1. 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

2. 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

3. 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

4. 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

5. Hypothesis
MASCs, when implanted in a biocompatible scaffold, will regenerate skin in full-thickness, non-healing wounds in adult rats.

6. Materials
Rat MASCs transduced with green fluorescent protein (GFP) passage 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 g adult rats

7. 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

8. 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

9. Wounds
Empty
MASCs
Day Day 28

10. 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

11. Histology
Empty defect Defect with MASCs

12. Immune Staining Red: keratinocytes Blue: nuclei Green: MASCs
Normal epidermis Empty defect Defect with MASCs

13. Immune Staining Yellow: endothelium Yellow: apocrine epithelium
Hair follicle within defect Microvasculature Apocrine gland within defect

14. 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

15. 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

17. 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

18. 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

19. Wound Area

20. 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:
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:
7. Taub PJ et al. Plastic Reconstructive Surgery 123(4): , 2009
8. Achor T et al. Tiss Engineering submitted 2015