Human Perivascular Stem Cells Research Articles

Human perivascular stem cells prevent bone graft resorption in osteoporotic contexts by inhibiting osteoclast formation.

The vascular wall stores mesenchymal progenitor cells which are able to induce bone regeneration, via direct and paracrine mechanisms. Although much is known regarding perivascular cell regulation of osteoblasts, their regulation of osteoclasts, and by extension utility in states of high bone resorption, is not known. Here, human perivascular stem cells (PSCs) were used as a means to prevent autograft resorption in a gonadectomy‐induced osteoporotic spine fusion model. Furthermore, the paracrine regulation by PSCs of osteoclast formation was evaluated, using coculture, conditioned medium, and purified extracellular vesicles. Results showed that PSCs when mixed with autograft bone induce an increase in osteoblast:osteoclast ratio, promote bone matrix formation, and prevent bone graft resorption. The confluence of these factors resulted in high rates of fusion in an ovariectomized rat lumbar spine fusion model. Application of PSCs was superior across metrics to either the use of unpurified, culture‐defined adipose‐derived stromal cells or autograft bone alone. Under coculture conditions, PSCs negatively regulated osteoclast formation and did so via secreted, nonvesicular paracrine factors. Total RNA sequencing identified secreted factors overexpressed by PSCs which may explain their negative regulation of graft resorption. In summary, PSCs reduce osteoclast formation and prevent bone graft resorption in high turnover states such as gonadectomy‐induced osteoporosis.

Perivascular Stem Cell-Derived Cyclophilin A Improves Uterine Environment with Asherman’s Syndrome via HIF1α-Dependent Angiogenesis

Asherman's syndrome (AS) is characterized by intrauterine adhesions or fibrosis resulting from scarring inside the endometrium. AS is associated with infertility, recurrent miscarriage, and placental abnormalities. Although mesenchymal stem cells show therapeutic promise for the treatment of AS, the molecular mechanisms underlying its pathophysiology remain unclear. We ascertained that mice with AS, like human patients with AS, suffer from extensive fibrosis, oligo/amenorrhea, and infertility. Human perivascular stem cells (hPVSCs) from umbilical cords repaired uterine damage in mice with AS, regardless of their delivery routes. In mice with AS, embryo implantation is aberrantly deferred, which leads to intrauterine growth restriction followed by no delivery at term. hPVSC administration significantly improved implantation defects and subsequent poor pregnancy outcomes via hypoxia inducible factor 1α (HIF1α)-dependent angiogenesis in a dose-dependent manner. Pharmacologic inhibition of HIF1α activity hindered hPVSC actions on pregnancy outcomes, whereas stabilization of HIF1α activity facilitated such actions. Furthermore, therapeutic effects of hPVSCs were not observed in uterine-specific HIF1α-knockout mice with AS. Secretome analyses of hPVSCs identified cyclophilin-A as the major paracrine factor for hPVSC therapy via HIF1α-dependent angiogenesis. Collectively, we demonstrate that hPVSCs-derived cyclophilin-A facilitates HIF1α-dependent angiogenesis to ameliorate compromised uterine environments in mice with AS, representing the major pathophysiologic features of humans with AS.

Human perivascular stem cell-derived extracellular vesicles mediate bone repair.

The vascular wall is a source of progenitor cells that are able to induce skeletal repair, primarily by paracrine mechanisms. Here, the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First, purified human perivascular stem cells (PSCs) were observed to induce mitogenic, pro-migratory, and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic, pro-migratory, and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins, as demonstrated by EV trypsinization, or neutralizing antibodies for CD9 or CD81. Moreover, shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally, PSC-EVs stimulated bone repair, and did so via stimulation of skeletal cell proliferation, migration, and osteodifferentiation. In sum, PSC-EVs mediate the same tissue repair effects of perivascular stem cells, and represent an 'off-the-shelf' alternative for bone tissue regeneration.

WISP-1 drives bone formation at the expense of fat formation in human perivascular stem cells

The vascular wall within adipose tissue is a source of mesenchymal progenitors, referred to as perivascular stem/stromal cells (PSC). PSC are isolated via fluorescence activated cell sorting (FACS), and defined as a bipartite population of pericytes and adventitial progenitor cells (APCs). Those factors that promote the differentiation of PSC into bone or fat cell types are not well understood. Here, we observed high expression of WISP-1 among human PSC in vivo, after purification, and upon transplantation in a bone defect. Next, modulation of WISP-1 expression was performed, using WISP-1 overexpression, WISP-1 protein, or WISP-1 siRNA. Results demonstrated that WISP-1 is expressed in the perivascular niche, and high expression is maintained after purification of PSC, and upon transplantation in a bone microenvironment. In vitro studies demonstrate that WISP-1 has pro-osteogenic/anti-adipocytic effects in human PSC, and that regulation of BMP signaling activity may underlie these effects. In summary, our results demonstrate the importance of the matricellular protein WISP-1 in regulation of the differentiation of human stem cell types within the perivascular niche. WISP-1 signaling upregulation may be of future benefit in cell therapy mediated bone tissue engineering, for the healing of bone defects or other orthopaedic applications.

Adipose-derived Human Perivascular Stem Cells May Improve Achilles Tendon Healing in Rats.

Achilles tendon rupture is a common injury and the best treatment option remains uncertain between surgical and nonoperative methods. Biologic approaches using multipotent stem cells such as perivascular stem cells pose a possible treatment option, although there is currently a paucity of evidence regarding their clinical therapeutic use. The purpose of this study was to determine whether injected perivascular stem cells (PSCs) would (1) improve histologic signs of tendon healing (such as percent area of collagen); and (2) improve biomechanical properties (peak load or stiffness) in a rat model of Achilles tendon transection. Two subtypes of PSCs were derived from human adipose tissue: pericytes (CD146CD34CD45CD31) and adventitial cells (CD146CD34CD45CD31). Thirty-two athymic rats underwent right Achilles transection and were randomized to receive injection with saline (eight tendons), hydrogel (four tendons), pericytes in hydrogel (four tendons), or adventitial cells in hydrogel (eight tendons) 3 days postoperatively with the left serving as an uninjured control. Additionally, a subset of pericytes was labeled with CM-diI to track cell viability and localization. At 3 weeks, the rats were euthanized, and investigators blinded to treatment group allocation evaluated tendon healing by peak load and stiffness using biomechanical testing and percent area of collagen using histologic analysis with picrosirius red staining. Histologic analysis showed a higher mean percent area collagen for pericytes (30%) and adventitial cells (28%) than hydrogel (21%) or saline (26%). However, a nonparametric statistical analysis yielded no statistical difference. Mechanical testing demonstrated that the pericyte group had a higher peak load than the saline group (41 ± 7 N versus 26 ± 9 N; mean difference 15 N; 95% confidence interval [CI], 4-27 N; p = 0.003) and a higher peak load than the hydrogel group (41 ± 7 N versus 25 ± 3 N; mean difference 16; 95% CI, 8-24 N; p = 0.001). The pericyte group demonstrated higher stiffness than the hydrogel group (36 ± 12 N/mm versus 17 ± 6 N/mm; mean difference 19 N/mm; 95% CI, 5-34 N/mm; p = 0.005). Our results suggest that injection of PSCs improves mechanical but not the histologic properties of early Achilles tendon healing. This is a preliminary study that provides more insight into the use of adipose-derived PSCs as a percutaneous therapy in the setting of Achilles tendon rupture. Further experiments to characterize the function of these cells may serve as a pathway to development of minimally invasive intervention aimed at improving nonoperative management while avoiding the complications associated with surgical treatment down the line.

Isolation and characterization of minipig perivascular stem cells for bone tissue engineering.

Human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC) in recent years. The current study was designed to sort the minipig (mp) perivascular stem cells (PSCs) and investigate the osteogenic potential. Purification of human PSCs was achieved via fluorescence-activated cell sorting (FACS) from human liposuction samples [cluster of differentiation (CD)45−CD34−CD146+ perithelial cells and CD45−CD34+CD146− adventitial cells]. Subsequently, PSCs were isolated from mp adipose tissue samples (n=9), characterized and, using purified mpPSCs (obtained by FACS, which is used in human PSC purification), the mpPSC osteogenic and adipogenic potential was evaluated by Alizarin Red S and Oil Red O staining in vitro, respectively. The cell morphometry was observed following cell isolation and culture, and hematoxylin and eosin staining was performed to identify the fat tissue structure and vascular distribution. Osteogenic and adipogenic differentiation-associated gene expression levels were analyzed by reverse transcription-quantitative polymerase chain reaction. The results demonstrated that the same antigens used for human PSC identification and isolation were working in mp tissue (CD45, CD146 and CD34). The two cell groups: CD45−CD34−CD146+ pericytes and CD45−CD34+CD146− adventitial cells were successfully isolated from the subcutaneous fat in the posterior neck of mps, mpPSCs accounted for 8.6% of the stromal vascular fraction (SVF) with 1.4% pericytes and 7.2% adventitial cells. mpPSCs demonstrated characteristics of MSCs, including cell surface marker expression, colony forming unit-fibroblast inclusion, and the stronger osteogenic and adipogenic differentiation potential than that of the non-selected vascular stromal cells. The mRNA expression levels of osteocalcin, collagen, type I, α1 and peroxisome proliferator-activated receptor-γ in the mpPSCs group were significantly higher than those of the unsorted pSVF group (P<0.05). Thus, the current study successfully isolated and cultured CD146+ and CD34+ cell populations from mp tissues, characterized the cells' PSC-like phenotype and identified their distinctly osteogenic and adipogenic potential.

WNT16 induces proliferation and osteogenic differentiation of human perivascular stem cells

Perivascular stem cells (PSC) are a progenitor population defined by their perivascular residence. Recent studies have examined the relative difference in Wnt ligands to induce PSC differentiation, including Wnt16. Here, we examine the role of Wnt16 in the proliferation and osteogenic differentiation of human PSC. Treatment of PSC with WNT16 significantly increased cell proliferation to a greater extent than did WNT3A. In addition, WNT16 showed a significant increase in osteogenic gene expression among PSC. These data demonstrate that WNT16 represents a combined mitogenic/pro-osteogenic stimulus that may play a functional role in human mesenchymal stem cell mediated bone repair.

Designing a 3D printed human derived artificial myo-structure for anal sphincter defects in anorectal malformations and adult secondary damage

Fecal incontinence (FI) is a major social and economic burden. Adults suffering from post-surgery consequences or trauma and children affected by congenital anorectal malformations are the main categories of patients suffering from FI due to defective sphincter complex. Current therapeutic options for FI are often only partially effective, require lifelong care and are resource consuming. Besides, in both congenital and acquired anal sphincter defects, muscle damage is often irregular, leading to a diffuse derangement not suitable for surgical repair.In this context, regenerative medicine offers an increasing number of theoretically suitable solutions to restore sphincter structures, but only a few preclinical studies have been published regarding the use of cell therapy, biomaterials or tissue engineered structures for FI application. Here we present, for the first time, the generation of an in vitro engineered muscular sphincter using a population of adult human perivascular stem cells (pericytes) and 3D bioprinting. 3D bioprinting is an emerging technology for fabricating artificial tissue and organ constructs, which lends itself perfectly to the design of muscle constructs for anal sphincter repair. In our approach, 3D functional constructs have been created by simultaneously depositing cells and supporting material in a spatially defined, layer-by-layer stacking organization, generating a biomimetic spatial arrangement. Specifically, we developed a 3D myo-structure starting from human muscle derived pericytes, perivascular stem cells able to undergo robust myogenesis while also guaranteeing a rapid vascularization supporting and recruiting new blood vessels. The results presented here represent a pre-clinic study that strongly supports the feasibility of this innovative approach to treat the forms of fecal incontinence that are unresponsive to conservative treatments.

Perivascular Stem Cells Diminish Muscle Atrophy and Retain Viability in a Rotator Cuff Tear Model

Objectives:Rotator cuff tears (RCTs) are a common cause of shoulder pain and often necessitate surgical repair. Muscle changes including atrophy, fibrosis, and fatty degeneration can develop after RCTs, which may compromise surgical repair and clinical outcomes. Lipoaspirate-derived human perivascular stem cells (PSCs) have demonstrated myogenic and angiogenic potential in other small animal models of muscle injury. We hypothesized that the administration of PSCs following massive RCTs may help to diminish these muscle changes in a small animal model.Methods:A total of 90 immunodeficient mice were used (15 groups, N=6). Each was assigned to one of three surgical groups: i) sham, ii) supraspinatus and infraspinatus tendon transection (TT), or iii) TT and suprascapular nerve denervation (TT+DN). PSCs were harvested from human lipoaspirate and sorted using fluorescence-activated cell sorting into small blood vessel residing pericytes (CD146+ CD34- CD45- CD31-) and large blood perivascular adventitial cells (CD146- CD34+ CD45- CD31-). Mice received either a) no injection, b) saline injection, c) pericyte injection, or d) adventitial cell injection at the time of the index procedure or at two weeks following index surgery. The supraspinatus muscles were harvested six weeks after the index procedure. Muscle atrophy was assessed by measuring percent wet muscle weight change for each sample. Muscle fiber cross-sectional area (CSA), fibrosis, and fatty degeneration were analyzed using Image J™. Additionally, pericytes and adventitial cells were transduced with a luciferase-containing construct. Animals were given injections of luciferin and imaged using IVIS to track in vivo bioluminescence following injections to assess cell viability.Results:Treatment with PSC injection after TT resulted in less wet weight loss and greater muscle fiber CSA than control groups (P<0.05). The TT+DN groups treated with PSC injections two weeks post-op also had less muscle weight loss and greater muscle fiber CSA than their respective control groups. The TT+DN groups treated with PSC injections at the time of surgery demonstrated no differences in weight loss, but had greater muscle fiber CSA than their respective controls. There was no difference in fibrosis between the TT groups. However, TT+DN groups treated with pericyte injections at both time points and adventitial cell injections two weeks post-op had less fibrosis than TT+DN controls. There was less fatty degeneration in the TT groups treated with pericyte injections at both time points and adventitial cells at the time of surgery compared to matched controls. There were no differences in the amount of fatty degeneration between the TT+DN groups. Bioluminescence imaging demonstrated viability of the injected cells at three weeks following injections (Figure 1).Conclusion:Our findings demonstrate significantly less muscle atrophy in the groups treated with PSC injections compared to respective controls for both TT and TT+DN procedures. These results suggest that the use of PSCs may have a role in the prevention of muscle atrophy by aiding in the maintenance of muscle bulk without leading to increased fibrosis or fatty infiltration. Additionally, bioluminescence data suggests that these cells maintain viability and engraft within the native muscle to improve muscle bulk. Improved muscle quality in the setting of rotator cuff tears may increase the success rates of rotator cuff repair and lead to superior clinical outcomes.

Accelerated Chondrogenic Differentiation of Human Perivascular Stem Cells with NELL-1.

Osteoarthritis is the leading cause of disability in the US. Consequently, there is a pressing need for restoring the structural and functional properties of diseased articular cartilage. Yet the search for the right combination of proper target cells and growth factors for cartilage regeneration remains challenging. In this study, we first tested the intrinsic chondrogenic differentiation ability of human perivascular stem cells (hPSCs), a novel source of mesenchymal stem cells (MSCs) isolated by fluorescence-activated cell sorting (FACS) from human adipose tissue. A putative prochondrogenic growth factor, NEL-like molecule-1 (NELL-1), was added to the hPSC pellets to upregulate gene expression of chondrogenic markers, including AGGRECAN, COLLAGEN II, and COMP. Furthermore, the addition of NELL-1 to a transforming growth factor beta 3 (TGF-β3) + bone morphogenetic protein-6 (BMP-6) "cocktail" resulted in the best combinatorial stimulation in accelerating the chondrogenic differentiation of hPSCs, as evidenced by increased gene and protein expression of chondrogenic markers in a shortened induction time without elevating expression of hypertrophic, fibrotic, and osteogenic markers. Mechanistically, this acceleration rendered by NELL-1 may be partially attributed to NELL-1's upregulation of BMP receptors and TGF-β receptor type I in hPSCs for increased responsiveness to BMPs + TGF-βs. In conclusion, lipoaspirate-derived hPSCs present a novel and abundant cell source of MSCs for cartilage regeneration, and the combinatorial application of NELL-1, TGF-β3, and BMP-6 with hPSCs may remarkably enhance and accelerate cartilage repair.

Brief Report: Human Perivascular Stem Cells and Nel-Like Protein-1 Synergistically Enhance Spinal Fusion in Osteoporotic Rats.

Autologous bone grafts (ABGs) are considered as the gold standard for spinal fusion. However, osteoporotic patients are poor candidates for ABGs due to limited osteogenic stem cell numbers and function of the bone microenvironment. There is a need for stem cell-based spinal fusion of proven efficacy under either osteoporotic or nonosteoporotic conditions. The purpose of this study is to determine the efficacy of human perivascular stem cells (hPSCs), a population of mesenchymal stem cells isolated from adipose tissue, in the presence and absence of NELL-1, an osteogenic protein, for spinal fusion in the osteoporosis. Osteogenic differentiation of hPSCs with and without NELL-1 was tested in vitro. The results indicated that NELL-1 significantly increased the osteogenic potential of hPSCs in both osteoporotic and nonosteoporotic donors. Next, spinal fusion was performed by implanting scaffolds with regular or high doses of hPSCs, with or without NELL-1 in ovariectomized rats (n = 41). Regular doses of hPSCs or NELL-1 achieved the fusion rates of only 20%-37.5% by manual palpation. These regular doses had previously been shown to be effective in nonosteoporotic rat spinal fusion. Remarkably, the high dose of hPSCs+NELL-1 significantly improved the fusion rates among osteoporotic rats up to approximately 83.3%. Microcomputed tomography imaging and quantification further confirmed solid bony fusion with high dose hPSCs+NELL-1. Finally, histologically, direct in situ involvement of hPSCs in ossification was shown using undecalcified samples. To conclude, hPSCs combined with NELL-1 synergistically enhances spinal fusion in osteoporotic rats and has great potential as a novel therapeutic strategy for osteoporotic patients.

Human perivascular stem cell-based bone graft substitute induces rat spinal fusion.

Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair.

Human perivascular stem cells show enhanced osteogenesis and vasculogenesis with Nel-like molecule I protein.

An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34-, and CD45-), which surround capillaries and microvessels, and (2) adventitial cells (CD146-, CD34+, and CD45-), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 μg/μL), hPSC (2.5×10(5) cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC+NELL-1 in the development of strategies for vascularized bone regeneration.

An Abundant Perivascular Source of Stem Cells for Bone Tissue Engineering

Adipose tissue is an ideal mesenchymal stem cell (MSC) source, as it is dispensable and accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which has disadvantages for tissue regeneration. In the present study, we prospectively purified human perivascular stem cells (PSCs) from n = 60 samples of human lipoaspirate and documented their frequency, viability, and variation with patient demographics. PSCs are a fluorescence-activated cell sorting-sorted population composed of pericytes (CD45-, CD146+, CD34-) and adventitial cells (CD45-, CD146-, CD34+), each of which we have previously reported to have properties of MSCs. Here, we found that PSCs make up, on average, 43.2% of SVF from human lipoaspirate (19.5% pericytes and 23.8% adventitial cells). These numbers were minimally changed by age, gender, or body mass index of the patient or by length of refrigerated storage time between liposuction and processing. In a previous publication, we observed that human PSCs (hPSCs) formed significantly more bone in vivo in comparison with unsorted human SVF (hSVF) in an intramuscular implantation model. We now extend this finding to a bone injury model, observing that purified hPSCs led to significantly greater healing of mouse critical-size calvarial defects than hSVF (60.9% healing as opposed to 15.4% healing at 2 weeks postoperative by microcomputed tomography analysis). These studies suggest that adipose-derived hPSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, hPSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy.

Perivascular Stem Cells: A Prospectively Purified Mesenchymal Stem Cell Population for Bone Tissue Engineering

Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone-forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence-activated cell sorting) of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient-matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel-like molecule 1 (NELL-1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose-derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL-1 is a candidate growth factor able to induce human PSC osteogenesis.

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

An Isolated Cryptic Peptide Influences Osteogenesis and Bone Remodeling in an Adult Mammalian Model of Digit Amputation

Biologic scaffolds composed of extracellular matrix (ECM) have been used successfully in preclinical models and humans for constructive remodeling of functional, site-appropriate tissue after injury. The mechanisms underlying ECM-mediated constructive remodeling are not completely understood, but scaffold degradation and site-directed recruitment of progenitor cells are thought to play critical roles. Previous studies have identified a cryptic peptide derived from the C-terminal telopeptide of collagen IIIα that has chemotactic activity for progenitor cells. The present study characterized the osteogenic activity of the same peptide in vitro and in vivo in an adult murine model of digit amputation. The present study showed that the cryptic peptide increased calcium deposition, alkaline phosphatase activity, and osteogenic gene expression in human perivascular stem cells in vitro. Treatment with the cryptic peptide in a murine model of mid-second phalanx digit amputation led to the formation of a bone nodule at the site of amputation. In addition to potential therapeutic implications for the treatment of bone injuries and facilitation of reconstructive surgical procedures, cryptic peptides with the ability to alter stem cell recruitment and differentiation at a site of injury may serve as powerful new tools for influencing stem cell fate in the local injury microenvironment.