19F MRI cellular tracer preserves the differentiation potential of hematopoietic stem cells Brooke Helfer, PhD Celsense, Inc Pittsburgh PA
Financial disclosure I have the following relationships to disclose: Employee of Celsense, Inc, the supplier of the Cell Sense reagent used herein.
Why image? Unsolved questions: Do transferred cells migrate to sites of disease or off-target sites? Is migration correlated with clinical effectiveness Can outcomes be improved by modifying migration/homing? Preclinical rodent models may not be predictive, and small animal imaging technologies are generally not translatable for clinical use
19F MRI for cell tracking Cell Sense (CS-1000): Self-delivering perfluorocarbonbased cellular label for Fluorine 19 (19F) MRI Ex vivo cell labeling, in vivo 19F MRI detection High specificity Quantitative Biologically inert Preserves cellular phenotype and function Built-in qualities to facilitate 19F MRI: Short T1, long T2, single major spectral peak Designed for clinical translation to enable seamless testing throughout development of a therapeutic product
Dendritic Cells Quantification of DC1 migration (Urban, Kalinski)
Neural stem cells Intracranial transplantation of therapeutic human neural stem cells (CTX0E03). (Courtesy of E Bible and M Modo, King s College, London. Manuscript in preparation, 2011)
Cell Sense General Protocol
What is the effect on stem cells?
Assessing HSC labeling in vitro Labeling efficiency Viability Cell function in vivo Cell pluripotency and reconstitution
Labeled (% FITC+) CD 34+ Labeling efficiency Increasing dose of FITC-MRI tracer 100 80 60 40 20 0 control 1 3 5 FITC-MRI tracer (mg/ml) 4.4 x 10 11 19F/cell
Percent of total CD34+ Viability 100 80 60 40 control MRI tracer 20 0 Viability Apoptosis Cell Yield
Colonies (#) Cellular function in vitro Colony forming assay 80 60 40 20 0 CFU-E BFU-E CFU-GM CFU-GEMM 0 1 3 5 Cell Sense (mg/ml)
In vivo repopulation studies Lethal TBI 950 cgy 12 days Cohorts No transplant 1.5 x 10 5 BM + Vehicle 1.5 x 10 5 BM + Cell Sense Day 12 CFU-Spleen Assay: A quantitative measurement of hematopoietic progenitors with longterm in vivo repopulating activity Long term BM reconstitution: A qualitative measurement of engraftment with transplanted primitive hematopoietic progenitors (into myeloablated recipients) with long-term in vivo repopulating activity (i.e. the capacity for self-renewal) Reconstitution in peripheral blood Endpoint: Engraftment (CBC) 10 7 BM + Cell Sense *** c-kit+, sca-1+, lineage - >70% labeled
Day 12 CFU-S CFU-S 12 days 30 25 20 15 10 5 0 No BM BM + vehicle BM + CS-1000 Treatment group (N = 8/cohort)
Murine hematopoietic reconstitution was maintained by labeled murine bone marrow. CBC panels indicate that all cell lineages were present including lymphocytes, monocytes and neutrophils. Normal Ranges Cell Sense BMT recipients Ave SD WBC 6-15 8.2 1.45 LYM 3.4-7.44 5.73 0.64 MON 0-0.6 0.35 0.14 NEU 0.5-3.8 2.12 1.08 LY% 57 93 70.7 7.64 MO% 0 7 4.35 1.84 NE% 8 48 24.92 7.43 RBC 7 12 9.84 0.16 HGB 12.2 16.2 14.57 0.58 HCT 35 45 42.45 1.74 MCV 45 55 43.17 0.98 MCH 11.1 12.7 14.82 0.56 MCHC 22.3 32 34.38 1.86 RDWc N/A 19.55 0.33 PLT 200-450 238* 175 PCT N/A 0.17 0.11 MPV N/A 7.13 0.70 PDWc N/A 33.35 4.71
Summary Cell labeling without transfection agents Non toxic to the cells No impact to cell phenotype or function Differentiation and proliferation maintained (in vitro and in vivo) Cell Sense reagent is commercially available
Acknowledgments Celsense Anthony Balducci Amy Wesa Charlie O Hanlon Carnegie Mellon University Eric Ahrens Roberto Gil