Name: rapid isolation of bmpr-ib+ adipose-derived stromal cells for use in a calvarial defect healing model
Uploaded: 2017-02-24T13:00:00
Description: Watch this Scientific Journal Video about Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model at JoVE.com

C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by NIH grants U01 HL099776, R01 DE021683-01, and RC2 DE020771. D.C.W. was supported by NIH grant 1K08DE024269, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.

NOTE: Samples were obtained from patients who gave informed consent. All protocols were reviewed and approved by the appropriate Stanford University Institutional Review Board.&#160;While handling human tissue and cells, always adhere to Biosafety Level 2 (BSL2) precautions, as specified by your institution.
1. Preparation of Reagents
<ol>
	<li>Prepare FACS buffer: Add 10 mL FBS, 5 mL Poloxamer 188 and 5 mL Pen-Strep to 500 mL sterile phosphate-buffered saline (PBS).</li>
	<li>Prepare digest...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+graft+substitutes.">Bone graft substitutes.</a> Expert Rev Med Devices. 3 (1), 49-57 (2006).</li><li>Walmsley, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+in+bone+tissue+engineering.">Nanotechnology in bone tissue engineering.</a> Nanomedicine. 11 (5), 1253-1263 (2015).</li><li>Chapekar, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering:+challenges+and+opportunities.">Tissue engineering: challenges and opportunities.</a> J Biomed Mater Res. 53 (6), 617-620 (2000).</li><li>Levi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+derived+stromal+cells+heal+critical+size+mouse+calvarial+defects.">Human adipose derived stromal cells heal critical size mouse calvarial defects.</a> PLoS One. 5 (6), e11177 (2010).</li><li>Gimble, J. M., Katz, A. J., Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+for+regenerative+medicine.">Adipose-derived stem cells for regenerative medicine.</a> Circ Res. 100 (9), 1249-1260 (2007).</li><li>Levi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD105+protein+depletion+enhances+human+adipose-derived+stromal+cell+osteogenesis+through+reduction+of+transforming+growth+factor+beta1+(TGF-beta1)+signaling.">CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor beta1 (TGF-beta1) signaling.</a> J Biol Chem. 286 (45), 39497-39509 (2011).</li><li>Chung, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD90+(Thy-1)-positive+selection+enhances+osteogenic+capacity+of+human+adipose-derived+stromal+cells.">CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells.</a> Tissue Eng Part A. 19 (7-8), 989-997 (2013).</li><li>Wozney, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+regulators+of+bone+formation:+molecular+clones+and+activities.">Novel regulators of bone formation: molecular clones and activities.</a> Science. 242 (4885), 1528-1534 (1988).</li><li>Wan, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+mouse+adipose-derived+adult+stromal+cells+requires+retinoic+acid+and+bone+morphogenetic+protein+receptor+type+IB+signaling.">Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling.</a> Proc Natl Acad Sci U S A. 103 (33), 12335-12340 (2006).</li><li>McArdle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+for+bone+morphogenetic+protein+receptor+type-IB+promotes+differentiation+and+specification+of+human+adipose-derived+stromal+cells+toward+an+osteogenic+lineage.">Positive selection for bone morphogenetic protein receptor type-IB promotes differentiation and specification of human adipose-derived stromal cells toward an osteogenic lineage.</a> Tissue Eng Part A. 20 (21-22), 3031-3040 (2014).</li><li>Sharon, Y., Alon, L., Glanz, S., Servais, C., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+normal+and+cancer-associated+fibroblasts+from+fresh+tissues+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS).</a> J Vis Exp. (71), e4425 (2013).</li><li>Lo, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+a+critical-sized+calvarial+defect+model+using+adipose-derived+stromal+cells+harvested+from+lipoaspirate.">Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate.</a> J Vis Exp. (68), (2012).</li><li>Lester, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of surgical pathology. , (2010).</li><li>Bunnell, B. 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Critical Steps within the Protocol
During the harvest of ASCs, the critical step is adequate digestion of fat with collagenase. Inadequate digestion will result in a low yield of ASCs. During FACS sorting of BMPR-IB+ cells, it is important to carefully define the gate for positivity. Defining gates too loosely may result in sorted populations that are not pure. During creation of the calvarial defect, it is critical to drill the defect through the bone of the skull but to not advance into the dur...

Major bone defects resulting from injury, infection, or invasive cancer have a significant impact on a patient&#39;s recovery and quality of life. Techniques exist to fill these defects with healthy bone from elsewhere in the patient&#39;s own body, but this transfer carries its own morbidity and risk of complications1,2,3. Furthermore, some defects are so large or complex that sufficient donor bone is not available to fill the defect. Prosthetic devices are a potential option for filling bony defects but these are associated with several disadvantages including infection risk, hardware failure, and foreign body reaction4.
For these reasons there is great interest in the possibility of engineering biological bone substitutes using a patient&#39;s own cells5. Adipose-derived stromal cells (ASCs) have potential for this application because they are abundantly available in the patient&#39;s own fat tissue and they have demonstrated the ability to heal bone defects by generating new bone tissue6,7. ASCs are a diverse population of cells and several studies have shown that selecting for specific cell surface markers can produce cell populations with enhanced osteogenic activity8,9. Selecting ASCs with the highest osteogenic potential would increase the likelihood that a scaffold seeded with these cells could regenerate a large bone defect.
Bone morphogenetic protein (BMP) signaling is critical for regulating bone differentiation and formation10 and the BMP Receptor type IB (BMPR-IB) is known to be important for osteogenesis in ASCs11. Recently, we have shown that expression of BMPR-IB can be used to select for ASCs with enhanced osteogenic activity12. Here we demonstrate a protocol for the isolation of BMPR-IB-expressing ASCs from human fat followed by an assay of their osteogenic activity using an in vivo calvarial defect model.

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rapid isolation of bmpr-ib+ adipose-derived stromal cells for use in a calvarial defect healing model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Micro CT scan done on the day of surgery will clearly show the skull defect. At this time there will be no ingrowth into the 4 mm defect. Subsequent scans are obtained over time to quantify the size of the defect over time compared with the baseline. Defects seeded with BMPR-IB+ cells should demonstrate more rapid closure of the defect when compared with BMPR-IB- and Unsorted cells (Figure 5). In addition, the portion of the skull containing the defect can be decalcified ...

Watch this Scientific Journal Video about Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model at JoVE.com

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

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