Resolving Stem Cell Heterogeneity Using Flow Cytometry

Transcription

1 Resolving Stem Cell Heterogeneity Using Flow Cytometry Mirko Corselli, PhD Senior Scientist

2 We have not fully utilized the power of flow cytometry to address biological questions in other systems Flow cytometry is essential for our understanding of the hematopoietic system

3 Challenges in MSC Research Phenotypic characterization: Heterogeneity Lack of biomarkers associated with MSC function Functional characterization: Need for standardized potency assays

4 Overview Minimal criteria to define MSCs Multicolor panels to identify MSC subpopulations Surface marker screening to define new stem cell signatures Multi-parametric assay to study MSC immunomodulatory activity

5 MSCs must be plastic adherent. MSCs must express CD73, CD105, and CD90 and lack expression of CD45, CD34, CD14, CD11b, CD79a, and CD19. MSCs must differentiate into osteoblasts, adipocytes, and chondrocytes in vitro.

6 Relative Cell Number Relative Cell Number Relative Cell Number Relative Cell Number Relative Cell Number Relative Cell Number Relative Cell Number Relative Cell Number Analysis of MSCs by Flow Cytometry BM-MSCs Lin PE CD90 FITC CD73 APC CD105 PerCP-Cy 5.5 ADSCs Lin PE CD90 FITC CD73 APC CD105 PerCP-Cy 5.5 BD Stemflow MSC Analysis Kit For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

7 MSCs are Functionally Heterogeneous Source: Bone marrow Adipose Tissue Umbilical Cord Intra-population heterogeneity: Subsets of MSCs with distinct functions/potential Early vs Late Passage in culture Normoxia vs Hypoxia Serum vs Serum free Resting vs Licensed The markers used for the minimal definition of MSCs do not discriminate functionally different subsets of MSCs.

8 MSC Functional Heterogeneity Can we complement the minimal definition MSC panel by designing a multi-color flow panel to characterize functional subsets of MSCs?

9 New Fluorochrome Landscape BV421 BV510 BV605 BV650 BV711 Violet 405nm BUV395 UV 355nm Goal: Design panel to complement the minimal definition MSC panel by adding markers to functional subsets of MSCs.

10 CD90 FITC Count Count Count Count Eight Color Flow Cytometry Analysis of BM MSCs Lineage cocktail PE CD73 APC CD105 PerCP-Cy5.5 CD90 FITC CD271 BV421 GD2 BV510 SSEA-4 BUV395 CD146 BV711 For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

11 CD90 FITC Count Count Count Count Eight Color Flow Cytometry Analysis of ADSCs Lineage cocktail PE CD73 APC CD105 PerCP-Cy5.5 CD90 FITC CD271 BV421 GD2 BV510 SSEA-4 BUV395 CD146 BV711 For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

12 CD90 FITC CD90 FITC Sub-Population Comparison: BM MSCs and ASCs ADSCS CD271 BV421 GD2 BV510 SSEA-4 BUV395 CD146 BV711 BM MSCs CD271 BV421 GD2 BV510 SSEA-4 BUV395 CD146 BV711 For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

13 Multi-Color Flow Cytometry and Stem Cells 2-4 color Minimal Compensation Panels are possible in various stem cell populations by spreading colors along multiple laser lines to avoid spectral overlap. Fluorophores amenable to panel design: BUV395 BV421 FITC/A488 APC/A647 Know your instrument: lasers and filters Always confirm panels with and without compensation

14 Use of Single Versus Multiple Lasers: Spectral Overlap FITC PE PE-CF594 BV421 FITC APC Use of Bright Polymer Dyes to spread colors along multiple laser lines to avoid spectral overlap.

15 FITC FITC Use of Single Versus Multiple Lasers: Spectral Overlap FITC PE Not Compensated Compensated PE PE

16 FITC FITC Use of Single Versus Multiple Lasers: Spectral Overlap FITC APC Not Compensated Compensated APC APC Goal: Design 4 color panel with minimal spectral overlap

17 Minimal Compensation Panels for BM MSCs SSC-A IgG FITC IgG FITC IgG FITC Laser Filter Fluorochrome MSC Panel Blue 488 nm 530/30 FITC CD90 Red 640 nm 670/30 Alexa Fluor 647 CD146 Violet 405 nm 450/40 BV421 CD271 Ultraviolet 355 nm 379/28 BUV395 SSEA-4 Isotype Controls FSC-A IgG BV421 IgG Alexa 647 IgG BUV395 For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

18 Minimal Compensation Panels for BM MSCs Compensated Comp. Matrix 89.9% 10.1% 33.7% 66.3% 12% 88% Fluor -% Fluor Comp FITC 0.01 APC BV BUV BV Not Compensated 89.5% 10.5% 33.7% 66.3% 11.8% 88.2% APC FITC 0.01 BUV BV FITC APC 0.26 BUV BV FITC BUV APC 0.05 For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

19 Summary: MSC Immunophenotype BD Stemflow MSC Analysis Kit is suitable for the minimal definition of MSCs The MSC Analysis Kit was complemented with bright polymer dyes to identify MSC sub-populations in an 8 color panel The same MSC sub-populations were detected using a 4 color panel requiring no compensation The markers used for MSC sub-population detection were selected based on literature data

20 Cell Surface Marker Screening to Identify Novel Stem Cell Signatures

21 March 2011, Volume 6, Issue 3

22 BD Lyoplate Screening Panels Enabling researchers to immunophenotype cell populations by flow cytometry or immunofluorescence microscopy using BD s portfolio of monoclonal antibodies Accelerate the discovery of unique cell-surface signatures A unique, cost-effective alternative to screening using hundreds of single-vial reagents Product Contents Size BD Lyoplate Human Cell Surface Marker Screening Panel Cat. No BD Lyoplate Mouse Cell Surface Marker Screening Panel Cat. No CD markers* Isotype controls Alexa Fluor 647 second step 176 CD markers Isotype controls Biotin second step Alexa Fluor 647 streptavidin third step *CD and other cell-surface molecules. One marker per well. 5 tests 5 tests For Research Use Only. Not for use in diagnostic or therapeutic procedures. Alexa Fluor is a registered trademark of Molecular Probes, Inc. The Alexa Fluor dye antibody conjugates in this product are sold under license from Molecular Probes, Inc., for research use only, excluding use in combination with microarrays, or as analyte specific reagents.

23 Immunophenotyping Workflow Flow Cytometry Prepare a single cell suspension and aliquot into 3x96 well plates Reconstitute lyoplates Transfer reconstituted antibodies to plates with cells Incubate Wash Secondary antibody Wash Collect Data Analyze data

24 Analysis % Positive Plate 1 % Positive Plate 2 MFI Plate 1 MFI Plate 2 /resources/stemcell/ For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

25 Isolation of Neurons by Sorting Neurons: CD184 - /CD44 - /CD15 lo /CD24 +

26 August 2011, Volume 6, Issue 8 October 2011, Volume 29, Issue 11

27 December 2012, Volume 7, Issue 12

28 Surface Marker Screening to Resolve MSC Heterogeneity Source: Bone marrow Adipose Tissue Umbilical Cord Intra-population heterogeneity: Subsets of MSCs with distinct functions/potential Early vs Late Passage in culture Normoxia vs Hypoxia Serum vs Serum free Resting vs Licensed

29 MSC Immunomodulation Assays MSCs suppress T-cell activation: Paracrine mechanism (PGE2, IDO, IL-6) Contact-dependent mechanism (CD54, CD274) Challenges: Batch-to-batch variability Lack of biomarkers to predict immunomodulatory activity Immunomodulatory molecule profile and potency assays: MSC licensing Response to inflammatory stimuli MSC-PBMC co-culture Functional changes in distinct cell components

30 MSC Licensing IFN-γ +/-TNF-α treatment is conventionally used to license MSCs IFN-γ TNF-α CD54 CD274 HLA-DR CD106 IL-6 CXCL-10 PGE2 IDO Flow Cytometry CD54 CD106 CD274 Elisa PCR Modified from: Di Trapani M. et al., 2013 Melief SM et al., 2013 English K. et al.., methodologies used to study surface marker, cytokine and intracellular protein expression: time and material consuming Unable to correlate expression of surface and intracellular molecules Multi-color flow assay to simultaneously measure different molecules

31 Flow Cytometric Screening of Immunomodulatory Molecules MSCs derived from bone marrow (BM MSCs) or fat (ADSCs) were licensed with IFN-γ +/- TNF-α Multi-parameter flow cytometry was used to correlate the expression of surface markers with: Cytokines: IL-3, IL-4, IL-6, IL-8 Chemokines: CXCL-10, MCP-1, RANTES PGE2-producing enzyme COX-2 Cells were incubated with BD Golgi Stop TM for 5 hours to prevent cytokine or chemokine secretion

32 CD54 Flow Cytometric Screening of Immunoregulators BM-MSCs Untreated ADSCs Untreated BM MSCs + IFN- ADSCs + IFN- BM MSCs + IFNy/TNF-α ADSCs + IFNy/TNF-α 30% 0% 86% 0% 94% 0% 85% 13.5% 0% 99% 0% 99% CXCL-10 BM MSCs ADSCs Ease of use: simultaneous analysis of surface and intracellular markers, and cytokine profile Comparative analysis of different batches of MSCs Differences between BM-MSCs and ADSCs before and after licensing Resting ADSCs display a more activated phenotype compared to BM-MSCs For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

33 Cross-talk Between MSCs and Immune cells Dendritic cell CD1a+ CXCL-10 TGF-b MCP-1 PGE2 IL-4 IL-6 IDO HLA-G IL-8 IL-1b IL-10 RANTES TNF-a IFN-y Monocyte CD14+ T-cell CD3+ M2 Macrophage CD14+CD206+ Melief SM et al., 2013 Francois M et al., 2012 Spiaggiari GM et al., 2009 ELISA or PCR are unable to identify the source of cytokines Unable to measure MSC activation in heterogeneous co-cultures Physical separation is required to study each cellular component

34 Flow Cytometric Analysis of Co-cultures Multi-parametric flow cytometry for the simultaneous measurement of: - T-cell proliferation - Activation of MSCs - Monocyte differentiation CD3/CD28 beads PBMCs BM MSCs or ADSCs T-cell/MSC panel CD45 V500 CD3 PerCP-Cy5.5 CD4 BUV395 IFN-y Alexa 488 Violet Proliferation Dye (VPD) CD73 BV605 CD54 APC IL-6 PE Monocyte/MSC panel CD45 V500 CD4 BUV395 CD14 PerCP-Cy5.5 CD206 FITC Violet Proliferation Dye (VPD) CD73 BV605 COX-2 PE

35 CD3 SSC-A Panel T-cell/MSC: Inhibition of T-cell Activation SSC-A CD45 CD4 % CD3+IFN- + cells % CD4+ proliferating cells CD45+CD3+ T-cells CD3+CD4+ T-cells PBMCs SSC low CD73- PBMCs FSC-A CD73 CD3 CD3 Resting PBMCs Stimulated PBMCs Stim. PBMCs + BM MSCs Stim. PBMCs + ADSCs 0% 20% 7% 3% IFN- Resting PBMCs Stimulated PBMCs Stim. PBMCs + BM MSCs Stim. PBMCs + ADSCs 4.5% 63.7% 29.8% 7% VPD For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

36 CD54 Panel T-cell/MSC: Activation of MSCs in Co-Culture SSC-A SSC-A MSCs SSC hi CD73+ MSCs FSC-A CD73 BM-MSCs Alone ADSCs Alone BM MSCs + Stim. PBMCs ADSCs + Stim. PBMCs 20% 9% 35% 56% 100% 100% 55% 16% 5% 4% BM MSCs and ADSCs up-regulate CD54 and IL-6 expression in co-culture within 24 hours IL-6 For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

37 CD14 Panel Mono/MSC: Analysis of M2 Macrophage Differentiation SSC-A SSC-A % CD14+CD206+ cells FSC-A PBMCs SSC low CD73- PBMCs CD73 Resting PBMCs Stimulated PBMCs Stim. PBMCs + BM MSCs Stim. PBMCs + ADSCs CD206 FITC Resting PBMCS Stimulated Stim. PBMCS Stim. PBMCS PBMCS + BM MSCs +ADSCs CD3/CD28 bead stimulation leads to rapid loss of M2 immunomodulatory macrophages (6-24hrs). M2 macrophages are maintained in stimulated cultures in the presence of MSCs For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

38 SSC-A Panel Mono/MSC: Simultaneous Analysis of Monocyte and MSC Activation BM-MSCs Alone ADSCs Alone BM MSCs + Stim. PBMCs ADSCs + Stim. PBMCs 7.7% 30% 99% 99% SSC hi CD73+ MSCs CD14 CD73 COX-2 SSC low CD73- PBMCs CD73 Resting PBMCs Stimulated PBMCs Stim. PBMCs + BM MSCs Stim. PBMCs + ADSCs 20% 0% 0.4% 0.1% 2% 4.5% 2% 4% COX-2 Both MSCs and monocytes up-regulate the expression of PGE2-producing enzyme in co-culture For Research Use Only. Not for Use in Diagnostic or Therapeutic Procedures

39 Conclusions Multi-parametric flow cytometry is a powerful technique for the analysis stem cells Multi-parametric flow cytometry enables: Comprehensive phenotypic analysis for the detection of multiple sub-populations in heterogeneous stem cell cultures Discovery of novel stem cell signatures Study of the kinetics of multiple molecule expression in distinct cell components of heterogeneous cultures Identification of biomarkers that correlate with potency

40 Acknowledgements BD Research and Development Christian Carson, Mirko Corselli, Nil Emre, Liana Goodwin, Ravi Hingorani, Jody Martin, Jurg Rohrer, Aaron Tyznik, Jason Vidal, Lissette Wilensky Collaborators: Larry Goldstein, Martin Masala, Eric D. O Connor (UCSD) Marc Moorman (ViaCyte, Inc.) Sylvia Evans, Ju Chen, Karina Palomares (UCSD) BD Colleagues Bob Balderas, Andrea Nguyen, Ranga Partha, Dennis Sasaki, Nicolas Quoix