Dermal Regeneration Using Multipotent Adult Stem Cells Miner Ross, MPH, MS3; Alexandra West, BA, MS4; Jessie Chan; Paul D. Marino, BS; Paul Lucas, PhD Department of Orthopaedic Surgery New York Medical College 11 February 2015
Background Full-thickness skin wounds are common in clinical practice Caused by trauma, infection, immobilization, etc. Treatment ranges from serial dressing changes to split- or full-thickness skin grafts Healing produces a fibrotic, contracted scar There is an unmet need for treatment that is more cost-effective and efficacious
Multipotent Adult Stem Cells Adult cells found in connective tissues Not transduced with any genes Distinct from mesenchymal stem cells (MSCs) Expand apparently indefinitely in cell culture Rat MASCs >120 doublings, human MASCs > 100 doublings Regenerate all three germ layers in vitro e.g. endothelial cells, keratinocytes, astrocytes, hepatocytes
Tissue Regeneration Cells are grown into biocompatible, biodegradable matrix Typically polyglycolic acid (PGA) mesh Cells are always implanted undifferentiated Observed regeneration in vivo: Cartilage Bone Peritoneum Vasculature
Hypothesis MASCs, when implanted in a biocompatible scaffold, will regenerate skin in full-thickness, non-healing wounds in adult rats.
Materials Rat MASCs transduced with green fluorescent protein (GFP) passage 19-23 were grown into 4mm thick polyglycolic acid (PGA) felt matrix 2 full-thickness skin wounds 3cm in diameter were made to the level of the underlying musculature in 400-450g adult rats
Methods Each skin wound was assigned to one of three treatments: Empty defect: 10 PGA alone : 10 PGA with MASCs: 10 All wounds were given standard care with Xeroform, Telfa, and VetRap Wounds were photographed at creation and every 7 days for 8 weeks
Data Obtained Defects were dissected at 8 weeks and processed for paraffin histology Samples were stained with H&E, Mallory- Heidenhain, or immunohistochemical markers Photographs were used to measure wound area changes over time
Wounds Empty MASCs Day 0 Day 28
Results Empty defects and those treated with PGA alone contract into small, triangular scars Wounds treated with PGA + MASCs show regeneration of epidermis without contraction
Histology Empty defect Defect with MASCs
Immune Staining Red: keratinocytes Blue: nuclei Green: MASCs Normal epidermis Empty defect Defect with MASCs
Immune Staining Yellow: endothelium Yellow: apocrine epithelium Hair follicle within defect Microvasculature Apocrine gland within defect
Histology Control defects demonstrate only fibrotic scar tissue without dermal or epidermal elements MASC-treated wounds demonstrate regenerating dermal and epidermal structures viz. keratinocytes, fibroblasts, endothelium, smooth muscle, epidermal adnexa MASC origin of regenerating cells is established by elaboration of GFP
Conclusion Treatment of full-thickness skin wounds with MASCs results in regeneration of dermis and epidermis with decreased fibrosis and contraction Immunohistochemistry demonstrates that MASCs differentiate into cell types likely determined by local factors Future studies: larger animals, e.g. porcine
Isolation of MASCs Enzymatic digestion Primary culture Freeze-thaw Allow progenitor cells to differentiate Pre-selected serum Freeze-thaw 7.5% DMSO slow freeze -80ºC MASCs survive
MASCs vs. MSCs Limited proliferation potential Unlimited proliferation potential NCAM+, APPL2+, ITGB3+, PECAM1+, nestin+ Differentiation into phenotypes of all germ layers Limited proliferation potential NCAM-, APPL2-, ITGB3- , PECAM1-, nestin- Some mesodermal phenotypes
Wound Area
References 1. Schultz SS et al. Wound Repair Regen. 14(2):224-31, 2006 2. Paul A. Lucas et al. U.S. Patent No. 7,259,011 B2 Issued Aug. 21, 2007 3. Lucas PA et al. J. Surg. Res., 62: 229-232 1996. 4. Arriero M et al. Am J Physiol Renal Physiol. 287(4):F621-7, 2004 5. Sealy, R, et al. ORS, 2004 6. Lucas PA et al. J. Surg. Res., 62: 229-232 1996 7. Taub PJ et al. Plastic Reconstructive Surgery 123(4): 1178-85, 2009 8. Achor T et al. Tiss Engineering submitted 2015