Multiple surface markers can identify several distinct populations with a clonogenic potential in the mouse hair follicle.

Transcription

1 Multiple surface markers can identify several distinct populations with a clonogenic potential in the mouse hair follicle. Uffe Birk Jensen Institute of Human Genetics And Department of Clinical Genetics Aarhus University Hospital

2 3 parts I: Background II: Sorting strategy of rare events III: Alternative approach to fixation of cells using nontoxic components

3 I: Background Why is a clinical geneticist interested in stem cells?

4 2/3 of the family cases are concerning predispotition to cancer 1/3 classical cases: Of these a large proportion concerns neurodegenerative disorders

5 Defining a stem cell Definition Stem cells cells are are capable of of extensive self-renewal extending to to at at least one one natural life life span of of the the organism. Stem cells cells are are relatively undifferentiated, i.e. i.e. They are are indeed differentiated but but may may be be less less differentiated than than their their decendants. They usually have the the ability to to produce daughter cells cells that that undergo terminal differentiation. Some stem cells cells has has a low low turn-over rate rate in in steady state. Professor Laszlo G.Lajtha, 1979

6 Dystrophin expression in the mdx mouse restored by stem cell transplantation EMANUELA GUSSONI*, YUKO SONEOKA, CORINNE D. STRICKLAND*, ELIZABETH A. BUZNEY*, MOHAMED K. KHAN, ALAN F. FLINT*, LOUIS M. KUNKEL* & RICHARD C. MULLIGAN Nature vol September 1999

7 Identification of sp-cells in the epidermis

8 Sca-1 distribution in the mouse skin SG

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10 CD34 Keratin 1 Sca-1

11 Struktur og lokalisation af stamceller i hårfollikle Og den interfollikulære epidermis Modified from Potten CS, Booth C. J Invest Dermatol. 22

12 FACS setup FACSAria I With either 45nm 488nm 633nm or 375nm 488nm 633nm

13 FACS setup FACSAria I with either 45nm 488nm 633nm DAPI dead cell exclusion GFP, FITC, PE, PE-Cy7, APC, Alexa 647, APC-Cy7, Alexa 68 or 375nm 488nm 633nm Hoechst GFP, FITC, PE, PE-Cy7, Pi APC, Alexa 647, APC-Cy7, Alexa 68

14 Jensen et al., Figure 2 A B C D P1: IFD + IFE P2: UI P3: Bulge # Cells # Cells α6 integrin Sca-1 CD34 Merge E CD34-APC 1 5 α6 all cells P3 all P1 all α6-fitc α6-fitc Sca-1-PE H 1 5 α6 L cells F I P2 G J α6 L+H /CD34 + /Sca-1 - (BG): 8% of live cell population α6 L+H /CD34 - /Sca-1 + (IFD + IFE) α6 L /CD34 - /Sca-1 - (UI): 5% of live cell population # Cells α6-fitc α6-fitc Sca-1-PE 1 K 5 α6 H cells L 1 5 M3b CD34- APC CD34- APC CD34- APC CD34- APC CD34- APC α6 H /CD34 - /Sca-1 - : 2.8% of live cell population α6-fitc α6-fitc Sca-1-PE

15 Colony forming effiency A H H L L H L α6 CD34 Sca-1 Colonies/dish CD34 - /Sca-1 - CD34 + /Sca-1 - CD34 - /Sca-1 + * * B D E F % colonies> 4 mm1 H H L L H L H H L L H L * C * α6 α6 α6 L /CD34 - /Sca-1 - α6 H /CD34 + /Sca-1 - α6 H /CD34 - /Sca-1 +

16 Jensen et al., Figure 4 A C E 2.5 x 1 5 cells 1.25 x 1 6 cells 2.5 x 1 5 cells 1.25 x 1 6 cells G P3: α6 L+H /CD34 + /Sca-1 - B P3: α6 L+H /CD34 + /Sca-1 - D P2: α6 L /CD34 - /Sca-1 - F P1: α6 all /CD34 - /Sca-1 + H P3: α6 L+H /CD34 + /Sca-1 - I P3: α6 L+H /CD34 + /Sca-1 - P2: α6 L /CD34 - /Sca-1 - J DF control Q Bl/6 H-2K b K Nude H-2K q L sg hair shaft club Bl/6 H-2K q Nude H-2K b M N O P dp hair shaft P2 graft EGFP T P2:H-2K b R P2:H-2K q P2:H-2K b S sl P2:H-2K q epid P1: H-2K q H-2K b epid U Nude EGFP P2: EGFP bl hf P1: EGFP bl

17 Jensen et al., Figure 5 H

18 Jensen et al., Figure S5 1 5 A 1 5 B 1 5 C % % % EdU (FITC) Forward scatter Bulge UI IFE+IFD 1 1

19 Jensen UB, Yan X, Triel C, Woo SH, Christensen R, Owens DM (28). J Cell Sci. Mar 1;121(Pt 5):

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23 II: Sorting strategy of rare events

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25 4 time increase which could be doubled by also removing CD34 pos cells

26 Stem cells are target for carcinogenesis!

27 III: Alternative approach to fixation of cells using nontoxic components

28 Aim ambition! We want a simple non-toxic fixation method that allows for the detection of: 1: DNA (quantification with acceptable profiles) 2: pulse labeled cells (EdU) 3: surface proteins 4: Intracellular proteins on the same sample! Challenge: penetrate the cell membrane denature? DNA without destroying surface proteins avoid degrading of DNA

29 Zn-fix Components of the fixation buffer: Tris-HCl (CH 3 COO) 2 Ca (CH 3 COO) 2 Zn ZnCl 2 Procedure: resuspend cells in 1µl PBS-- Add 1 ml Zn-fix while vortexing incubate at 4 C o.n. wash cells in buffered saline before analysis Add 1 part glycerol to store at -2 Advantages: simple cheap also regarding hands on time non-hazardous

30 # Cells HCl 46.8 DNAse protocol on lymphocytes: 1-15U DNAse in PBS ABC w..2% saponin and 1% BSA, 6 ice 3 ±Exo(18 U) 3 ice BrdU -exo 2 +exo 1 U # Cells 2 # Cells BrdU -exo BrdU +exo 15 5 U # Cells 1 5 # Cells BrdU -exo BrdU +exo 1 U # Cells # Cells BrdU 2 -exo BrdU +exo 15 U # Cells # Cells BrdU BrdU

31 Click-iT - a new technology for detection of cells labeled with nucleotide analogs

32 Detection of EdU labelled cells in EdU pulsed C57 mice PFA # Cells 6 CD CD EdU Sca alpha 6 integrin Zn # Cells 6 CD CD EdU Sca alpha 6 integrin

33 DNA profiles generated from Zn fixed cells using 375nm Near UV laser.

34 Comparison of live cells with Zn and PFA fixed cells Living cells CD CD Sca alpha 6 integrin PFA fixated CD CD Sca alpha 6 integrin Zn fixated CD CD Sca alpha 6 integrin

35 Samples can be stored 4 d 11 d

36 Aarhus group: Uffe Birk Jensen Rikke Christensen Anette Thomsen Charlotte Triel Collaborators at Columbia University: David M Owens Xiaohong Yan Seung-Hyun Woo

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