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1.  Flow Cytometric Cell Sorting and In Vitro Pre-Osteoinduction Are Not Requirements for In Vivo Bone Formation by Human Adipose-Derived Stromal Cells 
PLoS ONE  2013;8(2):e56002.
Human adipose-derived stromal cells (hASCs) are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI) and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS). The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI) or between purified hASCs and purified hASCs+OI (P>0.05). Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.
doi:10.1371/journal.pone.0056002
PMCID: PMC3569421  PMID: 23409111
2.  Enhancement of Human Adipose-Derived Stromal Cell Angiogenesis through knockdown of a BMP-2 inhibitor 
Introduction
When employing tissue engineering approaches to clinical problems, cells are often transplanted to a distant site on a scaffold into an environment different from their original niche. Previous studies have demonstrated the role of Noggin, a BMP inhibitor in vascular development and angiogenesis. We hypothesized that noggin suppression in human adipose derived stromal cells (hASCs) would enhance VEGF secretion and angiogenesis in vitro and in vivo to a greater extent than BMP-2 alone.
Methods
hASCs were isolated from human lipoaspirate (n=6) and transfected with a Noggin shRNA construct. Knockdown was confirmed and angiogenesis was assessed by tubule formation and qRT-PCR. Cells were seeded on scaffolds with or without BMP-2 and implanted into a 4mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluoresence and immunohistochemistry.
Results
hASCs with noggin suppression secreted significantly higher amounts of VEGF protein on ELISA (*p<0.05). hASCs with noggin knockdown expressed higher levels of angionegic gene markers by qRT-PCR (VE-cadherein, VEGFA, and HIF1A), and displayed enhanced vascular tubule formation in vitro. In vivo, calvarial defects seeded with noggin shRNA hASCs exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (*p<0.05). Additionally, BMP-2 releasing scaffolds significantly enhanced VEGF and PECAM protein levels in the defect site.
Conclusion
hASCs demonstrate significant increases in angiogenesis both in vitro and in vivo both with noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased BMP-2, we can create a more angiogenic niche.
doi:10.1097/PRS.0b013e3182361ff5
PMCID: PMC3246067  PMID: 21915082
Angiogenesis; vasculogenesis; Skeletal tissue engineering; Tissue regeneration; Multipotent stromal cells; Calvarial defect; Noggin; Bone morphogenetic protein; scaffold; adipose-derived stromal cells
3.  Functional Binding of Human Adipose-Derived Stromal Cells: Effects of Extraction Method & Hypoxia Pretreatment 
Annals of plastic surgery  2008;60(4):437-444.
Human adipose-derived stromal cells (hASCs) were evaluated in vitro for their ability to bind vascular adhesion and extracellular matrix proteins in order to arrest (firmly adhere) under physiological flow conditions. hASCs were flowed through a parallel plate flow chamber containing substrates presenting immobilized Type I Collagen, fibronectin, E-selectin, L-selectin, P-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) under static and laminar flow conditions (wall shear stress = 1 dyn/cm2). hASCs were able to firmly adhere to Type I Collagen, fibronectin, VCAM-1, and ICAM-1 substrates, but not to any of the selectins. Pretreatment with hypoxia increased the ability of hASCs isolated by liposuction to adhere to VCAM-1 and ICAM-1, but this effect was not seen in cells isolated by tissue excision. These results indicate that hASCs possess the ability to adhere key adhesion proteins, illustrate the importance of hASC harvest procedure, and suggest mechanisms for homing in a setting where interaction with inflamed or injured tissue is necessary.
doi:10.1097/SAP.0b013e318095a771
PMCID: PMC2829884  PMID: 18362576
Adipose-derived stromal cells; hypoxia; liposuction; parallel plate flow chamber; adhesion cascade
4.  Locally applied VEGFA increases the osteogenic healing capacity of human adipose derived stem cells by promoting osteogenic and endothelial differentiation 
Stem Cells (Dayton, Ohio)  2011;29(2):286-296.
Human adipose derived stem cells (hASCs) are known for their capability to promote bone healing when applied to bone defects. For bone tissue regeneration, both sufficient angiogenesis and osteogenesis is desirable. Vascular endothelial growth factor A (VEGFA) has the potential to promote differentiation of common progenitor cells to both lineages. To test this hypothesis, the effects of VEGFA on hASCs during osteogenic differentiation were tested in vitro. In addition, hASCs were seeded in murine critical-sized calvarial defects locally treated with VEGFA. Our results suggest that VEGFA improves osteogenic differentiation in vitro as indicated by alkaline phosphatase activity, alizarin red staining, and QRT-PCR analysis. Moreover, local application of VEGFA to hASCs significantly improved healing of critical sized calvarial defects in vivo. This repair was accompanied by a striking enhancement of angiogenesis. Both paracrine and, to a lesser degree, cell-autonomous effects of VEGFA treated hASCs were accountable for angiogenesis. These data were confirmed by utilization of CD31-/CD45- mouse ASCsGFP+ cells. In summary, we demonstrated that VEGFA increased osteogenic differentiation of hASCS in vitro and in vivo, which was accompanied by an enhancement of angiogenesis. Additionally, we showed that during bone regeneration, the increase in angiogenesis of hASCs upon treatment with VEGFA was attributable to both paracrine and cell-autonomous effects. Thus, locally applied VEGFA might prove to be a valuable growth factor that can mediate both osteogenesis and angiogenesis of multipotent hASCs in the context of bone regeneration.
doi:10.1002/stem.581
PMCID: PMC3400547  PMID: 21732486
VEGFA; Bone Regeneration; Angiogenesis; Endothel; Calvaria
5.  Human Adipose-Derived Stromal Cells Stimulate Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts 
Stem Cells and Development  2010;20(2):243-257.
Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair are not fully understood. In this study, we examined the potential for hASCs to stimulate autogenous repair of a mouse calvarial defect. Immunofluoresence, osteogenic stains, and surface electron microscopy were used to demonstrate osteogenic differentiation of hASCs. hASCs were engrafted into 4 mm calvarial defects in athymic mice using an osteoconductive scaffold. Analysis included microcomputed tomography, histology, in situ hybridization, and quantitative real-time–polymerase chain reaction. Next, the in vitro interaction between hASCs and mouse calvarial osteoblasts (mOBs) was assessed by the conditioned medium and coculture assays. The medium was supplemented with Hedgehog signaling modifiers, including recombinant N-terminal Sonic hedgehog, smoothened agonist, and cyclopamine. Finally, cyclopamine was delivered in vivo to hASC-engrafted defects. Significant calvarial healing was observed among hASC-engrafted defects compared with control groups (no treatment or scaffold alone) (*P < 0.05). hASCs showed evidence of stimulation of host mouse osteogenesis, including (1) increased expression of bone markers at the defect edge by in situ hybridization, and (2) increased host osteogenic gene expression by species-specific quantitative real-time polymerase chain reaction. Using the conditioned medium or coculture assays, hASCs stimulated mOB osteogenic differentiation, accompanied by Hedgehog signaling activation. N-terminal Sonic hedgehog or smoothened agonist replicated, while cyclopamine reversed, the pro-osteogenic effect of the conditioned medium on mOBs. Finally, cyclopamine injection arrested bone formation in vivo. hASCs heal critical-sized mouse calvarial defects, this is, at least in part, via stimulation of autogenous healing of the host defect. Our studies suggest that hASC-derived Hedgehog signaling may play a paracrine role in skeletal repair.
doi:10.1089/scd.2010.0250
PMCID: PMC3128781  PMID: 20698749
6.  Ovarian cancer-derived lysophosphatidic acid stimulates secretion of VEGF and stromal cell-derived factor-1α from human mesenchymal stem cells 
Experimental & Molecular Medicine  2010;42(4):280-293.
Lysophosphatidic acid (LPA) stimulates growth and invasion of ovarian cancer cells and tumor angiogenesis. Cancer-derived LPA induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts. Presently, we explored whether cancer-derived LPA regulates secretion of pro-angiogenic factors from hASCs. Conditioned medium (CM) from the OVCAR-3 and SKOV3 ovarian cancer cell lines stimulated secretion angiogenic factors such as stromal-derived factor-1α (SDF-1α) and VEGF from hASCs. Pretreatment with the LPA receptor inhibitor Ki16425 or short hairpin RNA lentiviral silencing of the LPA1 receptor abrogated the cancer CM-stimulated expression of α-SMA, SDF-1, and VEGF from hASCs. LPA induced expression of myocardin and myocardin-related transcription factor-A, transcription factors involved in smooth muscle differentiation, in hASCs. siRNA-mediated depletion of endogenous myocardin and MRTF-A abrogated the expression of α-SMA, but not SDF-1 and VEGF. LPA activated RhoA in hASCs and pretreatment with the Rho kinase inhibitor Y27632 completely abrogated the LPA-induced expression of α-SMA, SDF-1, and VEGF in hASCs. Moreover, LPA-induced α-SMA expression was abrogated by treatment with the ERK inhibitor U0126 or the phosphoinositide-3-kinase inhibitor LY294002, but not the PLC inhibitor U73122. LPA-induced VEGF secretion was inhibited by LY294002, whereas LPA-induced SDF-1 secretion was markedly attenuated by U0126, U73122, and LY294002. These results suggest that cancer-secreted LPA induces differentiation of hASCs to cancer-associated fibroblasts through multiple signaling pathways involving Rho kinase, ERK, PLC, and phosphoinositide-3-kinase.
doi:10.3858/emm.2010.42.4.027
PMCID: PMC2859327  PMID: 20177148
carcinoma; fibroblasts; lysophosphatidic acid; ovarian neoplasms; receptors, lysophosphatidic acid; rho-associated kinases; vascular endothelial growth factor A
7.  Angiopoietin-1-expressing adipose stem cells genetically modified with baculovirus nanocomplex: investigation in rat heart with acute infarction 
The objective of this study was to develop angiopoietin-1 (Ang1)-expressing genetically modified human adipose tissue derived stem cells (hASCs) for myocardial therapy. For this, an efficient gene delivery system using recombinant baculovirus complexed with cell penetrating transactivating transcriptional activator TAT peptide/deoxyribonucleic acid nanoparticles (Bac-NP), through ionic interactions, was used. It was hypothesized that the hybrid Bac- NPAng1 system can efficiently transduce hASCs and induces favorable therapeutic effects when transplanted in vivo. To evaluate this hypothesis, a rat model with acute myocardial infarction and intramyocardially transplanted Ang1-expressing hASCs (hASC-Ang1), genetically modified by Bac-NPAng1, was used. Ang1 is a crucial pro-angiogenic factor for vascular maturation and neovasculogenesis. The released hAng1 from hASC-Ang1 demonstrated profound mitotic and anti-apoptotic activities on endothelial cells and cardiomyocytes. The transplanted hASC-Ang1 group showed higher cell retention compared to hASC and control groups. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with hASC-Ang1 treatment compared to infarcted hearts treated with hASC or the untreated group. Furthermore, the hASC-Ang1 group showed significantly higher cardiac performance in echocardiography (ejection fraction 46.28% ± 6.3%, P < 0.001 versus control, n = 8) than the hASC group (36.35% ± 5.7%, P < 0.01, n = 8), 28 days post-infarction. The study identified Bac-NP complex as an advanced gene delivery vehicle for stem cells and demonstrated its potential to treat ischemic heart disease with high therapeutic index for combined stem cell-gene therapy strategy.
doi:10.2147/IJN.S26882
PMCID: PMC3278230  PMID: 22334788
combined stem cell-gene therapy; baculovirus; nanoparticle; myocardial therapy; angiogenesis; tissue engineering
8.  Regulation of Human Adipose-Derived Stromal Cell Osteogenic Differentiation by Insulin-like Growth Factor-1 and Platelet-Derived Growth Factor-Alpha 
Introduction
Human adipose-derived stromal cells (hASCs) possess a great potential for tissue engineering purposes. Our laboratory is interested in harnessing hASCs for skeletal tissue regeneration and identifying those factors that enhance hASC osteogenic differentiation. We hypothesized that Insulin-Like Growth Factor (IGF) and Platelet Derived Growth Factor (PDGF) would stimulate hASC osteogenesis and that IGF would stimulate adipogenesis.
Materials and Methods
ASCs were harvested from human lipoaspirate. Previously, a microarray analysis examined gene expression throughout osteogenic differentiation. In a candidate fashion we added recombinant Insulin-Like Growth Factor (IGF-1) and Platelet-Derived Growth Factor (PDGF)-α individually as well as in combination. Osteogenesis and adipogenesis were assessed by alkaline phosphatase, Alizarin red, and Oil red O staining, as well as qRT-PCR (RUNX2, ALP, OCN, IGF1, PPARG, LPL, AP2, GCP1). Finally, intersection between IGF and PDGF signaling pathways was evaluated.
Results
IGF-1 was observed to increase osteogenic differentiation by all markers (*p<0.01). However, PDGF-α when added alone primarily did not effect osteogenic markers. PDGF-α positively regulated transcription of IGF1. Addition of PDGF-α in combination with or prior to IGF-1 enhanced osteogenesis more than either alone. IGF-1 increased while PDGF-α diminished hASC adipogenesis.
Conclusion
IGF signaling significantly increased osteogenesis in human ASCs and may be used for tissue engineering purposes. The combination of PDGF and IGF may be more beneficial than either alone in driving ASC osteogenesis. Future in vivo applications will focus on the combination of ASCs, biomimetic scaffolds and recombinant IGF.
doi:10.1097/PRS.0b013e3181da8858
PMCID: PMC3016898  PMID: 20220555
Adipose derived stromal cells; Skeletal tissue engineering; Tissue regeneration; Multipotent stromal cells; IGF; PDGF
9.  Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo 
Stem Cells and Development  2010;20(3):427-439.
Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)–polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT–polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze–thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze–thaw process in vitro (*P < 0.01). In vivo, near complete healing was observed among calvarial defects engrafted with fresh hASCs. This was in comparison to groups engrafted with freeze–thaw hASCs that showed little healing (*P < 0.01). Finally, recombinant insulin-like growth factor 1 or recombinant bone morphogenetic protein 4 was observed to increase or rescue in vitro osteogenic differentiation among frozen hASCs (*P < 0.01). The freezing of ASCs for storage significantly impacts their biology, both in vitro and in vivo. The ability of ASCs to successfully undergo osteogenic differentiation after freeze–thaw is substantively muted, both in vitro and in vivo. The use of recombinant proteins, however, may be used to mitigate the deleterious effects of the freeze–thaw process.
doi:10.1089/scd.2010.0082
PMCID: PMC3128779  PMID: 20536327
10.  Human Adipose Tissue-Derived SSEA-4 Subpopulation Multi-Differentiation Potential Towards the Endothelial and Osteogenic Lineages 
Tissue Engineering. Part A  2012;19(1-2):235-246.
Human adipose tissue has been recently recognized as a potential source of stem cells for regenerative medicine applications, including bone tissue engineering (TE). Despite the gathered knowledge regarding the differentiation potential of human adipose tissue-derived stem cells (hASCs), in what concerns the endothelial lineage many uncertainties are still present. The existence of a cell subpopulation within the human adipose tissue that expresses a SSEA-4 marker, usually associated to pluripotency, raises expectations on the differentiation capacity of these cells (SSEA-4+hASCs). In the present study, the endothelial and osteogenic differentiation potential of the SSEA-4+hASCs was analyzed, aiming at proposing a single-cell source/subpopulation for the development of vascularized bone TE constructs. SSEA-4+hASCs were isolated using immunomagnetic sorting and cultured either in α-MEM, in EGM-2 MV (endothelial growth medium), or in osteogenic medium. SSEA-4+hASCs cultured in EGM-2 MV formed endothelial cell-like colonies characterized by a cobblestone morphology and expression of CD31, CD34, CD105, and von Willebrand factor as determined by quantitative reverse transcriptase (RT)-polymerase chain reaction, immunofluorescence, and flow cytometry. The endothelial phenotype was also confirmed by their ability to incorporate acetylated low-density lipoprotein and to form capillary-like structures when seeded on Matrigel. SSEA-4+hASCs cultured in α-MEM displayed fibroblastic-like morphology and exhibited a mesenchymal surface marker profile (>90% CD90+/CD73+/CD105+). After culture in osteogenic conditions, an overexpression of osteogenic-related markers (osteopontin and osteocalcin) was observed both at molecular and protein levels. Matrix mineralization detected by Alizarin Red staining confirmed SSEA-4+hASCs osteogenic differentiation. Herein, we demonstrate that from a single-cell source, human adipose tissue, and by selecting the appropriate subpopulation it is possible to obtain microvascular-like endothelial cells and osteoblasts, the most relevant cell types for the creation of vascularized bone tissue-engineered constructs.
doi:10.1089/ten.tea.2012.0092
PMCID: PMC3530944  PMID: 22924692
11.  Culture effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on cryopreserved human adipose-derived stromal/stem cell proliferation and adipogenesis 
Previous studies have demonstrated that EGF and bFGF maintain the stem cell properties of proliferating human adipose-derived stromal/stem cells (hASCs) in vitro. While the expansion and cryogenic preservation of isolated hASCs are routine, these manipulations can impact their proliferative and differentiation potential. This study examined cryogenically preserved hASCs (n = 4 donors), with respect to these functions, after culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at varying concentrations (0–10 ng/ml). Relative to the control, cells supplemented with EGF and bFGF significantly increased proliferation by up to three-fold over 7–8 days. Furthermore, cryopreserved hASCs expanded in the presence of EGF and bFGF displayed increased oil red O staining following adipogenic induction. This was accompanied by significantly increased levels of several adipogenesis-related mRNAs: aP2, C/EBPα, lipoprotein lipase (LPL), PPARγ and PPARγ co-activator-1 (PGC1). Adipocytes derived from EGF- and bFGF-cultured hASCs exhibited more robust functionality based on insulin-stimulated glucose uptake and atrial natriuretic peptide (ANP)-stimulated lipolysis. These findings indicate that bFGF and EGF can be used as culture supplements to optimize the proliferative capacity of cryopreserved human ASCs and their adipogenic differentiation potential.
doi:10.1002/term.198
PMCID: PMC2763318  PMID: 19670348
adipogenesis; adipose-derived stem cells; edipermal growth factor; basic fibroblast growth factor; cryopreservation; differentiation
12.  Adipogenic Human Adenovirus Ad-36 Induces Commitment, Differentiation, and Lipid Accumulation in Human Adipose-Derived Stem Cells 
Stem cells (Dayton, Ohio)  2008;26(4):969-978.
Human adenovirus Ad-36 is causatively and correlatively linked with animal and human obesity, respectively. Ad-36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad-36-induced adipogenesis in human obesity, the effect of the virus on commitment, differentiation, and lipid accumulation was investigated in vitro in primary human adipose-derived stem/stromal cells (hASC). Ad-36 infected hASC in a time- and dose-dependent manner. Even in the presence of osteogenic media, Ad-36-infected hASC showed significantly greater lipid accumulation, suggestive of their commitment to the adipocyte lineage. Even in the absence of adipogenic inducers, Ad-36 significantly increased hASC differentiation, as indicated by a time-dependent expression of genes within the adipogenic cascade—CCAAT/Enhancer binding protein-β, peroxisome proliferator-activated receptor-γ, and fatty acid-binding protein—and consequentially increased lipid accumulation in a time- and viral dose-dependent manner. Induction of hASC to the adipocyte state by Ad-36 was further supported by increased expression of lipoprotein lipase and the accumulation of its extracellular fraction. hASC from subjects harboring Ad-36 DNA in their adipose tissue due to natural infection had significantly greater ability to differentiate compared with Ad-36 DNA-negative counterparts, which offers a proof of concept. Thus, Ad-36 has the potential to induce adipogenesis in hASC, which may contribute to adiposity induced by the virus.
doi:10.1634/stemcells.2007-0868
PMCID: PMC2747294  PMID: 18203674
Obesity; Adiposity; Adipogenesis; Infectobesity; Lipogenesis; Adipocyte progenitors
13.  Acute Skeletal Injury is Necessary for Human Adipose-Derived Stromal Cell Mediated Calvarial Regeneration 
Plastic and reconstructive surgery  2011;127(3):1118-1129.
Background
Studies have demonstrated that human adipose derived stromal cells (hASCs) are able to repair acute calvarial injuries. However, the more clinically relevant repair of an established skeletal defect has not been addressed. We sought to determine whether hASCs could heal chronic (established) calvarial defects.
Methods
Critical-sized (4mm) mouse parietal defects were created. hASCs were either engrafted immediately postoperatively (acute defect), or 8 weeks following defect creation (established defect). Methods of analysis included microCT scans, histology, and in situ hybridization. Finally, hASCs were treated in vitro with PRP to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (Alkaline phosphatase, Alizarin red, and qRT-PCR).
Results
Near complete osseous healing was observed when calvarial defects were immediately engrafted with hASCs. In contrast, when hASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microCT, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in a established hASC engrafted defect. In situ hybridization and qRT PCR showed an increase in BMP expression (Bmp2, Bmp4 and Bmp7) acutely following calvarial defect creation. Finally, in vitro treatment of hASCs with PRP enhanced osteogenic differentiation and increased Bmp2 expression.
Conclusions
While hASCs can be utilized to heal an acute mouse calvarial defect, hASCs do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated post-injury may explain these differences in healing. Platelet rich plasma enhances osteogenic differentiation of hASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.
doi:10.1097/PRS.0b013e318205f274
PMCID: PMC3073240  PMID: 21364415
Osteogenesis; Adipose derived mesenchymal cells; Multipotent stromal cells; Platelet rich plasma, Bone morphogenetic protein, Adult Stem Cells
14.  Ligament-Derived Matrix Stimulates a Ligamentous Phenotype in Human Adipose-Derived Stem Cells 
Tissue Engineering. Part A  2010;16(7):2307-2319.
Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 × 106, 1 × 106, or 2 × 106 hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p ≤ 0.05). Increased seeding density increased DNA content incrementally over 28 days in culture for LDM but not COL constructs (p ≤ 0.05). These findings suggest that LDM can stimulate a ligament phenotype by hASCs, and may provide a novel scaffold material for ligament engineering applications.
doi:10.1089/ten.tea.2009.0720
PMCID: PMC2947935  PMID: 20406104
15.  Microarray Analysis of Human Adipose-Derived Stem Cells in Three-Dimensional Collagen Culture: Osteogenesis Inhibits Bone Morphogenic Protein and Wnt Signaling Pathways, and Cyclic Tensile Strain Causes Upregulation of Proinflammatory Cytokine Regulators and Angiogenic Factors 
Tissue Engineering. Part A  2011;17(21-22):2615-2627.
Human adipose-derived stem cells (hASC) have shown great potential for bone tissue engineering. However, the molecular mechanisms underlying this potential are not yet known, in particular the separate and combined effects of three-dimensional (3D) culture and mechanical loading on hASC osteogenesis. Mechanical stimuli play a pivotal role in bone formation, remodeling, and fracture repair. To further understand hASC osteogenic differentiation and response to mechanical stimuli, gene expression profiles of proliferating or osteogenically induced hASC in 3D collagen I culture in the presence and absence of 10% uniaxial cyclic tensile strain were examined using microarray analysis. About 847 genes and 95 canonical pathways were affected during osteogenesis of hASC in 3D culture. Pathway analysis indicated the potential roles of Wnt/β-catenin signaling, bone morphogenic protein (BMP) signaling, platelet-derived growth factor (PDGF) signaling, and insulin-like growth factor 1 (IGF-1) signaling in hASC during osteogenic differentiation. Application of 10% uniaxial cyclic tensile strain suggested synergistic effects of strain with osteogenic differentiation media on hASC osteogenesis as indicated by significantly increased calcium accretion of hASC. There was no significant further alteration in the four major pathways (Wnt/β-catenin, BMP, PDGF, and IGF-1). However, 184 transcripts were affected by 10% cyclic tensile strain. Function and network analysis of these transcripts suggested that 10% cyclic tensile strain may play a role during hASC osteogenic differentiation by upregulating two crucial factors in bone regeneration: (1) proinflammatory cytokine regulators interleukin 1 receptor antagonist and suppressor of cytokine signaling 3; (2) known angiogenic inductors fibroblast growth factor 2, matrix metalloproteinase 2, and vascular endothelial growth factor A. This is the first study to investigate the effects of both 3D culture and mechanical load on hASC osteogenic differentiation. A complete microarray analysis investigating both the separate effect of soluble osteogenic inductive factors and the combined effects of chemical and mechanical stimulation was performed on hASC undergoing osteogenic differentiation. We have identified specific genes and pathways associated with mechanical response and osteogenic potential of hASC, thus providing significant information toward improved understanding of our use of hASC for functional bone tissue engineering applications.
doi:10.1089/ten.tea.2011.0107
PMCID: PMC3204199  PMID: 21767168
16.  Human Adipose-Derived Stem Cells Impair Natural Killer Cell Function and Exhibit Low Susceptibility to Natural Killer-Mediated Lysis 
Stem Cells and Development  2011;21(8):1333-1343.
Human adipose-derived stem cells (hASCs) have been successfully used in treating numerous diseases. However, several aspects need to be considered, particularly in the context of allogeneic cell therapy. To better understand hASCs-host interactions, we studied the phenotype of hASCs and their modulatory effect on natural killer (NK) cells by using bone marrow-mesenchymal stem cells (hBM-MSCs) as a reference. The hASCs displayed a lower susceptibility to NK cell-mediated lysis and a lower expression of ligands for DNAM-1 when compared with hBM-MSCs. Moreover, here we demonstrated that hASCs and hBM-MSCs can modulate NK cells through the action of soluble factors such as indoleamine 2,3-dioxygenase. Altogether, these results suggest that for an adoptive cell therapy based on the transfer of allogeneic hASCs, the NK-hASCs crosstalk will not result in an immediate recognition of the transferred cells. Thus, hASCs may remain in the tissue long enough to balance the immune response before being cleared.
doi:10.1089/scd.2011.0139
PMCID: PMC3353740  PMID: 21867426
17.  Differentiation of Human Adipose-derived Stem Cells along the Keratocyte Lineage In vitro 
Purpose
To evaluate differentiation of human adipose-derived stem cells (hASCs) to the keratocyte lineage by co-culture with primary keratocytes in vitro.
Materials and Methods
A co-culture system using transwell inserts to grow hASCs on bottom and keratocytes on top in keratocyte differentiating medium (KDM) was developed. hASCs that were cultured in complete culture medium (CCM) and KDM were used as control. After 16 days, hASCs were examined for morphologic changes and proliferation by cell count. qRT-PCR and flow cytometry were used to detect the expression of aldehyde dehydrogenase 3 family, member A1 (ALDH3A1) and keratocan.
Results
hASCs became more dendritic and elongated in co-culture system relative to CCM and KDM. The doubling time of the cells was longer as differentiation progressed. qRT-PCR showed a definite trend towards increased expression of both ALDH3A1 and keratocan in co-culture system despite statistically non-significant p-values. Flow cytometry showed significantly increased protein levels of ALDH3A1 and keratocan in co-culture system relative to CCM group (p < 0.001) and even relative to KDM group (p < 0.001 for ALDH3A1 and p < 0.01 for keratocan).
Conclusion
The co-culture method is a promising approach to induce differentiation of stem cell populations prior to in vivo applications. This study reveals an important potential for bioengineering of corneal tissue using autologous multi-potential stem cells.
doi:10.4172/2155-9570.1000270
PMCID: PMC3737075  PMID: 23936748
Human adipose-derived stem cells; Co-culture system; Keratocyte; Bioengineered cornea
18.  Primary Cilia: The Chemical Antenna Regulating Human Adipose-Derived Stem Cell Osteogenesis 
PLoS ONE  2013;8(5):e62554.
Adipose-derived stem cells (ASC) are multipotent stem cells that show great potential as a cell source for osteogenic tissue replacements and it is critical to understand the underlying mechanisms of lineage specification. Here we explore the role of primary cilia in human ASC (hASC) differentiation. This study focuses on the chemosensitivity of the primary cilium and the action of its associated proteins: polycystin-1 (PC1), polycystin-2 (PC2) and intraflagellar transport protein-88 (IFT88), in hASC osteogenesis. To elucidate cilia-mediated mechanisms of hASC differentiation, siRNA knockdown of PC1, PC2 and IFT88 was performed to disrupt cilia-associated protein function. Immunostaining of the primary cilium structure indicated phenotypic-dependent changes in cilia morphology. hASC cultured in osteogenic differentiation media yielded cilia of a more elongated conformation than those cultured in expansion media, indicating cilia-sensitivity to the chemical environment and a relationship between the cilium structure and phenotypic determination. Abrogation of PC1, PC2 and IFT88 effected changes in both hASC proliferation and differentiation activity, as measured through proliferative activity, expression of osteogenic gene markers, calcium accretion and endogenous alkaline phosphatase activity. Results indicated that IFT88 may be an early mediator of the hASC differentiation process with its knockdown increasing hASC proliferation and decreasing Runx2, alkaline phosphatase and BMP-2 mRNA expression. PC1 and PC2 knockdown affected later osteogenic gene and end-product expression. PC1 knockdown resulted in downregulation of alkaline phosphatase and osteocalcin gene expression, diminished calcium accretion and reduced alkaline phosphatase enzymatic activity. Taken together our results indicate that the structure of the primary cilium is intimately associated with the process of hASC osteogenic differentiation and that its associated proteins are critical players in this process. Elucidating the dynamic role of the primary cilium and its associated proteins will help advance the application of hASC in generating autologous tissue engineered therapies in critical defect bone injuries.
doi:10.1371/journal.pone.0062554
PMCID: PMC3656889  PMID: 23690943
19.  Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1 
PLoS ONE  2012;7(9):e46087.
Background
Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible factor 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF.
Methodology/Principal Findings
Immunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot.
Conclusion
In hASCs trypsin and hypoxia induce VEGF expression through separate pathways.
doi:10.1371/journal.pone.0046087
PMCID: PMC3457954  PMID: 23049945
20.  Engineering Adipose-like Tissue in vitro and in vivo Utilizing Human Bone Marrow and Adipose-derived Mesenchymal Stem Cells with Silk Fibroin 3D Scaffolds 
Biomaterials  2007;28(35):5280-5290.
Biomaterials derived from silk fibrion prepared by aqueous (AB) and organic (HFIP) solvent based processes, along with collagen (COL) and poly-lactic acid (PLA) based scaffolds were studied in vitro and in vivo for their utility in adipose tissue engineering strategies. For in vitro studies, human bone marrow and adipose-derived mesenchymal stem cells (hMSCs and hASCs) were seeded on the various biomaterials and cultured for 21 days in the presence of adipogenic stimulants (AD) or maintained as noninduced controls. Alamar Blue analysis revealed each biomaterial supported initial attachment of hMSCs and hASCs to similar levels for all matrices except COL in which higher levels were observed. hASCs and hMSCs cultured on all biomaterials in the presence of AD showed significant upregulation of adipogenic mRNA transcript levels (LPL, GLUT4, FABP4, PPARγ, adipsin, ACS) to similar extents when compared to noninduced controls. Similarly Oil-Red O analysis of hASC or hMSC-seeded scaffolds displayed substantial amounts of lipid accumulating adipocytes following cultivation with AD. The data revealed AB and HFIP scaffolds supported similar extents of lipid accumulating cells while PLA and COL scaffolds qualitatively displayed lower and higher extents by comparison, respectively. Following a 4 week implantation period in a rat muscle pouch defect model, both AB and HFIP scaffolds supported in vivo adipogenesis either alone or seeded with hASCs or hMSCs as assessed by Oil-Red O analysis, however the presence of exogenous cell sources substantially increased the extent and frequency of adipogenesis observed. In contrast, COL and PLA scaffolds underwent rapid scaffold degradation and were irretrievable following the implantation period. The results suggest that macroporous 3D AB and HFIP silk fibroin scaffolds offer an important platform for cell-based adipose tissue engineering applications, and in particular, provide longer-term structural integrity to promote the maintenance of soft tissue in vivo.
doi:10.1016/j.biomaterials.2007.08.017
PMCID: PMC2695965  PMID: 17765303
21.  A Xenogeneic-Free Protocol for Isolation and Expansion of Human Adipose Stem Cells for Clinical Uses 
PLoS ONE  2013;8(7):e67870.
Human adipose stem cells (hASCs) play a crucial role in the fields of regenerative medicine and tissue engineering for different reasons: the abundance of adipose tissue, their easy harvesting, the ability to multipotent differentiation and the fact that they do not trigger allogeneic blood response or secrete cytokines that act as immunosuppressants. The vast majority of protocols use animal origin reagents, with the underlying risk of transmitting infections by non-human pathogens. We have designed a protocol to isolate and maintain the properties of hASCs avoiding xenogeneic reagents. These changes not only preserve hASCs morphology, but also increase cell proliferation and maintain their stem cell marker profile. On the other hand, human serum albumin (HSA), Tryple® and human Serum (HS), do not affect hASCs multipotent differentiation ability. The amendments introduced do not trigger modifications in the transcriptional profile of hASCs, alterations in key biochemical pathways or malignization. Thus, we have proven that it is possible to isolate and maintain hASCs avoiding animal reagents and, at the same time, preserving crucial culture parameters during long term culture. Thereby we have revealed a novel and effective tool for the improvement of clinical, cell-based therapies.
doi:10.1371/journal.pone.0067870
PMCID: PMC3706484  PMID: 23874459
22.  Agent-Based Model of Therapeutic Adipose-Derived Stromal Cell Trafficking during Ischemia Predicts Ability To Roll on P-Selectin 
PLoS Computational Biology  2009;5(2):e1000294.
Intravenous delivery of human adipose-derived stromal cells (hASCs) is a promising option for the treatment of ischemia. After delivery, hASCs that reside and persist in the injured extravascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limit the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic hASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting bottlenecks in the homing process. This process, however, is incredibly complex, which merited the use of computational techniques to speed the rate of discovery. We developed a multicell agent-based model (ABM) of hASC trafficking during acute skeletal muscle ischemia, based on over 150 literature-based rules instituted in Netlogo and MatLab software programs. In silico, trafficking phenomena within cell populations emerged as a result of the dynamic interactions between adhesion molecule expression, chemokine secretion, integrin affinity states, hemodynamics and microvascular network architectures. As verification, the model reasonably reproduced key aspects of ischemia and trafficking behavior including increases in wall shear stress, upregulation of key cellular adhesion molecules expressed on injured endothelium, increased secretion of inflammatory chemokines and cytokines, quantified levels of monocyte extravasation in selectin knockouts, and circulating monocyte rolling distances. Successful ABM verification prompted us to conduct a series of systematic knockouts in silico aimed at identifying the most critical parameters mediating hASC trafficking. Simulations predicted the necessity of an unknown selectin-binding molecule to achieve hASC extravasation, in addition to any rolling behavior mediated by hASC surface expression of CD15s, CD34, CD62e, CD62p, or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 µm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications.
Author Summary
Ischemic pathologies, such as acute myocardial infarction and peripheral vascular disease, continue to be associated with high morbidities and mortalities. Recently, therapies wherein adult stem cells are injected into the circulation have been shown to increase blood flow and help to restore tissue function following injury. Pre-clinical animal models and human trials have shown successes utilizing this approach, but variable trafficking efficiencies and low incorporation of cells into the injured tissue severely limit effectiveness and may preclude clinical adoption. To address this, we sought to study the complex process of how injected stem cells traffic through the microcirculation and home to sites of injury, in an effort to identify bottlenecks in this process that could be manipulated for therapeutic gain. We developed an agent-based computer model to speed the rate of discovery, and we identified a key cell–cell adhesion interaction that could be targeted to enhance stem cell homing efficiencies during injectable stem cell therapies.
doi:10.1371/journal.pcbi.1000294
PMCID: PMC2636895  PMID: 19247427
23.  Alignment and Elongation of Human Adipose-Derived Stem Cells in Response to Direct-Current Electrical Stimulation 
In vivo, direct current electric fields are present during embryonic development and wound healing. In vitro, direct current (DC) electric fields induce directional cell migration and elongation. For the first time, we demonstrate that cultured human adipose tissue-derived stem cells (hASCs) respond to the presence of direct-current electric fields. Cells were stimulated for 2–4 hours with DC electric fields of 6 V/cm that were similar to those encountered in vivo post-injury. Upon stimulation, hASCs were observed to elongate and align perpendicularly to the applied electric field, disassemble gap junctions, and upregulate the expression of genes for connexin-43, thrombomodulin, vascular endothelial growth factor, and fibroblast growth factor. In separate related studies, human epicardial fat-derived stem cells (heASCs) were also observed to align and elongate. It is interesting that the morphological and phenotypic characteristics of mesenchymal stem cells derived both from liposuction aspirates and from cardiac fat can be modulated by direct current electric fields. In further studies, we will quantify the effects of the electrical fields in the context of wound healing.
doi:10.1109/IEMBS.2009.5333142
PMCID: PMC2791914  PMID: 19964171
24.  Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects 
PLoS ONE  2010;5(6):e11177.
Background
Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect.
Methodology/Principal Findings
Human ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo.
Conclusions/Significance
Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.
doi:10.1371/journal.pone.0011177
PMCID: PMC2887361  PMID: 20567510
25.  Proteomic Identification of ADAM12 as a Regulator for TGF-β1-Induced Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle Cells 
PLoS ONE  2012;7(7):e40820.
Background
Transforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.
Methods and Results
Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells.
Conclusions
These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.
doi:10.1371/journal.pone.0040820
PMCID: PMC3396647  PMID: 22808268

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