression, fresh adipose tissue was dissected from two individual canines by an 7

8 independent veterinarian in Pennsylvania. The first canine was a 6 year old neutered 9 10

male Labrador and the second canine was a 10 year old neutered male German

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13 Shepard mix. Stromal vascular fractions were processed by company M. A portion of 14 15 each freshly isolated SVF preparation underwent PRP/photo-activation treatment or 16

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18 was left untreated. All samples were subsequently delivered to the University of 19

20 Kentucky on ice within 2 hours of preparation, where both PRP/photo-activated and 21 22 untreated ASC preparations were incubated for 6 hours in a 370C water bath prior to 23 24

flow cytometry or RNA purification. RNA from all samples was stored at -800C until

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27 gene profiling analysis was performed as described above. 28 29 30 Flow cytometric analysis of SVF 31 32

33 To analyze the cell surface marker phenotype of cells in SVF preparations, 34 35

aliquots of each SVF preparation were stained with fluorescently-labeled antibodies

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38 specific for the following proteins: PE-conjugated mouse anti-human CD29 (Invitrogen, 39 40 Grand Island, NY), PE/Cy7-conjugated rat anti-human CD44 (Biolegend, San Diego, 41 42

43 CA), Alexa Fluor 488-conjugated rat anti-dog CD45 (AbD Serotec, Raleigh, NC), and 44

45 APC-conjugated rat anti-dog CD90 (eBioscience, San Diego, CA). All antibodies used 46 47 in these studies were confirmed by flow cytometry by us to bind to canine PBMCs. SVF 48 49

50 cells (1 x 106 cells) were incubated for 15 min at 370C in PBS containing 5% bovine 51 52 serum albumin (BSA) plus Hoechst 33342 dye to label nucleated cells. Samples were 53 54

then incubated an additional 30 min on ice with saturating concentrations of each

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then analyzed on a Sony-iCyt Synergy cell sorter to determine the percentage of cells 4 5

6 expressing each marker. An initial gate was set on paraformaldehyde-fixed canine 7

8 PBMCs to exclude events smaller than PBMCs. That cell size gate was applied to all 9 10

SVF samples. Cells were subsequently gated on Hoechst positive events to identify

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13 nucleated cells prior to analysis for marker expression. Analysis of flow cytometric data 14 15 was performed using WinList analytical software (Verity Software House, Topsham, 16 17

18 ME). 19

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21 Microarray gene profiling 22

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24 RNA quality and concentration of each sample was determined on an Agilent 25

26 2100 Bioanalyzer and then analyzed for gene expression on an Affymetrix GCS 3000 27 28

7G scanner using Affymetrix Canine Gene 1.0 ST Array chips (Affymetrix, Inc., Santa 29 30

31 Clara, CA). Affymetrix CEL files were imported into Affymetrix Expression Console 32

33 software for quality analysis and subsequently were imported into Partek Genomic Suite 34 35

Software (Partek, Inc., St. Louis, MO) to determine normalized log2 expression values

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38 for all genes. Gene expression was analyzed statistically by ANOVA to obtain P values. 39 40 Fold changes for comparisons of interest were calculated by dividing the least-square 41 42

43 mean expression values for one group by the least-square mean of the expression 44

45 values of the comparison group. Fold changes greater than 2.0 are presented. 46 47 Biological pathway analysis was performed using Ingenuity IPA software package 48 49

50 (Ingenuity Systems, Inc., Redwood City, CA). RNA quantification and microarray 51 52 analyses were performed by the University of Kentucky Microarray Core Facility. 53

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displayed the highest viability both at the time of ASC preparation (98% viable) and after

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Results 4 5

6 Flow cytometric characterization of ASCs in canine SVF.

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9 Surface marker expression was analyzed on freshly prepared SVF samples to

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12 determine the percentage of ASCs. In analysis of multiple SVF samples, 1 – 3% of 13

14 cells were found to express CD29, CD44, and CD90, but not CD45 (Figure 1). Of the 15 16

17 CD44 positive cells, approximately 85% were found to co-express both CD29 and 18

19 CD90. These data are consistent with the phenotype of adult canine mesenchymal 20

21 stem cells described by other groups in SVF preparations [26-28]. Expression of 22 23

24 CD105 was evaluated using anti-human CD105 monoclonal antibody (clone 43A3); 25

26 however, expression of this marker was never observed on canine SVF cells (not 27 28

shown). Since CD105 is an expected marker for ASCs, we suspect that this antibody 29 30

31 did not cross react with canine CD105, despite a previous publication indicating cross 32

33 reactivity [29]. Importantly, the SVF preparations analyzed never contained more than 34 35

1% CD45+ hematopoietic cells. These results demonstrate the SVF preparations

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38 contained ASCs that were CD29+, CD44+, CD90+, CD45neg. 39 40 41 Cell Viability and Gene Expression Profiling of Canine ASC Preparations 42 43 44 The viability of the ASC preparations was evaluated both at the time of ASC 45

46 preparation and after cryopreservation. Note that the freshly prepared ASC-P was used 47 48

49 for other studies1, but viabilities of those preparations also are reported for this study. 50 51 Viabilities, as determined by trypan blue staining, varied substantially depending on the 52 53

source of the adipose cell preparation (Figure 2). ASC preparations from company M

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expressed by ASC-P from company M compared to company S. Table 8 shows genes

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rapid thawing of cryopreserved ASC-P (88% viable). In contrast, viability of the ASC-P 4 5

6 from company V was 74% viable after ASC preparation and only 30% viable after 7

8 thawing the cryopreserved sample. Similarly, the ASC-P from company S was 74% 9 10

viable after ASC preparation but only 8% viable after thawing.

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Analysis of microarray results of ASC-P from a single adipose tissue sample

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16 prepared by three different companies showed expression of 17, 854 annotated genes 17 18 in the canine ASC preparations. At total of 1357 genes were more highly expressed in 19 20

21 ASC-P from company M compared to ASC-P from Company S (157 fold to 2 fold 22

23 greater expression) and 315 genes were more highly expressed in ASC-P from 24

25 company M compared to ASC-P from Company V (65 fold to 2 fold greater expression). 26 27

28 A comparison of Company S to Company M showed that 1265 genes were more highly 29 30 expressed (2 fold or greater) by ASC-P from Company S. Finally, a comparison of 31 32

Company V to Company M showed increased expression of 1008 genes (2 fold or

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35 greater). Differential expression of these genes between the three ASC preparations 36 37 was genuine, since expression of commonly used housekeeping genes did not vary 38 39

between the ASC preparations (Table 1). None of these 5 housekeeping genes varied

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42 more than two standard deviations for any ASC preparation from their mean expression 43 44 level. Biological pathway analysis was performed on the overexpressed genes to 45 46

47 identify the subset of genes that coded for secreted proteins known to be important for 48

49 differentiation of MSCs into different types of tissues [30-106]. The greatest number of 50 51

MSC-relevant changes occurred in ASC-P from Company M, compared to either of the 52 53

54 other companies. Tables 2 - 7 show genes of secreted proteins which were more highly

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protease genes encode proteins which promote MSC migration and differentiation into

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of secreted proteins which were more highly expressed by ASC-P from company M 4 5

6 compared to company V. Genes for 19 distinct cell growth factors critical for lineage 7

8 differentiation of MSCs were more highly expressed by ASC-P from company M 9 10

compared to company S (Table 2), including platelet-derived growth factors, insulin-like

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13 growth factor, colony stimulating factors, bone morphogenic proteins, and fibroblast 14

15 growth factors. 16 17 18 Eleven cytokine genes (interleukins) were more highly expressed by ASC-P from 19 20

21 company M, some of which were over 100 times greater than the ASC-P from company 22

23 S (Table 3). These included cytokines known to attract MSCs to tissue sites, promote 24

25 MSC proliferation and wound healing and to inhibit ongoing inflammation at tissue sites. 26 27

28 The anti-inflammatory effects of SVF cells and MSCs play important roles in the 29 30 efficiency of stem cell therapy by reducing inflammation at the injection site which 31 32

provides for better stem cell survival and differentiation [72]. Eleven additional

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35 cytokines were found to be more highly expressed by ASC-P from company M 36 37 compared to company S, which have been shown to be involved in MSC migration and 38 39

lineage differentiation (Table 4). These included genes for factors known to promote

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42 MSC migration and differentiation toward chondrocytes, and osteoblasts. An additional 43 44 13 chemokine genes were found to be more highly expressed by ASC-P from company 45 46

47 M compared to company S (Table 5). These included both CC and CXC chemokines, 48

49 which have been shown not only to mediate MSC migration, but also to promote bone 50 51

repair (CCL7) and angiogenesis (CCL8). A number of protease and glycoprotein genes 52 53

54 were also more highly expressed by ASC-P from company M (Table 6). Several of the

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chondrocytes and osteoblasts. Finally, 7 additional glycoprotein genes were more 4 5

6 highly expressed by ASC-P from company M compared to company S and some of 7

8 these have been shown not only to promote MSC differentiation, but also to mediate 9 10

some of the immunosuppressive properties of MSCs (Table 7).

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In the comparison study of gene expression with ASC cell preparations from

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16 company M versus company V, the 315 genes that were more highly expressed by the 17 18 ASC-P from company M also showed a variety of secreted factors which are important 19 20

21 for regenerative stem cell therapy, including factors involved in MSC migration, 22

23 proliferation and lineage differentiation as well as several anti-inflammatory factors 24

25 (Table 8). Thus, genome-wide analysis of gene expression in ASC cell preparations 26 27

28 from three different companies demonstrated that ASCs prepared by different 29 30 proprietary protocols can vary substantially in terms of expression level of genes known 31 32

to be critical for effective therapeutic use of adipose-derived MSCs [34-36, 46, 51, 62,

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35 65-67, 71, 72, 74, 75, 78, 81, 84-93, 107-116]. 36

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38 Of the 1265 genes more highly expressed by ASC-P from Company S compared 39

40 to Company M, only 4 genes coded for cytokines or growth factors: IL-34 (2.1 41 42

43 fold),TNFSF15 (2.5 fold), osteopontin SPP1 (2.1 fold), and granulin (2.0 fold). Similarly, 44

45 only 5 of the 1008 genes that were overexpressed (2 fold or greater) by ASC-P from 46 47 Company V compared to Company M were cytokine or growth factor genes: CCL19 48 49

50 (2.2 fold), IL-16 (2.1 fold), platelet-derived growth factor beta (2.1 fold), thyroid 51 52 stimulating hormone, beta (2.1 fold), and angiopoietin 2 (2.1 fold). 53 54

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expressing these MSC markers. Compared to untreated cells, SVF preparations

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Effect of PRP/Photo-Activation Treatment on Gene Expression in SVF 4 5

6 preparations 7 8

9 Platelet-rich plasma contains a variety of growth factors and cytokines that can 10 11 promote the viability, proliferation and differentiation capacity of ASCs [117, 118]. These 12 13

factors are contained within the alpha granules of platelets and are rapidly released

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16 upon activation of the platelets by any of a number of activation stimuli, including photo- 17 18 activation [119, 120]. Treatment of ASC preparations with photo-activated PRP is 19 20

21 commonly used by veterinary stem cell companies in the preparation of ASCs from 22

23 SVF. However, the effects that photo-activated PRP has on expression of genes that 24

25 may be critical for the viability and tissue regenerative potential of veterinary ASCs is 26 27

28 relatively unknown. To address this issue, gene profiling studies were performed on 29 30 canine ASC-P which were treated or untreated with PRP/photo-activation. ASCs were 31 32

prepared by company M from canine adipose tissue and a portion of each preparation

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35 was either treated with their proprietary PRP/photo-activation procedures or left 36 37 untreated. Upon receipt of these samples from the company, both treated and 38 39

untreated preparations were incubated at 370C for 6 hours to allow genes induced by

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42 PRP/photo-activation to be fully expressed. Total RNA was isolated and analyzed by 43 44 flow cytometry for expression of ASC surface markers for by microarray for differential 45 46

47 gene expression. 48 49

50 For flow cytometric studies, SVF preparations were stained with fluorochrome- 51 52 labeled antibodies against CD45, CD29, CD44 and CD90 and replicates of the CD45- 53 54

negative fractions were analyzed by flow cytometry for the percentage of cells

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preparation and therapeutic protocols that have been used. As a field, mesenchymal

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treated by PRP/photo-activation displayed significant increases in the percentage of 4 5

6 cells expressing all of the MSC markers tested (Figure 3). Thus, PRP/photo-activation 7

8 dramatically improved the number of identifiable MSC cells in the ASC preparations. 9 10 11 Gene expression profiling indicated that 190 genes in PRP/photo-activated SVF 12 13

showed at least a 2 fold increase in expression compared to untreated SVF and 726

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16 additional genes showed at least a 1.5 fold increase following PRP/photo-activation. Of 17 18 the genes with increased expression, 46 were secreted proteins that included a number 19 20

21 of cytokines and growth factors involved in MSC migration, proliferation and lineage 22

23 differentiation, such as colony stimulating factors, chemokines, bone morphogenetic 24

25 proteins and proteases (Table 9). PRP/photo-activation caused extensive activation of 26 27

28 cells in the ASC preparations as evidenced by increased expression of 37 distinct 29 30 intracellular signaling molecules and 50 different transcriptional regulators (not shown). 31 32

Thus, treatment of ASC-P with combined platelet-rich plasma and photo-activation

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35 leads to increased expression of secreted genes known to be critical for effective ASC 36 37 therapy. 38 39

40 Discussion 41 42 Highly effective stem cell therapy has become the golden ring for which many 43 44

45 clinicians strive in the areas of human and veterinary tissue engineering and 46 47 regenerative medicine. The rate of progress using mesenchymal stem cell therapeutic 48 49 approaches has been increasing but results from the limited number of clinical trials and 50 51

52 veterinary studies have been mixed. These mixed results do not necessarily imply that 53

54 MSC therapy overall is ineffective, but more likely reflects the wide array of MSC

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cryopreservation, all samples were received frozen by the investigator’s laboratory for

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stem cell therapy remains an art in search of a science. Uniform protocols are 4 5

6 desperately needed, but must await confirmation of the most effective protocols. Even 7

8 the nomenclature for MSCs has remained confusing in spite of criteria for MSC 9 10

designation proposed by the International Society for Cellular Therapy [121]. More

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13 importantly, the source of MSCs used in clinical and research settings remain quite 14 15 variable and rarely have different preparation protocols been compared for their 16 17

18 effectiveness. 19

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21 Use of stem cell therapy is rapidly increasing in clinical veterinary medicine and 22

23 several companies now offer in house services and kits for the isolation of MSCs from 24

25 fresh adipose tissue. Proprietary methods for adipose tissue processing are offered by 26 27

28 each company and each purports that their isolation procedures generate ASCs that are 29 30 therapeutically effective both as fresh or cryopreserved cell preparations. Commercially 31 32

available adipose stem cell preparations for veterinary medicine are all based on

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35 isolation of the stromal vascular fraction from fresh adipose tissue. Adipose SVF is a 36 37 very heterogeneous tissue preparation which contains not only variable numbers of 38 39

MSCs, but also cell types with can produce growth factors and cytokines that can

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42 positively affect MSCs in a paracrine fashion. The goal of this study was to evaluate 43 44 expression of genes in adipose SVF preparations that code for secreted products 45 46

47 known to promote the therapeutic effectiveness of adipose MSCs. SVF stem cells 48

49 preparations were prepared commercially by three different veterinary stem cell 50 51

companies from a single source of canine adipose tissue. Each ASC-P was 52 53

54 cryopreserved by the company which prepared it and approximately one month after

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M. While the number of differences between ASC-P from Company M and Company V

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analysis. Thus, the only variables between ASC-P samples were the proprietary 4 5

6 isolation procedure and cryopreservation performed by each company. 7 8

9 A difference in viability of the cryopreserved samples after thawing was quite 10 11 evident with the rank order of most viable preparations being Company M (88%) ˃ 12 13

Company V (30%) ˃ Company S (8%). To some extent, these differences were

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16 reflected in the viability of the freshly isolated ASC-P samples as well. Freshly prepared 17 18 ASC-P from the same adipose tissue also was received by the investigator’s laboratory 19 20

21 at the same time that cryopreserved samples were prepared. Viability of these fresh 22

23 ASC-P samples (which were used for other studies in the lab) was determined. Initial 24

25 viability of ASC-P prepared by Company M was the greatest (98%) compared to 26 27

28 Companies V (74%) and S (74%). Whether a lower initial viability reflects cell injury at 29 30 the time of isolation which may have had a negative impact on viability of cryopreserved 31 32

samples is currently unknown.

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Gene expression profiling of the cryopreserved ASC-P samples showed several

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38 important differences in expression of genes that are known to be critical for MSC 39

40 regenerative function. ASC-P prepared by Company M had, by far, the greatest 41 42

43 number of genes that were overexpressed compared to either Company S or Company 44

45 V which coded for cytokines or growth factors known to be important for adipose MSC 46 47 function. Moreover, the degree by which these genes were overexpressed was 48 49

50 dramatically greater in ASC-P from Company M, especially when compared to ASC-P 51 52 from Company S. A total of 20 growth factor genes, 22 cytokine/interleukin genes and 53 54

13 chemokine genes were expressed at least 2 fold greater by ASC-P from Company

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and colony stimulation factors CSF2 and CSF3 3 – 12 fold, all of which have been

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was not as great, 23 genes for factors known to be important for MSC function were 4 5

6 overexpressed by ASC-P from Company M, while only 5 genes MSC-relevant genes 7

8 were overexpressed by ASC-P from Company V. The cellular source of the growth 9 10

factors and cytokine produced by ASC-P cannot be determined from these studies due

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13 to the heterogeneous nature of adipose SVF. However, studies by Taleb, et al [122] 14 15 have shown that the bulk of factors are produced by non-MSCs in ASC-P. The 16

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18 paracrine production of many of these factors suggests that non-MSCs likely serve a 19

20 critical role in the regenerative capacity of MSCs in the ASC-P. Overall, results of this 21 22 gene profiling study indicate that the method of adipose tissue preparation used to 23 24

25 prepare ASCs in SVF has an impact on the gene expression pattern in the final cell 26

27 preparation. While the specific protocols for processing adipose tissue by the three 28 29

respective companies remains proprietary, gene profiling of cryopreserved ASC-P 30 31

32 indicated that the quality of ASC-P was not equal for all company preparations, with 33

34 Company M ˃ Company V ˃ Company S. 35 36 37 Microarray analysis of ASC-P treated with PRP/photo-activation demonstrated 38 39

several important changes in expression of genes known to be important for the

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42 regenerative functions of MSCs. These included increase expression of IL-11 and bone 43 44 morphogenetic protein 2, both of which can promote bone regeneration [98, 123, 124]. 45 46

47 A recent clinical trial of co-infusion with mesenchymal stromal cells and hematopoietic 48

49 stem cells in leukemia patients showed both enhanced stem cell engraftment and 50 51

increased IL-11 production in sera of patients [125]. PRP/photo-activation also 52 53

54 enhanced expression of the chemokine genes, CCL3, CCL7 and CXCL7, 3 – 11 fold

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shown to promote chemotaxis of MSCs [42, 50, 126, 127]. Expression of 4 different 4 5

6 matrix metalloproteases, which are involved in migration of MSCs, was enhanced by 7

8 PRP/photo-activation as much as 13 fold over untreated ASC-P. Interestingly, the 9 10

proangiogenic factors, relaxin 1 and urotensin 2 [128, 129], as well as the anti-

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13 angiogenic factor, vasohibin 2 [130], were all upregulated by PRP/photo-activation. 14 15 Angiogenesis is an important component of tissue regeneration. The effect of 16

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18 enhanced expression of these pro- and anti-angiogenic factors is currently unknown. 19

20 The overall range of genes that displayed enhanced expression after PRP/photo- 21 22 activation of ASC-P was similar to those seen in another MSC gene profiling study of 23 24

25 activated bone marrow MSC [127]. 26 27

28 In conclusion, gene profiling studies of cryopreserved canine ASC preparations 29 30 demonstrated that the procedures used to isolate adipose SVF can affect the overall 31 32

viability of the ASC preparation. Moreover, the ability of cells in the ASC-P to express

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35 genes that are known to be important for the regenerative functions of MSCs in the 36 37 ASC-P is also dependent on the initial procedures to isolate adipose SVF. Finally, 38 39

activation of ASC-P with PRP/photo-activation appears to be beneficial for the

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42 regenerative capacity of ASC-P in that a number of genes which encode for proteins 43 44 that promote MSC tissue regeneration are expressed at higher levels following this 45 46

47 treatment. 48

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8. Huang, Y.C., et al., Isolation of mesenchymal stem cells from human placental decidua basalis and resistance to hypoxia and serum deprivation. Stem Cell Rev, 2009. 5(3): p. 247‐55. Fan, J., et al., Synovium‐derived mesenchymal stem cells: a new cell source for musculoskeletal regeneration. Tissue Eng Part B Rev, 2009. 15(1): p. 75‐86.

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Acknowledgments 4 5 I wish to extend my gratitude to the University of Kentucky Microarray core facility for 6 7

8 assistance in performing and evaluating gene profiling results. I also wish to thank the 9 10 University of Kentucky Flow Cytometry and Cell Sorting core facility for help in 11 12

performing flow cytometric analysis of cells.

14 15

Author Disclosure Statement

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18 The author is a full-time faculty member of the University of Kentucky and is a 19

20 consultant for MediVet-America, LLC. Funding for these studies were provided by 21 22

23 MediVet-America, LLC. 24

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26 Footnotes: 27

28 1 Validation of Stromal Vascular Cell Yields from Canine Adipose Tissue and the 29 Significance of PRP and Photo-activation. Veterinary Practice News 25(1):14-16, 2013. 30 31 32

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34 References 35

36 1. Black, L.L., et al., Effect of intraarticular injection of autologous adipose‐derived mesenchymal 37 stem and regenerative cells on clinical signs of chronic osteoarthritis of the elbow joint in dogs. 38 Vet Ther, 2008. 9(3): p. 192‐200. 39 2. Black, L.L., et al., Effect of adipose‐derived mesenchymal stem and regenerative cells on 40

lameness in dogs with chronic osteoarthritis of the coxofemoral joints: a randomized, double‐ 41

42 blinded, multicenter, controlled trial. Vet Ther, 2007. 8(4): p. 272‐84. 43 3. Reed, S.A. and E.R. Leahy, Growth and Development Symposium: Stem cell therapy in equine 44 tendon injury. J Anim Sci, 2013. 91(1): p. 59‐65. 45 4. Richardson, L.E., et al., Stem cells in veterinary medicine‐‐attempts at regenerating equine 46 tendon after injury. Trends Biotechnol, 2007. 25(9): p. 409‐16. 47 5. Mizuno, H., M. Tobita, and A.C. Uysal, Concise review: Adipose‐derived stem cells as a novel tool 48

for future regenerative medicine. Stem Cells, 2012. 30(5): p. 804‐10.

50 6. Mohal, J.S., H.D. Tailor, and W.S. Khan, Sources of adult mesenchymal stem cells and their 51 applicability for musculoskeletal applications. Curr Stem Cell Res Ther, 2012. 7(2): p. 103‐9. 52 7. Fan, C.G., Q.J. Zhang, and J.R. Zhou, Therapeutic potentials of mesenchymal stem cells derived 53 from human umbilical cord. Stem Cell Rev, 2011. 7(1): p. 195‐207.

54 55 56 57 58 59 60

28(8): p. 682‐90. Bischoff, D.S., et al., Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage. J Cell Biochem, 2008. 103(3): p. 812‐ 24.

31.

20

43

of 38 1

2

3 10. Al Battah, F., et al., Current status of human adipose‐derived stem cells: differentiation into 4

5 hepatocyte‐like cells. ScientificWorldJournal, 2011. 11: p. 1568‐81.

6 11. Mazo, M., et al., Adipose‐derived stem cells for myocardial infarction. J Cardiovasc Transl Res, 7 2011. 4(2): p. 145‐53. 8 12. Lindroos, B., R. Suuronen, and S. Miettinen, The potential of adipose stem cells in regenerative 9 medicine. Stem Cell Rev, 2011. 7(2): p. 269‐91. 10 13. Zavan, B., et al., Neural potential of adipose stem cells. Discov Med, 2010. 10(50): p. 37‐43. 11 14. Hong, S.J., D.O. Traktuev, and K.L. March, Therapeutic potential of adipose‐derived stem cells in 12

13 vascular growth and tissue repair. Curr Opin Organ Transplant, 2010. 15(1): p. 86‐91. 14 15. Uysal, A.C. and H. Mizuno, Tendon regeneration and repair with adipose derived stem cells. Curr 15 Stem Cell Res Ther, 2010. 5(2): p. 161‐7. 16 16. Kim, S.C., D.J. Han, and J.Y. Lee, Adipose tissue derived stem cells for regeneration and 17 differentiation into insulin‐producing cells. Curr Stem Cell Res Ther, 2010. 5(2): p. 190‐4. 18 17. de Villiers, J.A., N. Houreld, and H. Abrahamse, Adipose derived stem cells and smooth muscle 19

cells: implications for regenerative medicine. Stem Cell Rev, 2009. 5(3): p. 256‐65.

21 18. Rada, T., R.L. Reis, and M.E. Gomes, Adipose tissue‐derived stem cells and their application in 22 bone and cartilage tissue engineering. Tissue Eng Part B Rev, 2009. 15(2): p. 113‐25. 23 19. Hoogendoorn, R.J., et al., Adipose stem cells for intervertebral disc regeneration: current status 24 and concepts for the future. J Cell Mol Med, 2008. 12(6A): p. 2205‐16. 25 20. Riordan, N.H., et al., Non‐expanded adipose stromal vascular fraction cell therapy for multiple 26

sclerosis. Journal of translational medicine, 2009. 7: p. 29. 27

28 21. Astori, G., et al., "In vitro" and multicolor phenotypic characterization of cell subpopulations 29 identified in fresh human adipose tissue stromal vascular fraction and in the derived 30 mesenchymal stem cells. Journal of translational medicine, 2007. 5: p. 55. 31 22. Sheng, L., et al., Transplantation of stromal vascular fraction as an alternative for accelerating 32 tissue expansion. Journal of plastic, reconstructive & aesthetic surgery : JPRAS, 2013. 66(4): p. 33 551‐7. 34 23. Mazo, M., et al., Adipose stromal vascular fraction improves cardiac function in chronic 35

36 myocardial infarction through differentiation and paracrine activity. Cell transplantation, 2012. 37 21(5): p. 1023‐37. 38 24. Blaber, S.P., et al., Analysis of in vitro secretion profiles from adipose‐derived cell populations. 39 Journal of translational medicine, 2012. 10: p. 172. 40 25. Gimble, J.M., et al., Adipose‐derived stromal/stem cells: A primer. Organogenesis, 2013. 9(1). 41 26. Takemitsu, H., et al., Comparison of bone marrow and adipose tissue‐derived canine 42

mesenchymal stem cells. BMC Vet Res, 2012. 8: p. 150.

44 27. Martinello, T., et al., Canine adipose‐derived‐mesenchymal stem cells do not lose stem features 45 after a long‐term cryopreservation. Res Vet Sci, 2011. 91(1): p. 18‐24. 46 28. Vieira, N.M., et al., Isolation, characterization, and differentiation potential of canine adipose‐ 47 derived stem cells. Cell transplantation, 2010. 19(3): p. 279‐89. 48 29. Iohara, K., et al., Complete pulp regeneration after pulpectomy by transplantation of CD105+ 49 stem cells with stromal cell‐derived factor‐1. Tissue Eng Part A, 2011. 17(15‐16): p. 1911‐20. 50

51 30. Noh, Y.H., et al., Correlation between chemokines released from umbilical cord blood‐derived 52 mesenchymal stem cells and engraftment of hematopoietic stem cells in nonobese 53 diabetic/severe combined immunodeficient (NOD/SCID) mice. Pediatr Hematol Oncol, 2011.

106(2): p. 419‐27. Kalwitz, G., et al., Chemokine profile of human serum from whole blood: migratory effects of CXCL‐10 and CXCL‐11 on human mesenchymal stem cells. Connect Tissue Res, 2010. 51(2): p. 113‐22.

53

54 55 56 57 58 59 60

51.

20

43

of 38 1

2

3 32. Adams, J.C. and J. Lawler, The thrombospondins. Cold Spring Harb Perspect Biol, 2011. 3(10): p. 4

5 a009712.

6 33. Hankenson, K.D., et al., Thrombospondins and novel TSR‐containing proteins, R‐spondins, 7 regulate bone formation and remodeling. Curr Osteoporos Rep, 2010. 8(2): p. 68‐76. 8 34. Zhao, X., et al., Progesterone enhances immunoregulatory activity of human mesenchymal stem 9 cells via PGE2 and IL‐6. Am J Reprod Immunol, 2012. 68(4): p. 290‐300. 10 35. Bouffi, C., et al., IL‐6‐dependent PGE2 secretion by mesenchymal stem cells inhibits local 11 inflammation in experimental arthritis. PLoS One, 2010. 5(12): p. e14247. 12

13 36. Kalwitz, G., et al., Gene expression profile of adult human bone marrow‐derived mesenchymal 14 stem cells stimulated by the chemokine CXCL7. Int J Biochem Cell Biol, 2009. 41(3): p. 649‐58. 15 37. Tu, Z., et al., Mesenchymal stem cells inhibit complement activation by secreting factor H. Stem 16 Cells Dev, 2010. 19(11): p. 1803‐9. 17 38. van Buul, G.M., et al., Mesenchymal stem cells secrete factors that inhibit inflammatory 18 processes in short‐term osteoarthritic synovium and cartilage explant culture. Osteoarthritis 19

Cartilage, 2012. 20(10): p. 1186‐96.

21 39. Ortiz, L.A., et al., Interleukin 1 receptor antagonist mediates the antiinflammatory and 22 antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci U S A, 2007. 23 104(26): p. 11002‐7. 24 40. Hara, T. and K. Tanegashima, Pleiotropic functions of the CXC‐type chemokine CXCL14 in 25 mammals. J Biochem, 2012. 151(5): p. 469‐76. 26

41. Sallenave, J.M., Antimicrobial activity of antiproteinases. Biochem Soc Trans, 2002. 30(2): p. 111‐ 27

28 5. 29 42. Shinohara, K., et al., Stromal cell‐derived factor‐1 and monocyte chemotactic protein‐3 improve 30 recruitment of osteogenic cells into sites of musculoskeletal repair. J Orthop Res, 2011. 29(7): p. 31 1064‐9. 32 43. Lu, Z., et al., Short‐term exposure to tumor necrosis factor‐alpha enables human osteoblasts to 33 direct adipose tissue‐derived mesenchymal stem cells into osteogenic differentiation. Stem Cells 34 Dev, 2012. 21(13): p. 2420‐9. 35

36 44. Huang, H., et al., Dose‐specific effects of tumor necrosis factor alpha on osteogenic 37 differentiation of mesenchymal stem cells. Cell Prolif, 2011. 44(5): p. 420‐7. 38 45. Cheng, J., et al., Interleukin‐1alpha induces immunosuppression by mesenchymal stem cells 39 promoting the growth of prostate cancer cells. Mol Med Rep, 2012. 6(5): p. 955‐60. 40 46. Spaeth, E., et al., Inflammation and tumor microenvironments: defining the migratory itinerary 41 of mesenchymal stem cells. Gene Ther, 2008. 15(10): p. 730‐8. 42

47. Qiu, P., et al., Platelet‐derived growth factor promotes the proliferation of human umbilical cord‐

44 derived mesenchymal stem cells. Cell Biochem Funct, 2013. 31(2): p. 159‐65. 45 48. Ball, S.G., C.A. Shuttleworth, and C.M. Kielty, Mesenchymal stem cells and neovascularization: 46 role of platelet‐derived growth factor receptors. J Cell Mol Med, 2007. 11(5): p. 1012‐30. 47 49. Ringe, J., et al., Towards in situ tissue repair: human mesenchymal stem cells express chemokine 48 receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2. J Cell 49 Biochem, 2007. 101(1): p. 135‐46. 50

51 50. Sordi, V., et al., Bone marrow mesenchymal stem cells express a restricted set of functionally 52 active chemokine receptors capable of promoting migration to pancreatic islets. Blood, 2005.

66. Watkins, D.J., et al., Heparin‐binding epidermal growth factor‐like growth factor protects mesenchymal stem cells. J Surg Res, 2012. 177(2): p. 359‐64. Krampera, M., et al., HB‐EGF/HER‐1 signaling in bone marrow mesenchymal stem cells: inducing cell expansion and reversibly preventing multilineage differentiation. Blood, 2005. 106(1): p. 59‐ 66. Huang, Y.L., et al., Effects of insulin‐like growth factor‐1 on the properties of mesenchymal stem cells in vitro. J Zhejiang Univ Sci B, 2012. 13(1): p. 20‐8. Granero‐Molto, F., et al., Mesenchymal stem cells expressing insulin‐like growth factor‐I (MSCIGF) promote fracture healing and restore new bone formation in Irs1 knockout mice: analyses of MSCIGF autocrine and paracrine regenerative effects. Stem Cells, 2011. 29(10): p. 1537‐48.

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

67.

68.

69.

20

43

of 38 1

2

3 52. Pulukuri, S.M., et al., Epigenetic upregulation of urokinase plasminogen activator promotes the 4

5 tropism of mesenchymal stem cells for tumor cells. Mol Cancer Res, 2010. 8(8): p. 1074‐83.

6 53. Gutova, M., et al., Urokinase plasminogen activator and urokinase plasminogen activator 7 receptor mediate human stem cell tropism to malignant solid tumors. Stem Cells, 2008. 26(6): p. 8 1406‐13. 9 54. Wang, M., et al., IL‐18 binding protein‐expressing mesenchymal stem cells improve myocardial 10 protection after ischemia or infarction. Proc Natl Acad Sci U S A, 2009. 106(41): p. 17499‐504. 11 55. Xu, J., et al., Chondrogenic differentiation of human mesenchymal stem cells in three‐ 12

13 dimensional alginate gels. Tissue Eng Part A, 2008. 14(5): p. 667‐80. 14 56. Barry, F., et al., Chondrogenic differentiation of mesenchymal stem cells from bone marrow: 15 differentiation‐dependent gene expression of matrix components. Exp Cell Res, 2001. 268(2): p. 16 189‐200. 17 57. Neuss, S., et al., Secretion of fibrinolytic enzymes facilitates human mesenchymal stem cell 18 invasion into fibrin clots. Cells Tissues Organs, 2010. 191(1): p. 36‐46. 19

58. Kachgal, S. and A.J. Putnam, Mesenchymal stem cells from adipose and bone marrow promote

21 angiogenesis via distinct cytokine and protease expression mechanisms. Angiogenesis, 2011. 22 14(1): p. 47‐59. 23 59. Velpula, K.K., V.R. Dasari, and J.S. Rao, The homing of human cord blood stem cells to sites of 24 inflammation: unfolding mysteries of a novel therapeutic paradigm for glioblastoma multiforme. 25 Cell Cycle, 2012. 11(12): p. 2303‐13. 26

60. Mishima, S., et al., Effective ex vivo expansion of hematopoietic stem cells using osteoblast‐ 27

28 differentiated mesenchymal stem cells is CXCL12 dependent. Eur J Haematol, 2010. 84(6): p. 538‐ 29 46. 30 61. Zhuang, Y., et al., Chemokine stromal cell‐derived factor 1/CXCL12 increases homing of 31 mesenchymal stem cells to injured myocardium and neovascularization following myocardial 32 infarction. Chin Med J (Engl), 2009. 122(2): p. 183‐7. 33 62. Nguyen, B.K., et al., Improved function and myocardial repair of infarcted heart by intracoronary 34 injection of mesenchymal stem cell‐derived growth factors. J Cardiovasc Transl Res, 2010. 3(5): p. 35

36 547‐58. 37 63. Hwang, J.H., et al., Comparison of cytokine expression in mesenchymal stem cells from human 38 placenta, cord blood, and bone marrow. J Korean Med Sci, 2009. 24(4): p. 547‐54. 39 64. Chamberlain, G., et al., Mesenchymal stem cells exhibit firm adhesion, crawling, spreading and 40 transmigration across aortic endothelial cells: effects of chemokines and shear. PLoS One, 2011. 41 6(9): p. e25663. 42

65. Weiss, S., et al., Impact of growth factors and PTHrP on early and late chondrogenic

44 differentiation of human mesenchymal stem cells. J Cell Physiol, 2010. 223(1): p. 84‐93.

human bone marrow mesenchymal stem cells. Differentiation, 2008. 76(10): p. 1057‐67. Guihard, P., et al., Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling. Stem Cells, 2012. 30(4): p. 762‐72. Song, H.Y., et al., Oncostatin M promotes osteogenesis and suppresses adipogenic differentiation of human adipose tissue‐derived mesenchymal stem cells. J Cell Biochem, 2007. 101(5): p. 1238‐ 51. Lee, M.J., et al., Oncostatin M stimulates expression of stromal‐derived factor‐1 in human mesenchymal stem cells. Int J Biochem Cell Biol, 2007. 39(3): p. 650‐9. Song, H.Y., et al., Oncostatin M induces proliferation of human adipose tissue‐derived mesenchymal stem cells. Int J Biochem Cell Biol, 2005. 37(11): p. 2357‐65. Zhou, Z., et al., Leptin differentially regulate STAT3 activation in ob/ob mouse adipose mesenchymal stem cells. Nutr Metab (Lond), 2012. 9(1): p. 109.

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

86.

87.

88.

89.

90.

20

43

of 38 1

2

3 70. Li, Y., et al., Insulin‐like growth factor 1 enhances the migratory capacity of mesenchymal stem 4

5 cells. Biochem Biophys Res Commun, 2007. 356(3): p. 780‐4.

6 71. Wang, N., et al., Mesenchymal stem cells attenuate peritoneal injury through secretion of TSG‐6. 7 PLoS One, 2012. 7(8): p. e43768. 8 72. Prockop, D.J. and J.Y. Oh, Mesenchymal stem/stromal cells (MSCs): role as guardians of 9 inflammation. Mol Ther, 2012. 20(1): p. 14‐20. 10 73. Lopetuso, L.R., F. Scaldaferri, and T.T. Pizarro, Emerging role of the interleukin (IL)‐33/ST2 axis in 11 gut mucosal wound healing and fibrosis. Fibrogenesis Tissue Repair, 2012. 5(1): p. 18. 12

13 74. Boeuf, S., et al., Regulation of aggrecanases from the ADAMTS family and aggrecan neoepitope 14 formation during in vitro chondrogenesis of human mesenchymal stem cells. Eur Cell Mater, 15 2012. 23: p. 320‐32. 16 75. Chou, S.C., et al., Identification of genetic networks during mesenchymal stem cell 17 transformation into neurons. Chin J Physiol, 2008. 51(4): p. 230‐46. 18 76. Zhao, L. and B.M. Hantash, TGF‐beta1 regulates differentiation of bone marrow mesenchymal 19

stem cells. Vitam Horm, 2011. 87: p. 127‐41.

21 77. Tang, Y., et al., TGF‐beta1‐induced migration of bone mesenchymal stem cells couples bone 22 resorption with formation. Nat Med, 2009. 15(7): p. 757‐65. 23 78. Bailey Dubose, K., M. Zayzafoon, and J.E. Murphy‐Ullrich, Thrombospondin‐1 inhibits osteogenic 24 differentiation of human mesenchymal stem cells through latent TGF‐beta activation. Biochem 25 Biophys Res Commun, 2012. 422(3): p. 488‐93. 26

79. Efimenko, A., et al., Angiogenic properties of aged adipose derived mesenchymal stem cells after 27

28 hypoxic conditioning. Journal of translational medicine, 2011. 9: p. 10. 29 80. Dicker, A., et al., Functional studies of mesenchymal stem cells derived from adult human 30 adipose tissue. Exp Cell Res, 2005. 308(2): p. 283‐90. 31 81. Lai, W.T., V. Krishnappa, and D.G. Phinney, Fibroblast growth factor 2 (Fgf2) inhibits 32 differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular 33 regulated kinase activation, and altering Fgf receptor expression levels. Stem Cells, 2011. 29(7): 34 p. 1102‐11. 35

36 82. Benisch, P., et al., The transcriptional profile of mesenchymal stem cell populations in primary 37 osteoporosis is distinct and shows overexpression of osteogenic inhibitors. PLoS One, 2012. 7(9): 38 p. e45142. 39 83. Huang, H., et al., IL‐17 stimulates the proliferation and differentiation of human mesenchymal 40 stem cells: implications for bone remodeling. Cell Death Differ, 2009. 16(10): p. 1332‐43. 41 84. Nasef, A., et al., Leukemia inhibitory factor: Role in human mesenchymal stem cells mediated 42

immunosuppression. Cell Immunol, 2008. 253(1‐2): p. 16‐22.

44 85. Lysy, P.A., et al., Leukemia inhibitory factor contributes to hepatocyte‐like differentiation of

105. Zhang, A., et al., Mechanism of TNF‐alpha‐induced migration and hepatocyte growth factor production in human mesenchymal stem cells. J Cell Biochem, 2010. 111(2): p. 469‐75. Vogel, S., et al., Hepatocyte growth factor‐mediated attraction of mesenchymal stem cells for apoptotic neuronal and cardiomyocytic cells. Cell Mol Life Sci, 2010. 67(2): p. 295‐303. Nagaya, N., et al., Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation, 2005. 112(8): p. 1128‐35. Linscheid, P., et al., Autocrine/paracrine role of inflammation‐mediated calcitonin gene‐related peptide and adrenomedullin expression in human adipose tissue. Endocrinology, 2005. 146(6): p. 2699‐708. Xiong, C.J., et al., Macrophage migration inhibitory factor inhibits the migration of cartilage end plate‐derived stem cells by reacting with CD74. PLoS One, 2012. 7(8): p. e43984.

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

106.

107.

108.

109.

20

43

of 38 1

2

3 91. Zeddou, M., et al., Differential signalling through ALK‐1 and ALK‐5 regulates leptin expression in 4

5 mesenchymal stem cells. Stem Cells Dev, 2012. 21(11): p. 1948‐55.

6 92. Noh, M., Interleukin‐17A increases leptin production in human bone marrow mesenchymal stem 7 cells. Biochem Pharmacol, 2012. 83(5): p. 661‐70. 8 93. Kim, S.W., et al., Amniotic mesenchymal stem cells with robust chemotactic properties are 9 effective in the treatment of a myocardial infarction model. Int J Cardiol, 2012. 10 94. Potapova, I.A., I.S. Cohen, and S.V. Doronin, Von willebrand factor increases endothelial cell 11 adhesiveness for human mesenchymal stem cells by activating p38 mitogen‐activated protein 12

13 kinase. Stem Cell Res Ther, 2010. 1(5): p. 35. 14 95. Yu, B., et al., Parathyroid hormone induces differentiation of mesenchymal stromal/stem cells by 15 enhancing bone morphogenetic protein signaling. J Bone Miner Res, 2012. 27(9): p. 2001‐14. 16 96. Friedman, M.S., M.W. Long, and K.D. Hankenson, Osteogenic differentiation of human 17 mesenchymal stem cells is regulated by bone morphogenetic protein‐6. J Cell Biochem, 2006. 18 98(3): p. 538‐54. 19

97. Pountos, I., et al., The effect of bone morphogenetic protein‐2, bone morphogenetic protein‐7,

21 parathyroid hormone, and platelet‐derived growth factor on the proliferation and osteogenic 22 differentiation of mesenchymal stem cells derived from osteoporotic bone. J Orthop Trauma, 23 2010. 24(9): p. 552‐6. 24 98. An, C., et al., IGF‐1 and BMP‐2 induces differentiation of adipose‐derived mesenchymal stem cells 25 into chondrocytes‐like cells. Ann Biomed Eng, 2010. 38(4): p. 1647‐54. 26

99. Haider, H., et al., IGF‐1‐overexpressing mesenchymal stem cells accelerate bone marrow stem 27

28 cell mobilization via paracrine activation of SDF‐1alpha/CXCR4 signaling to promote myocardial 29 repair. Circ Res, 2008. 103(11): p. 1300‐8. 30 100. Longobardi, L., et al., Effect of IGF‐I in the chondrogenesis of bone marrow mesenchymal stem 31 cells in the presence or absence of TGF‐beta signaling. J Bone Miner Res, 2006. 21(4): p. 626‐36. 32 101. Kim, D.H., et al., Gene expression profile of cytokine and growth factor during differentiation of 33 bone marrow‐derived mesenchymal stem cell. Cytokine, 2005. 31(2): p. 119‐26. 34 102. Silva, W.A., Jr., et al., The profile of gene expression of human marrow mesenchymal stem cells. 35

36 Stem Cells, 2003. 21(6): p. 661‐9. 37 103. Ng, F., et al., PDGF, TGF‐beta, and FGF signaling is important for differentiation and growth of 38 mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling 39 pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic 40 lineages. Blood, 2008. 112(2): p. 295‐307. 41 104. Kang, Y.J., et al., Role of c‐Jun N‐terminal kinase in the PDGF‐induced proliferation and migration 42

of human adipose tissue‐derived mesenchymal stem cells. J Cell Biochem, 2005. 95(6): p. 1135‐

44 45.

randomized, controlled clinical study. Stem Cells Dev, 2011. 20(10): p. 1679‐85. Schenk, S., et al., Monocyte chemotactic protein‐3 is a myocardial mesenchymal stem cell homing factor. Stem Cells, 2007. 25(1): p. 245‐51. Carrero, R., et al., IL1beta induces mesenchymal stem cells migration and leucocyte chemotaxis through NF‐kappaB. Stem Cell Rev, 2012. 8(3): p. 905‐16. Xu, S., H. Wen, and H. Jiang, Urotensin II promotes the proliferation of endothelial progenitor cells through p38 and p44/42 MAPK activation. Molecular medicine reports, 2012. 6(1): p. 197‐ 200. Segal, M.S., et al., Relaxin increases human endothelial progenitor cell NO and migration and vasculogenesis in mice. Blood, 2012. 119(2): p. 629‐36. Naito, H., et al., Induction and expression of anti‐angiogenic vasohibins in the hematopoietic stem/progenitor cell population. Journal of biochemistry, 2009. 145(5): p. 653‐9.

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

126.

127.

128.

129.

130.

20

43

of 38 1

2

3 110. Barrilleaux, B.L., et al., Activation of CD74 inhibits migration of human mesenchymal stem cells. 4

5 In Vitro Cell Dev Biol Anim, 2010. 46(6): p. 566‐72.

6 111. Du, J., et al., Demethylation of epiregulin gene by histone demethylase FBXL11 and BCL6 7 corepressor inhibits osteo/dentinogenic differentiation. Stem Cells, 2013. 31(1): p. 126‐36. 8 112. Oh, J.Y., et al., Cytokine secretion by human mesenchymal stem cells cocultured with damaged 9 corneal epithelial cells. Cytokine, 2009. 46(1): p. 100‐3. 10 113. Yang, J., et al., Heparin‐binding epidermal growth factor‐like growth factor and mesenchymal 11 stem cells act synergistically to prevent experimental necrotizing enterocolitis. J Am Coll Surg, 12

13 2012. 215(4): p. 534‐45. 14 114. Gupta, N., et al., Mesenchymal stem cells enhance survival and bacterial clearance in murine 15 Escherichia coli pneumonia. Thorax, 2012. 67(6): p. 533‐9. 16 115. Walenda, T., et al., Synergistic effects of growth factors and mesenchymal stromal cells for 17 expansion of hematopoietic stem and progenitor cells. Exp Hematol, 2011. 39(6): p. 617‐28. 18 116. Khoury, M., et al., Mesenchymal stem cells secreting angiopoietin‐like‐5 support efficient 19

expansion of human hematopoietic stem cells without compromising their repopulating

21 potential. Stem Cells Dev, 2011. 20(8): p. 1371‐81. 22 117. Gentile, P., et al., Concise review: adipose‐derived stromal vascular fraction cells and platelet‐ 23 rich plasma: basic and clinical implications for tissue engineering therapies in regenerative 24 surgery. Stem Cells Transl Med, 2012. 1(3): p. 230‐6. 25 118. Sanchez‐Gonzalez, D.J., E. Mendez‐Bolaina, and N.I. Trejo‐Bahena, Platelet‐rich plasma peptides: 26

key for regeneration. Int J Pept, 2012. 2012: p. 532519. 27

28 119. Davis, V.L., et al., Platelet‐Rich Preparations to Improve Healing. Part II: Platelet Activation and 29 Enrichment, Leukocyte Inclusion, and Other Selection Criteria. J Oral Implantol, 2012. 30 120. Freitag, J., A. Barnard, and A. Rotstein, Photoactivated platelet‐rich plasma therapy for a 31 traumatic knee chondral lesion. BMJ Case Rep, 2012. 2012. 32 121. Dominici, M., et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The 33 International Society for Cellular Therapy position statement. Cytotherapy, 2006. 8(4): p. 315‐7. 34 122. Taleb, S., et al., Microarray profiling of human white adipose tissue after exogenous leptin 35

36 injection. Eur J Clin Invest, 2006. 36(3): p. 153‐63. 37 123. Matsumoto, T., et al., Regulation of osteoblast differentiation by interleukin‐11 via AP‐1 and 38 Smad signaling. Endocr J, 2012. 59(2): p. 91‐101. 39 124. Chen, L., et al., Sustained delivery of BMP‐2 and platelet‐rich plasma‐released growth factors 40 contributes to osteogenesis of human adipose‐derived stem cells. Orthopedics, 2012. 35(9): p. 41 e1402‐9. 42

125. Liu, K., et al., Coinfusion of mesenchymal stromal cells facilitates platelet recovery without

44 increasing leukemia recurrence in haploidentical hematopoietic stem cell transplantation: a

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7 Figure Legends 8 9 Figure 1. Flow cytometric analysis of ASC marker expression in SVF preparations. 10

Freshly isolated ASC preparations were incubated with Hoechst 33342 dye to label 11

12 nucleated cells and subsequently stained with fluorochrome-labeled antibodies against 13 CD29, CD44, CD45 and CD90. Samples were gated on nucleated cells and the CD45- 14 negative fraction was analyzed for expression of the indicated markers. 15 16 17 18 Figure 2. Comparison of the viability of fresh and cryopreserved ASC preparations 19

prepared by three different protocols. Viability of freshly prepared and cryopreserved

21 ASC-P was determined by trypan blue staining and hemocytometry counting. 22 23

24

25 Figure 3. Effect of PRP/Photo-activation on Expression of MSC Markers by Canine 26 SVF Cells. ASC-P samples (n=3 per group) that were either treated with platelet-rich 27 plasma and photo-activation or were left untreated were incubated with Hoechst 33342 28

dye to label nucleated cells and subsequently stained with fluorochrome-labeled

30 antibodies against CD29, CD44, CD45 and CD90. Samples were gated on nucleated 31 cells and the CD45-negative fraction was analyzed for percent expression of the 32 indicated markers. 33 34 35 36 37

38 39 40 41 42 43 44

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Figure 1: Flow cytometric analysis of ASC marker expression in SVF preparations 4 5 6 7 8 9 10 11 12 13 14

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Figure 4

2: Comparison of the viability of

fresh and cryopreserved ASC

5 preparations prepared by three differ 6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31 32 33 34 35

36 37 38 39 40 41 42 43 44

nt protocols

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Figure 3. Effect of PRP/Photo-activation on Expression of MSC Markers by 4

5 Canine SVF Cells 6

7 8 9 10 11 12 13 14

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Table 1. Expression of Housekeeping Genes in ASC Preparations 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27

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Table 2. Effect of ASC preparation procedures on growth factor gene expression 4 5 6 7 8 9 10 11 12 13 14

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Table 3. Effect of ASC preparation procedures on interleukin gene expression 4 5 6 7 8 9 10 11 12 13 14

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of 38 1

2 3

Table 4. Effect of ASC preparation procedures on expression cytokine genes 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30 31 32 33 34 35 36 37 38 39 40 41

42 43 44

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

r R

vi

of 38 1

2 3

Table 5. Effect of ASC preparation procedures on expression of chemokine genes 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30 31 32 33 34 35 36 37 38 39 40 41

42 43 44

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

r R

vi

of 38 1

2 3

Table 6. Effect of ASC preparation procedures on expression of protease genes 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30 31 32 33 34 35 36 37 38 39 40 41

42 43 44

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

of 38 1

2 3

Table 7. Effect of ASC preparation procedures on expression of glycoprotein 4

5 genes 6

7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30 31 32 33 34 35 36 37 38 39 40

41 42 43 44

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

R vi e w

of 38 1

2 3

Table 4

8. Comparison gene expression by ASC preparations company M and

5 Company V 6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31 32 33 34 35

36 37 38 39 40 41 42 43 44

45 46 47 48 49 50 51

52 53 54 55 56 57 58 59 60

r R

vi w

of 38 1

2 3

Table 9. Effect of PRP/Photoactivation on gene expression by canine ASC-P 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30 31 32 33 34 35 36 37 38 39 40 41

42 43 44

2 Gene expression profiling of canine adipose-derived stem...

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