Isolation and Analysis of Pluripotent, Neural, and Hematopoietic Stem Cells

Transcription

1 Isolation and Analysis of Pluripotent, Neural, and Hematopoietic Stem Cells Christian Carson BD Biosciences R&D Scientist Stem Cell

2 Overview Introduction Challenges in stem cell research Antibody portfolio of stem cell markers Cell sorting of neural stem cells (NSCs) and neurons BD Lyoplate CD screening panel BD FACS CAP service Cell sorting of human embryonic stem cells (hescs) Flow cytometry kits for pluripotent stem cell research Compensation beads for larger cells and bright markers Mouse hematopoietic stem cell (mhsc) isolation Overview of stem cell flow kits 3

3 Stem Cell Background Embryonic ips Adult Oct3/4 Sox2 Klf4 c-myc Nanog LIN28 4 Schematic adapted from Wobus AM. and Boheler, KR., Physiol. Rev.2005; 85:

4 Stem Cell Background 5 Schematic adapted from

5 Challenges in Stem Cell Research Identify and isolate cells of interest from a heterogeneous pool Analyze cells for quality and purity Analyze cell function 6

6 Biomarkers are Crucial for Stem Cell Analysis and Isolation Growth Factors & Cytokines Growth Factors & Cytokines Undifferentiated Stem Cell Progenitor Cell Differentiated Cell Biomarker Identification DNA/RNA microarrays, sequencing, PCR, antibody arrays Growth Factors & Cytokines Growth Factors & Cytokines Undifferentiated Stem Cell Progenitor Cell Differentiated Cell Isolate stem cells of interest using flow cytometry or magnetic beads Defined media, surfaces, and supplement to control cell fate Biomarkers to define differentiation status: cell analysis using flow cytometry, imaging, WB, IHC 7 Functional in vitro assays (apoptosis, cell cycle, cell proliferation, BD Phosflow) In vivo animal studies Cell therapy Research

7 BD Biosciences First to commercialize tools for isolating and analyzing hematopoietic stem cells, now bringing this expertise to the broader stem cell field 8

8 Portfolio of High Quality Antibodies 9 For Research Use Only. Not for use in diagnostic or therapeutic procedures.

9 Purified mabs for Stem Cell Research human Nanog Nestin GATA4 hesc hesc-derived neural stem cells hesc-derived cardiomyocytes 10

10 Fluorochrome-Conjugated mabs for Imaging H9 grown on BD Matrigel hesc-qualified matrix with mtesr TM 1 Sox2 Alexa Fluor 647 Oct3/4 Alexa Fluor 555 Merge SSEA-4 Alexa Fluor 488 Hoechst 11

11 Fluorochrome-Conjugated mabs for Imaging Differentiated H9 hescs SSEA-1 Hoechst Cdx2 SSEA-1 Oct3/4 Hoechst SSEA-1 GATA4 Cdx2 Hoechst SSEA-1 (CD15): Multi-lineage Cdx2: Trophectoderm Oct3/4: Pluripotency GATA4: Endoderm 12

12 Fluorochrome-conjugated mabs for Flow Cytometry H9 hescs grown on BD Matrigel hesc-qualified matrix with mtesr TM 1 SSEA-4 Alexa Fluor % Sox2 Alexa Fluor % Oct3/4 PE 13

13 Fluorochrome-conjugated mabs for Flow Cytometry Differentiated H9 hescs Oct3/4 PE 12% 1.8% 2.2% 75% 13% 83% 13% SSEA-1 Alexa Fluor 488 Cdx2 Alexa Fluor 6 47 SSEA-1 (CD15): Multi-lineage Cdx2: Trophectoderm Oct3/4: Pluripotency 14

14 Neural Stem Cells: Background Found during embryonic development and in restricted regions of the adult brain (NSC) NSCs can be isolated and cultured in vitro Fetal and adult brain Differentiated from hescs Glial progenitor (GP) Neural progenitor (NP) Promises of NSCs Transplantation therapy In vitro models of human development In vitro models of human diseases Drug screening Toxicology Basic research Neuron 15 For Research Use Only. Not for use in diagnostic or therapeutic procedures.

15 Generation of Neural Cells from hescs Day hescs EBs Rosettes NSCs Neurons, Glia mtesr 1 withdraw bfgf N2 N2, B27, bfgf N2, B27, dbcamp 16

16 Closer Look at hesc-derived NSCs Sox2 Nestin Ki67 Hoechst 99% 99% Sox2 PE Sox2 PE Oct3/4 Alexa Fluor 488 Nestin Alexa Fluor 647 Ki-67 Alexa Fluor % 39% Sox2: hescs, NSCs Nestin: NSCs Oct3/4: hescs Ki67: proliferation Sox2 Alexa Fluor

17 Generation of Neural Cells from hesc Day hescs EBs Rosettes NSCs Neurons, Glia mtesr 1 withdraw bfgf N2 N2, B27, bfgf N2, B27, dbcamp Current Challenges Protocols are difficult Batch-to-batch variability of NSCs Neuronal differentiation is heterogeneous Need pure populations for applications: transplantation, arrays/sequencing, in vitro disease models 18

18 Generation of Neural Cells from hescs Day hescs EBs Rosettes NSCs Neurons, Glia mtesr 1 withdraw bfgf N2 N2, B27, bfgf N2, B27, dbcamp Performed a screen using 192 human CD markers by flow cytometry and bioimaging Define cell surface signatures of hescs, NSCs, NPs (neural progenitors), neurons, and glia Develop a method to isolate near-pure populations of NSCs, neurons, and glia 19

19 Cell Surface Marker Screening with BD Lyoplate Human CD Marker Panel CD marker screening in 96-well format by flow cytometry NSC hesc

20 Cell Surface Marker Screening with BD Lyoplate Human CD Marker Panel CD marker screening in 96-well format by imaging CD Marker Tuj1 Merge 21

21 Generation of Neural Cells from hescs Day hescs EBs Rosettes NSCs Neurons, Glia mtesr 1 withdraw bfgf N2 N2, B27, bfgf N2, B27, dbcamp Performed a screen using 192 human CD markers by flow cytometry and bioimaging Define cell surface signatures of hescs, NSCs, NPs (neural progenitors), neurons, and glia Develop a method to isolate near-pure populations of NSCs, neurons, and glia 22

22 Isolation of Neural Subtypes from hesc-derived NSCs FSC-A NSCs were differentiated 2 weeks prior to sorting BD FACSAria II sorter 70 PSI, 70-µm nozzle Ki-67 Nestin Map2b Hoechst Ki-67 SSC-A Presort Sorted: P2 Sorted: P3 9% 45% 1% 70% 29% 70% 20% 8.5% 46% 94% Sox2 1% 3.5% 23

23 Isolation of Neural Subtypes from Differentiating hesc-derived NSCs NSCs were differentiated 4 weeks prior to sorting BD FACSAria II sorter, 25 PSI, 100-µm nozzle GFAP Hoechst Glia Tuj1 Hoechst Neurons 24

24 Generation of Neural Cells from hescs Day hescs EBs Rosettes NSCs Neurons, Glia mtesr 1 withdraw bfgf N2 N2, B27, bfgf N2, B27, dbcamp Performed a screen using 192 human CD markers by flow cytometry and bioimaging Define cell surface signatures of hescs, NSCs, NPs (neural progenitors), neurons, and glia Develop a method to isolate near-pure populations of NSCs, neurons, and glia 25

25 Isolation of NSCs from Embryoid Bodies by Flow Cytometry EB NSCs Sorted from EBs Sox2 Nestin Hoechst Differentiation of NSCs Map2b Tuj1 Ki-67 Hoechst Neurons Sorted from Diff NSCs Map2b Tuj1 GFAP Hoechst Co-cultured on Astrocytes 26

26 BD Lyoscreen Human Cell Surface Kit Approximately 260 human cell surface markers in 96-well plates Each kit contains five tests of each antibody Customer dilutes antibodies to desired concentration into daughter plates Other kit components Fluorchrome-conjugated secondary antibodies for flow cytometry and imaging Isotype controls Analysis template

27 BD FACS CAP Cell Surface Phenotyping Service CD3 CD10 CD34 Custom service leverages BD s expertise as the world leader in flow cytometry Flexible format can integrate customer s specific markers Continuous addition of new important monoclonal antibodies Delivers flow cytometry-based combinatorial antibody profile 28 CAP = Combinatorial Antibody Profile

28 BD FACS CAP Service Luminal-like cells and basal-like cells show distinct cell surface marker expression BD FACS CAP technology may be used on a variety of human cell types 29

29 Pluripotent Stem Cell Research

30 Sorting and Analysis of Pluripotent Stem Cells Are sorted hescs viable? Fong et al. Stem Cell Rev Bajpai et al. Mol Reprod Dev Nicholas et al. Stem Cells Dev Sidhu et al. Stem Cells Dev Do sorted cells still express markers of pluripotency? Are sorted cells capable of further differentiation? No commercial, standardized methods for sorting or analysis by flow cytometry. 31

31 Sorting Experimental Design Cell surface markers SSEA-1 negative (differentiation) SSEA-3 positive (pluripotency) Tra-1-81 positive (pluripotency) Cell sorting with BD FACSAria II system 25 PSI, 100-µm nozzle hescs used: H9 P48 Grown on BD Matrigel hesc-qualified Matrix with mtesr 1 H9 P41 Cultured in KOSR on MEFs HUES9 Cultured in HUES on MEFs (Goldstein Lab, UCSD) 32

32 Sorting Based on Markers for Pluripotency and Differentiation SSEA-1 FITC SSEA-3 PE SSEA-3 PE TRA-1-81 Alexa Fluor

33 Sorting is Possible Under Feeder and Feeder-free Culturing Conditions H9 P48 day 2 post-sort H9 P41, P5 Day 3 post-sort HUES9 Day 4 post-sort mtesr TM 1, BD Matrigel, Accutase KOSR, MEF, Coll IV HUES, MEF, Trypsin Goldstein Lab, UCSD 34

34 Sorted H9 hescs Express Pluripotency Markers H9 P42 P6 post-sort TRA1-81 SSEA-1 Hoechst SSEA-4 Oct-3/4 SSEA-1 Hoechst Oct3/4 Percp-Cy % 1.1% 93.8% 5.2% SSEA-4 Alexa Fluor % 1.0% 3.6% 0.1% 35 SSEA-4 Alexa Fluor 647 SSEA-1 PE

35 Sorted H9 hescs Retain Differentiation Potential H9 P43, P7 sort EBs Mesoderm Ectoderm Endoderm Sox17 Hoechst 36

36 Sorting and Analysis of Pluripotent Stem Cells Are sorted hescs viable? Fong et al. Stem Cell Rev Bajpai et al. Mol Reprod Dev Nicholas et al. Stem Cells Dev Sidhu et al. Stem Cells Dev Do sorted cells still express markers of pluripotency? Are sorted cells capable of further differentiation? No commercial, standardized methods for sorting or analysis by flow cytometry. 37

37 Flow Cytometry Kits for Pluripotent Stem Cell Research Comprehensive, easy-to-use Compensation beads Verified protocols and software analysis guidelines Analysis and sorting Multicolor Pre-conjugated antibodies to markers for self-renewal and differentiation Open, modular Compatible for dropping-in additional antibodies to cell surface markers, transcription factors, cytokines, and phosphorylated proteins Compatible with GFP-expressing cells 38

38 BD CompBead Plus Overlays of unstained cells and cells and beads stained with SSEA-4 conjugates SSC-A SSEA-4 FITC SSEA-4 PE 39 SSEA-4 AlexaFluor 647 H9 hesc CompBead CompBead Plus SSEA-4 BD Horizon V450 FSC-A Autofluorescence of beads tracks hescs Facilitate scatter setup Compensation for any mouse or rat antibody

39 Dropping in Additional Antibodies Drop-ins add to the number and type of markers to analyze per sample Enable detailed analysis of cell fate and function of single cells: Correlation of marker expression (up regulation and down regulation) Simultaneous analysis of transcription factors, cell surface markers, cellular processes (cell cycle, cell signaling, cell death) Example: Mouse ES (E14) 5-day differentiation time-course (10 μm RA) Analysis of mnanog, Oct3/4, Sox2 (Mouse Pluripotency Analysis Kit TF) + GATA4- Alexa Fluor 488 drop-in 40

40 Dropping in Additional Antibodies Mouse ES (E14) 5-day differentiation time course (10 μm RA) 0.2% 1.1% 0.0% 2.2% 0.4% 0.7% Day 0 7.0% 91.6% 0.3% 97.4% 10.6% 88.2% Nanog PE Oct3/4 PerCP-Cy5.5 Sox2 Alexa Fluor 647 Day 2 GATA4 Alexa Fluor % 0.8% 85.5% 12.8% Nanog PE 0.5% 1.6% 41.6% 56.3% Oct3/4 Percp-Cy % 0.8% 11.5% 87% Sox2 Alexa Fluor % 1.8% 12.6% 3.2% 14.6% 1.0% Day % 0.4% 83% 1.2% 83.5% 1.0% 41 Nanog PE Oct3/4 PerCP-Cy5.5 Sox2 Alexa Fluor 647

41 Dropping in Additional Antibodies Mouse ES (E14) 5-day differentiation time course (10 μm RA) hnanog PE Oct3/4 PerCP-Cy5.5 Sox2 Alexa Fluor 647 GATA4 Alexa Fluor % Day % 92.6% 0.7% Day % 55.3% 19% 1.4% Day 5 4.6% 4.5% 5.2% 15.0% 42

42 43

43 Mouse HSC Isolation Kit Contents: APC Lineage Cocktail PE c-kit FITC CD34 PE-Cy 7 Sca-1 Matched Isotype Controls CD16/CD32 (Fc III/II Rec) 7-AAD vital dye BD CompBeads Verified protocols Utility: Sorting CD34 +/- KLS from mouse bone marrow 100 mice ~ 10 sorts Compatible with magnetic enrichment Compatible with side population KLS (SPKLS) LT-HSC: CD34 -, SCA-1 +, C-kit + ST-HSC: CD34 +, SCA-1 +, C-kit + MPP: CD34 +, SCA-1 +, C-kit + 44

44 Mouse HSC Isolation Kit C57 BM magnetic bead-enriched SSC-A Lin cocktail APC C-kit PE FSC-A C-kit PE Sca-1 PE-Cy7 CD34lo/- CD34hi Count 45 CD34 FITC

45 Mouse HSC Isolation Kit Mitomycin-C treated M2-10B4 stromal cells Unfractionated Bone Marrow KLS CD34 hi KLS CD34 lo/- Cobblestone cells Cobblestone cells 46

46 Mouse HSC Isolation Kit Colony forming assay KSL CD34 hi CFU Cobblestone cells 47

47 Mouse HSC Isolation Kit Side population, Kit +, Sca-1 +, Lin -, CD34 - (SPKLS) Viable Cells Lin - CFU-G Lin- APC C-Kit PE CFU-M C-Kit PE Lin - Sca-1 PE-Cy7 All Cells Sca-1 PE-Cy7 Hoechst Blue CFU-GM 48 CD34 FITC Hoechst Red

48 Kits for Stem Cell Research Human Pluripotent Stem Cell Sorting and Analysis Kit Human and Mouse Pluripotency Analysis Kit Human Pluripotency Analysis Kit-TF Mouse Pluripotency Analysis Kit-TF Mouse HSC Isolation Kit Kit Sp Antibodies Hu Hu Ms Hu Ms Ms SSEA-3 PE Tra-1-81 AlexaFluor 647 SSEA-1 FITC Oct3/4 PerCP-Cy5.5 SSEA-4 AlexaFluor 647 SSEA-1 PE Oct3/4 PerCP-Cy5.5 hnanog PE Sox2 AlexaFluor 647 Oct3/4 PerCP-Cy5.5 mnanog PE Sox2 AlexaFluor 647 c-kit PE Sca-1 PE-Cy TM 7 CD34 FITC Lineage Cocktail APC CS Analysis IC Analysis Sorting Drop-ins GFP All kits contain BD CompBead Plus, matched isotype controls, and verified protocols IC Analysis kits contain fix and perm buffers 49

49 Acknowledgments Stem Cell Research Bob Balderas Jurg Rohrer Rosanto Paramban Jason Vidal Julia Ember Jeanne Elia Nil Emre TAS Sue Reynolds R&D Cytometry Lab Dennis Sasaki Andrea Nguyen UCSD Larry Goldstein 50

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