I presented my ongoing research from Massachusetts General Hospital at the American Society of Transplantation Annual Scientific Exchange meeting in Orlando, FL.
WEBINAR Characterisation of human pluripotent stem cells (ESCs and IPSC) and ...
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1. Luis P. Fernández December 5, 2009 Image from Ken Shortman and Yong-Jun Liu, Nature Reviews Immunology 2, 151-161 (2002). “ Embryonic stem cell-based therapy for transplantation tolerance"
2. Relevant Financial Relationship Disclosure Statement Luis P. Fernández, PhD Transplantation Biology Research Center/ Massachusetts General Hospital I have no financial relationships to disclose within the past 12 months relevant to my presentation AND My presentation does not include discussion of off-label or investigational use
9. H&E CD11c , I-Ab Ig isotype 400x 100x kd cap 100x kd cap 100x kd cap cap kd P15 ESC Implanted ES-DCp BM-DC MEF spleen PU.1 CX3CR1 CD11b CD11c Oct3/4 -Actin water GM-CSF IL-3 ES-DCp Follow up on weeks 1, 2, 3, 6 ES-DCp in immunosuppressed mice: test of sustained commitment 1 mg anti-CD4 & -CD8 ESC
10. K b I-A b actin LN Thy Spn (anti-CD4) LN Thy Spn (anti-CD4 &-CD8) LN Thy Spn (anti-CD4) LN Thy (anti-CD8) 0.5 mg 0.25 mg Spn ES-DCp ESC Thymus, week 3 CD11c (red) I-Ab (green) I-Ab actin I-Ab -actin Kb ESC ES-DCp BM-DC Water Responder mice injected with: ES-DCp ESC % of ESC-treated BALB/c response to B6 p= 0.001 25 50 75 100
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13. ACKNOWLEDGEMENTS TBRC/Massachusetts General Hospital Christian Leguern Paulo Martins Sharon Germana Kaela Goldstein Yuanto Wang Guotong Man Akira Shimizu Childrens Hospital Boston George Q. Daley
Editor's Notes
The experimental design is two-fold. One is to direct differentiation of ES cells to the DC lineage but identify the timepoint at which DC lineage-committed ES cells, which we call ES-DC precursors, or ES-DCp, emerge. These are not to be confused with immature ES-DC, which derive from them. The second part is to implant these ES-DCp in allogeneic mice and test the hypothesis that accepted precursors differentiate and colonize lymphoid organs, potentially affecting host immunity to donor alloantigens. These cells should phenotypically be more like ES cells than DCs. To monitor their appearance, we check for transcription factors that guide their commitment, and not necessarily for surface markers. We wanted to establish proof of concept that these precursors can differentiate in vivo in an allogeneic host. We were not sure if commitment was reversible, but more importantly, if they were sustainable in the absence of cytokines. Emphasize “in the absence of cytokines” in the second question.
The mice were given either 0.5 mg or 0.25 of antibody. We homogenized lymph nodes, thymus, and spleen and checked for donor Class I and Class II by rt-PCR. Detection of donor Class I was improved with combined treatment of anti-CD4 and anti-CD8. Except for this signal, Class II signal was not strong in thymus, most likely because the tissue was not treated with collagenase prior to homogenization. Nevertheless, detection of Class II in thymus of the double Ab-treated mouse was promising, and this needed to be confirmed by IHC.
