頭蓋冠欠損治癒モデルで使用するためのBMPR-IB +脂肪由来間質細胞の迅速な単離
Summary
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.
Abstract
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.
Introduction
傷害、感染症、または浸潤がんに起因する主な骨欠損は患者の回復と生活の質に大きな影響を与えます。技術は、他の場所で、患者自身の体内でより健康な骨でこれらの欠陥を埋めるために存在するが、この転送は、独自の罹患率および合併症の1、2、3のリスクを伴います。さらに、いくつかの欠陥が十分なドナー骨が欠損を埋めるために使用できないほど大規模または複雑です。補綴デバイスは、骨欠損を充填するための潜在的なオプションですが、これらは、感染リスク、ハードウェア障害、および異物反応4を含むいくつかの欠点と関連しています。
これらの理由のために患者自身の細胞5を用いて、生物学的代用骨を操作する可能性に大きな関心があります。脂肪由来間質細胞（ASCに）彼らは、患者自身の脂肪組織で豊富に入手可能であり、それらは新しい骨組織6,7を生成することにより骨欠損を治癒する能力が実証されているため、このアプリケーションの可能性を有しています。 ASCを、細胞の多様な集団であり、いくつかの研究は、特定の細胞表面マーカーについて選択する強化骨形成活性8,9で細胞集団を生成することができることを示しています。最も高い骨形成能とのASCを選択するこれらの細胞を播種した足場は、大きな骨欠損を再生することができると可能性を増加させます。
骨形成タンパク質（BMP）シグナル伝達は骨分化と形成10及びBMPレセプタータイプIB（BMPR-IB）を調節するために重要であるが、ASCを11で骨形成に重要であることが知られています。最近、我々は、BMPR-IBの発現をbができることを示していますeは強化された骨形成活性12とのASCを選択するために使用します。ここでは、in vivoでの頭蓋冠欠損モデルを用いて、それらの骨形成活性のアッセイが続く人間の脂肪からBMPR-IB発現のASCを単離するためのプロトコルを示しています。
Protocol
Representative Results
Discussion
プロトコル内の重要なステップ
ASCの収穫時には、重要なステップは、コラゲナーゼと脂肪の適切な消化です。不十分な消化は、ASCを低収量になります。 BMPR-IB +細胞のFACSソーティングの際には、慎重に陽性のためのゲートを定義することが重要です。あまりにも緩くゲートを定義すると、純粋ではないソートされた集団になることがあります。頭蓋冠欠損の作成時に、頭蓋…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 100 micron cell strainer | Falcon | 352360 | |
| 15 blade scalpel | Miltex | 4-515 | |
| 24 well plate | Corning | 3524 | |
| 40 micron cell strainer | Falcon | 352340 | |
| 50 mL conical centrifuge tubes | Falcon | 352098 | |
| 6-0 Ethilon nylon suture, 18", P-3 needle, | Ethicon | 1698G | |
| Anti-BMPR-IB primary antibody | R&D systems | FAB5051A | |
| BioGel PI surgical gloves | Mölnlycke Health Care | ALA42675Z | |
| Buprenorphine SR | ZooPharm | ||
| Castro-Viejo needle driver | Fine Science Tools | 12565-14 | |
| CD1 nude mouse | Charles River | 086 | |
| Collagenase Type II powder | Gibco | 17101-015 | |
| DMEM medium | Gibco | 10564-011 | |
| Drill: Circular knife 4.0 mm | Xemax Surgical | CK40 | |
| Drill: Z500 Brushless Micromotor | NSK | NSKZ500 | |
| FBS | Gicbo | 10437-077 | |
| Fisherbrand Absorbent Underpads, 20" x 24" | Fisher Scientific | 14-206-62 | |
| Fisherbrand Sterile cotton gauze pad, 4" x 4" | Fisher Scientific | 22-415-469 | |
| Heating pad | Kent Scientific | DCT-20 | |
| Hyclone 199/EBSS medium | GE Life Sciences | SH30253.01 | |
| Isothesia isoflurane | Henry Schein | 050033 | |
| Micro Forceps with teeth | Roboz | RS-5150 | |
| Micro Forceps with teeth | Roboz | RS-5150 | |
| Paraffin film (Parafilm) | Bemis | PM996 | |
| PBS | Gibco | 10010-023 | |
| Pen-Strep | Gibco | 15140-122 | |
| PLGA scaffolds | Proprietary Formulation | ||
| Poloxamer 188, 10% | Sigma | P5556-100ML | |
| Polylined Sterile Field, 18" x 24" | Busse Hospital Disposables | 696 | Cut a rectangular hole of the appropriate size |
| Polysucrose Solution: Histopaque 1119 | Sigma | 11191 | |
| Povidone Iodine Prep Solution | Medline | MDS093944H | |
| Puralube petrolatum ophthalmic ointment, 1/8 oz. tube | Dechra Veterinary Products | ||
| RBC lysis buffer | Sigma | 11814389001 | |
| Webcol alcohol prep swabs | Covidien | 6818 |
References
- Silber, J. S., et al. Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion. Spine (Phila Pa 1976). 28 (2), 134-139 (2003).
- Giannoudis, P. V., Dinopoulos, H., Tsiridis, E. Bone substitutes: an update. Injury. 36, S20-S27 (2005).
- Laurencin, C., Khan, Y., El-Amin, S. F. Bone graft substitutes. Expert Rev Med Devices. 3 (1), 49-57 (2006).
- Walmsley, G. G., et al. Nanotechnology in bone tissue engineering. Nanomedicine. 11 (5), 1253-1263 (2015).
- Chapekar, M. S. Tissue engineering: challenges and opportunities. J Biomed Mater Res. 53 (6), 617-620 (2000).
- Levi, B., et al. Human adipose derived stromal cells heal critical size mouse calvarial defects. PLoS One. 5 (6), e11177 (2010).
- Gimble, J. M., Katz, A. J., Bunnell, B. A. Adipose-derived stem cells for regenerative medicine. Circ Res. 100 (9), 1249-1260 (2007).
- Levi, B., et al. CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor beta1 (TGF-beta1) signaling. J Biol Chem. 286 (45), 39497-39509 (2011).
- Chung, M. T., et al. CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells. Tissue Eng Part A. 19 (7-8), 989-997 (2013).
- Wozney, J. M., et al. Novel regulators of bone formation: molecular clones and activities. Science. 242 (4885), 1528-1534 (1988).
- Wan, D. C., et al. Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling. Proc Natl Acad Sci U S A. 103 (33), 12335-12340 (2006).
- McArdle, A., et al. Positive selection for bone morphogenetic protein receptor type-IB promotes differentiation and specification of human adipose-derived stromal cells toward an osteogenic lineage. Tissue Eng Part A. 20 (21-22), 3031-3040 (2014).
- Sharon, Y., Alon, L., Glanz, S., Servais, C., Erez, N. Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS). J Vis Exp. (71), e4425 (2013).
- Lo, D. D., et al. Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate. J Vis Exp. (68), (2012).
- Lester, S. C. . Manual of surgical pathology. , (2010).
- Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. Adipose-derived stem cells: isolation, expansion and differentiation. Methods. 45 (2), 115-120 (2008).
