Background. Background. Background. Background. Background 3/4/2013
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1 3/4/213 Human Very Small Embryonic-like Stem Cells (s) Capacity to Regenerate Bone Tissue: A Potential Cell-based Therapy for Autogenous Bone Grafting Aaron M. Havens, DMD Need to discover an improved autogenous cellular population with regenerative capabilities Identify donor cells within the recipient Harvest and isolate these cells Optimize and implant for regenerate therapy Bone grafting in the craniofacial region often requires multiple grafting procedures Challenge in timing and materials Xenografts, Allografts, Autografts, rh-bmp-2 Limitations Incomplete healing, rejection, vascularization, etc. Autogenous bone grafts are superior to the other choices Supply vital, immunocompatible, osteoregenerative cells to the site Most harvested from the iliac crest Large quantities of osteogenic precursor cells Mesenchymal stem cells (MSC) Contained within bone marrow stromal cell populations Capable of limited self-renewal 1
2 3/4/213 Limitations of MSCs Require bone marrow aspirate Require in vitro culture Requires time, expensive, potential contamination, lose or gain their differentiation potential, logistics Need to search for alternatives cellular populations Very Small Embryonic-like Stem Cells Extremely small (3-5µm) Very rare (.2% of total bone marrow) Express markers characteristic of epiblast/germ line and pluripotent stem cell markers CXCR4(+)CD34(+)Lin(-)CD45(-) Express Oct-4 and Nanog Give rise to all three germ-layer cellular populations Reside in bone marrow Isolated from cord blood May serve as a reserve population of repair cells In steady-state conditions, VSEL cells at very low levels within the peripheral blood Mobilized within the peripheral blood and organ tissues Myocardial infarct Cerebral infarct Retinal injury Statement of Purpose Hypothesis To lay the foundation for a new approach to osseous tissue regeneration based on a human marrow population Human very small embryonic-like stem cells (VSELs) represent a multi-potent population that are able to undergo multi-lineage differentiation for osseous repair in a cranial defect 2
3 3/4/213 Specific Aims Determine the extent to which cells can lead to osseous regeneration of craniotomy defects within an animal model Determine the fate of cells within a calvarial defect Collection and Processing In collaboration with NeoStem (Boston, MA) Human subjects (male) Injected for two consecutive days with G-CSF Peripheral blood collected day 3 Staining and cell sorting to isolate VSEL population phenotype Calvarial defects Female 5- to 7-week-old SCID mice 3mm Trephine bur in SS handpiece Craniotomy preformed in parietal bone Gelfoam matrix Sutured for primary closure 3-month incubation period Negative Control (sponge alone) Positive Control (mbmsc + BMP7 OE) Human VSELs 2, 1, 3, 5, Radiographic Studies Micro-CT scans of the calvaria MicroView 2.2 Individually assessed for the region of interest (ROI) Fixed threshold Tissue mineral content Bone volume fraction (BVF) Bone mineral density (BMD) 3
4 Tissue Mineral Content 3/4/213 Histological Studies Hematoxylin and Eosin Staining Masson s Trichrome staining staining Human-specific Osteocalcin CD31 Nestin PPAR-gamma Real-Time PCR Murine Tissues (halu) Whole blood Liver Spleen Femur Normalized against mβ-actin Statistical analysis Descriptive analysis (Mean, SE) Kruskal Wallis Test; Mann Whitney U Test ANOVA; Tukey's HSD Intra-operator reliability 1 samples, 2 months apart, Interclass correlations coefficients Differences with a p-value <.5 will be considered statistically significant Results Average of All Donors.6 Positive Negative Control Control 2, 1, 3, 5, Positive Control Negative Control 2, 1, 3, 5, Figure 1 Cells / Defect p <.5 Figure 2 4
5 Tissue Mineral Content H & E Stain Trichrome Stain Human Osteocalcin/Total Protein (ng/ml) Number of Human Cells (ALU/B-Actin) 3/4/213 Donor 1 Donor Donor 3 Neg. Control 2, 2, 2, Positive Control Negative Control 2, 2, 2, p <.5 Figure 2 Figure 3 A B Neg. Control 3 25 C D Positve Control Negative Murine Sponge Alone 2, 1, 3, 5, Control Positive BMP Figure 4 p <.5 Figure 5 A B C D 12 OC CD31 Nestin PPARγ 1 8 Neg. Control Neg. Control Neg. Control Neg. Control 6 4 OC CD31 Nestin PPARγ 2 Negative Control Positive Control Femur Spleen Liver Blood Figure 6 p <.5 Figure 7 5
6 Exp 1 Exp 2 Duration of Study Group VSEL Cells Comment Exp. Design Sample Size (Months) Negative Control Vehicle c.d. 8 3 Cells 3 Purified c.d. 5 3 Cells Vehicle c.d. 5 3 Cells 1 Enriched c.d. 1 3 Cells Vehicle c.d. 1 3 Cells 3 Enriched c.d. 5 3 Cells 3 Enriched + hcd34 c.d. 5 3 Cells 2 Purified c.d Cells Vehicle c.d Cells 2 Enriched c.d. 1 3 hcd34 Cells 2 hcd34 c.d. 1 3 Negative Control Vehicle Alone c.d. 4 3 Postive Control hbmsc c.d. 4 3 Cells 1 Purified c.d. 4 3 Cells 1 Enriched c.d. 4 3 Cells 1 Enriched + hcd34 c.d. 5 3 Cells 5 Enriched c.d. 5 3 Cells 5 Enriched + hcd34 c.d. 5 3 Cells PTH c.d. 5 3 Tumor Growth (%) Cells 1 Enriched + PTH c.d. 5 3 Exp 3 Positive Control Ad-BMP7 mmc3t3 c.d Exp 4 Negative Control Vehicle Alone c.d. 5 3 Cells 2 Purified c.d. 5 3 Cells 2 Purified c.d. 5 3 Cells 2 Enriched c.d. 5 3 Cells 2 Enriched c.d. 5 3 Cells 2 Enriched s.c. 5 3 Negative Control 2 Lin-Sca+CD45+ s.c mvsel Cells 5 Small Lin-Sca+CD45- s.c mvsel Cells 5 Small Lin-Sca+CD45- s.c Exp. 5 mvsel Cells 2 Established ossicle s.c Exp 6 Negative Control Established ossicle s.c Negative Control During ossicle formation s.c mvsel Cells 5 Established ossicle s.c mvsel Cells 5 During ossicle formation s.c to 1 to Exp 7 mvsel Cells 4-12 Serial transplant into ossicles s.c. 5 3 x 3 months Exp 8 hpca 2 PC3, C4-2B s.c Exp 9 hbca 2 MCF-7 S.c /4/213 Reconstitution of Skeletal Defects Formed well-organized human structures characteristic of osteoblasts, vascular structures, neurons and adipose tissue Limited reconstitution of large skeletal defects Human VSEL cells may require a longer experimental period for differentiation into osteogenic cells Did not correlate with the number of cells placed in defect Stem cell-to-stem cell communication May need accessory cells Scaffolds Mineralized matrix for proper osseous formation Ability to maintain sufficient space May not have been optimized to hold the cells in the wound site Identified human Alu signatures in the peripheral blood of the animals, not present in tissues Donor Differences Different numbers isolated from different donors Significant differences in osseous tissue formation with equivalent cell dosing (i.e. 2, cells) Potential inherent differences in the abilities of VSEL cells isolated from different individuals Frozen vs. fresh isolation Further Study?? Tumor 6
7 3/4/213 Conclusion Thank You For the first time, human VSEL cells have been isolated and shown to regenerate tissue in a mouse model of skeletal repair We now have the ability to harvest and deliver these cells as a therapeutic agent to enhance bone formation in states of craniofacial clefts and malformations, trauma and degenerative conditions such as osteoporosis and fracture repair 7
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