The Bone Marrow in Type 1 Diabetes

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Erica Flowers
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1 The Bone Marrow in Type 1 Diabetes Feasibility of Flow Cytometric Analysis of npod Bone Marrow specimens Paolo Madeddu, MD FAHA University of Bristol UK Miami January,

2 2 BACKGROUND Cardiovascular complications are responsible for about 50% of deaths in diabetic patients. Clinical outcome is worsened because of impairment of cellular and molecular mechanisms of vascular repair. U n r a v e l l i n g t h e c a u s e s o f defective repair mechanisms may lead to new therapies 124,000 heart attacks in UK every year In the DDCT/EDIC study, prevalence of myocardial scar was 4.3% by cardiac MRI and 1.4% by clinical adjudication of MI BHF data and Circulation 2011

3 3 The central role of bone marrow in cardiovascular repair Bone marrow stem cells 1. Slow, circadian release under homeostasis 2. Rapid, protease-dependent following injury Spnetti et al Cardiovasc Res 2011

4 The cause of all complications? 4

5 Reversing the paradigm 5

6 BM as a target of microangiopathy 6 C T1D Hoechst blue Endothelial cells Total BM cells Lin-SK cells Hoechst red Oikawa et al, ATVB 2010

7 Human BM as a target of micro-macro-angiopathy (a prospective study on tissue leftovers) 7 Controls T2D T2D+CLI Spinetti et al, unpublished

8 8 Enumeration of stem cells in T2D BM Spinetti et al, unpublished

9 Diabetic BM neuropathy 9 Ferraro et al Sci Transl Med 2012 Di Persio NEJM 2012

10 Consequences of BM remodelling 10 Infection BM Delayed healing NeuroPathy MicroAng Altered immunity MacroAng Delayed engraftment

11 11 Objective To characterize BM remodelling at histological cellular and molecular level in npod diabetic patients

12 The freshness and quality of the samples are relevant for cell enumeration and function B P= hr 24 hr P=0.81 C 0.4 CD P< P< % in lymphocytes % in lymphocytes E KDR CD P< hr D % in lymphocytes % in lymphocytes % A 24 hr 1 hr 24 hr CD P< hr hr 24 hr 1 hr 24 hr Cui et al PlosOne 2012

13 Use of lysis buffer Lysis wash/fixative frequently blocked the fluid system Cui et al PlosOne 2012

14 Interface of the multiple antibodies conjugated with different fluorophores 0.04 KDR CD133 CD34 P=0.023; F-test 0.10 P=0.16; F-test 0.25 P=0.70; F-test % in lymphocytes % in lymphocytes % in lymphocytes Null CXCR4 CD164 CD117 TrkA 0.00 Null CXCR4 CD164 CD117 TrkA 0.00 Null CXCR4 CD164 CD117 TrkA Tested if different PE-conjugated antibodies(cxcr4, CD164, CD117 and TrkA) have an effect on the enumeration of three commonly-used progenitor cells markers, KDR-FITC, CD133-APC and CD34-PECY7 Cui et al PlosOne 2012

15 Titration of each individual antibody is essential before combination for cell enumeration A B C CD34-PE-Cy7 D E F

16 16 Methods The effect of diabetes on BM PCs was assessed in three npod iliac crest BM cases: 1) case 6126, not diabedc (ND) male (M), age 25 years 2) case 6161, Type 1 diabedc (T1D), M, age 19 years 3) case 6132, T2D, female, age 52 years. Frozen specimens were thawed following npod instrucdons in DMEM+10% FBS. In addidon, cells were leu 2 hours at 37C, 5% CO 2 to recover from thawing before FACS analysis.

17 Recovery a1er thawing out of shipped frozen npod BM MNC A Frezeing/Thawing effect on BM-MNCs number B 7AAD negative cells BM-MNCsX Frozen ND T1D T2D Thawed out % indicated population on tot BM-MNCs ND T1D T2D A) number of tripan blue posidve BM MNCs. B) Percentage of 7ADDneg alive BM MNCs auer thawing.

18 DefiniEon HematopoieEc PCs T lymphocytes B lymphocytes Natural Killer (NKs) Mesenchymal Cells (MSCs) AnEgenic profile CD34 pos, CD133 pos, and c kit pos CD45 pos /CD3 pos CD45 pos /CD19 pos CD3 neg /CD56 pos /CD16 pos CD73 pos /CD105 pos /CD90 pos /CD34 neg /CD45 neg Endothelial cells (ECs) Early endothelial PCs CD45 neg /CD31 pos /CD144 pos CD34 pos /CD14 pos /CD45 dim /KDR pos /CXCR4 pos Late EPCs (lepcs) CD34 pos /CD14 neg /CD45 neg /KDR pos /CXCR4 pos

19 Hematopoietic stem cells 7AAD neg CD45 Hematopoietic progenitors 3 CD34 CD133 ckit % indicated population on tot BM-MNCs CD34 ND T1D T2D CD133 ckit

20 Lymphocytes Lymphocytes 7AAD neg T Lymphos CD3 CD45 B Lymphos CD19 NKs CD56/CD16 % indicated population on tot BM-MNCs T Lymphos B Lymphos ND T1D T2D NKs

21 Mesenchymal stem cells 7AAD neg CD45 neg CD34 neg CD105 CD73 CD90 Mesenchimal Cells % indicated population on tot BM-MNCs ND T1D T2D

22 Endothelial cells 7AAD neg CD45 neg Endothelial Cells CD31 CD144 ECs % indicated population on tot BM-MNCs ND T1D T2D

23 Early EPCs Early EPCs 7AAD neg KDR/CXCR4 Endothelial Progenitor Cells CD45 dim 0.3 CD34 CD14 neg pos CD45 neg KDR/CXCR4 % indicated population on tot BM-MNCs early EPCs late EPCs ND T1D T2D Late EPCs

24 Conclusions We successfully verified a SOP to thaw npod BM specimens for cytometric analysis. Samples were adequate to measure the percentages of different populations of BM cells. However, the proportion of cells can be skewed because of distinctive cell loss during freezing/thawing.

25 Perspectives BM Collect info on clinical correlates Compare with fresh samples Focus on certain populations (CD34+, CD34+KDR, lineage) to study cell specification CVD Extend npod to cardiovascular complications (heart, large vessels, limb muscle) Focus on similar molecular mechanisms Links Connection with other thematic areas

26 Acknowledgments Uni Bristol Atsuiko Oikawa Giuseppe Mangialardi Carlotta Reni Rajesh Katare MultiMedica Gaia Spinetti Daniela Cordella Orazio Fortunato Uni Bristol Costanza Emanueli Andrea Caporali Marco Meloni Diabetes UK

Demystifying Stem Cells. Brent Bost M.D., CPA, MBA, FACOG

Demystifying Stem Cells. Brent Bost M.D., CPA, MBA, FACOG Demystifying Stem Cells Brent Bost M.D., CPA, MBA, FACOG Disclaimers Author currently serves on Board of Directors of CordTrack Most applications discussed today are not currently FDA approved and are,