Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo

James, Aaron W.; Lévi, Benjamin; Nelson, Emily R.; Peng, Michelle; Commons, George W.; Lee, Min; Wu, Benjamin M.; Longaker, Michael T.

doi:10.1089/scd.2010.0082

Cited by 56 publications

(54 citation statements)

References 56 publications

(89 reference statements)

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“…Osteogenic differentiation of cells followed a previously published protocol. 26,27 The medium was supplemented with 300-600 ng/mL of NELL-1, as previously described. 28 Osteogenic differentiation was assessed using alkaline phosphatase (ALP) and alizarin red (AR) staining at 5 and 10 days of osteogenic differentiation, respectively (with or without 300 ng/mL NELL-1).…”

Section: Cell Culturementioning

confidence: 99%

Human Perivascular Stem Cells Show Enhanced Osteogenesis and Vasculogenesis with Nel-Like Molecule I Protein

Askarinam¹,

James

Zara

et al. 2013

Tissue Engineering Part A

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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 mg/mL), 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 patientmatched 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.

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Section: Cell Culturementioning

confidence: 99%

Human Perivascular Stem Cells Show Enhanced Osteogenesis and Vasculogenesis with Nel-Like Molecule I Protein

Askarinam¹,

James

Zara

et al. 2013

Tissue Engineering Part A

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“…Proliferation was assessed by bromodeoxyuridine (BrdU) incorporation assays as previously described [34], exposed to recombinant human (rh) SHH-N (250 and 500 ng/mL; R&D Systems) and rhNELL-1 (100 and 300 ng/mL). Dosages were used based on previous publications [12,33].…”

Section: Bromodeoxyuridine Incorporationmentioning

confidence: 99%

Additive Effects of Sonic Hedgehog and Nell-1 Signaling in Osteogenic Versus Adipogenic Differentiation of Human Adipose-Derived Stromal Cells

James

Pang

Askarinam

et al. 2012

Stem Cells and Development

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A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the proosteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N + NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.

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“…Scaffolds are custom-made per previously published protocol from poly(lactic-co-glycolic acid) (PLGA, Burmingham Polymer) with hydroxyapatite coating [4][5][6] . Apatite-coated PLGA scaffolds are fabricated from 85/15 PLGA by solvent casting and a particulate leaching process.…”

Section: Scaffold Creationmentioning

confidence: 99%

Use of Human Perivascular Stem Cells for Bone Regeneration

James¹,

Zara

Corselli

et al. 2012

JoVE

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Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering [1][2][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 cytokines 1,2 . 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-wires 4 . The FSD described is also a critical size defect, which does not significantly heal on its own 4 . 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. Video LinkThe video component of this article can be found at https://www.jove.com/video/2952/ Protocol Perivascular Stem Cell IsolationThis is described in details in the adjacent article "Purification of Perivascular Stem Cells from Human White Adipose tissue", by M. Corselli et al. Scaffold Creation1. Scaffolds are custom-made per previously published protocol from poly(lactic-co-glycolic acid) (PLGA, Burmingham Polymer) with hydroxyapatite coating [4][5][6] . Apatite-coated PLGA scaffolds are fabricated from 85/15 PLGA by solvent casting and a particulate leaching process. Scaffolds are created in a spherical shape (2-mm diameter) for muscle pouch implantation, a discoid shape (4 mm in diameter) for calvarial implantation, or cylindrical (4 mm in diameter, 6 mm in length) for femoral segmental defects. 2. Briefly, PLGA/chloroform solutions mixed with sucrose (polymer/sucrose ratio 5/95, w/w) are cast into a 200-300-μm diameter Teflon mold to create the custom-made construct. After freeze-drying overnight, scaffolds are removed from the Teflon mold and immersed in ddH 2 O to dissolve the sucrose. Scaffolds are disinfected by immersion in 70% ethanol for 30 min, followed by three rinses of ddH 2 O. 3. For apatite coating, a simulated body fluid (SBF) solution is prepared by sequentially dissolving CaCl 2 , MgCl2•6H 2 O, NaHCO 3 , and K 2 HPO 4 •3H 2 O in ddH 2 O. Solution pH is lowered to 6 by adding 1M hydrochloric acid to increase the solubility. Na 2 SO 4 , KCl, and NaCl are added and the final pH is adjusted to 6.5 (SBF 1). 4. Mg2+ and HCO 3 -free SBF (SBF 2) is prepared by adding CaCl 2 and K 2 HPO 4 •3H 2 O in ddH 2 O and pH is lowered to 6. KCl and NaC...

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Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo

Cited by 56 publications

References 56 publications

Human Perivascular Stem Cells Show Enhanced Osteogenesis and Vasculogenesis with Nel-Like Molecule I Protein

Human Perivascular Stem Cells Show Enhanced Osteogenesis and Vasculogenesis with Nel-Like Molecule I Protein

Additive Effects of Sonic Hedgehog and Nell-1 Signaling in Osteogenic Versus Adipogenic Differentiation of Human Adipose-Derived Stromal Cells

Use of Human Perivascular Stem Cells for Bone Regeneration

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