Stem Cells and Regenerative Medicine
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1 Stem Cells and Regenerative Medicine 纪俊峰 May 07, 2015
2 Definition of a Stem Cell
3 Potency of Stem Cells
4 Totipotent Pluripotent Multipotent /mucosa /enamel /cartilage/denti n
5 Types of Stem Cells Pluripotent - Embryonic stem cells (inner cell mass) ES - Induced pluripotent stem cells (somatic cells) ipscs - Embryonic germ cells (embryonic gonad) EG - Embryonic Carcinoma (P19s) EC Fetal - Umbilical l cord, placenta, amniotic fluid Adult - Bone marrow (hematopoietic & mesenchymal) - Tissue: neural, skin, skeletal muscle (satellite cells), fat
6 Derivation of Pluripotent Stem Cells ipsc
7 Fetal Stem Cells
8 Adult Stem Cells
9 Discovery of Adult Stem Cells
10 2005 Albert Lasker Basic Medical Research Award Ernest McCulloch and James Till for ingenious experiments that first identified a stem cell the blood-forming stem cell which set the stage for all current research on adult and embryonic stem cells
11 Colony Forming Unit-Spleen (CFU-S) assay
12 In the early 1960s, McCulloch and Till started a series of experiments that involved injecting bone marrow cells into irradiated mice. Visible nodules were observed in the spleens s of the mice, in proportion o to the number of bone marrow cells injected. Till and McCulloch called the nodules 'spleen colonies', and speculated that each nodule arose from a single marrow cell: perhaps a stem cell.
13 McCulloch, E.A., Till, J.E. (1960) The radiation sensitivity of normal mouse bone marrow cells, determined by quantitative marrow transplantation into irradiated mice. Radiation Research 13(1): Till, J.E., McCulloch, E.A. (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research 14: Becker, A.J., McCulloch, E.A., Till, J.E. (1963) Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197: Siminovitch, L., McCulloch, E.A., Till, J.E. (1963) The distribution of colony- forming cells among spleen colonies. Journal of Cellular and Comparative Physiology 62: Till, J.E., McCulloch, E.A., Siminovitch, L. (1964) A stochastic model of stem cell ( ) proliferation, based on the growth of spleen colony-forming cells. Proceedings of the National Academy of Sciences (USA) 51(1):29-36.
14 Purification of Hematopoietic stem cells (HSCs) with surface marker Lin-Thy-1.1lowSca
15 1998
16 Identification of c-kit as HSC marker
17 Identification of c-kit as HSC marker
18 Discovery of new Hematopoietic stem cells surface markers
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24 HSCs Isolation 2006 Terese Winslow
25 HSC Stemness assays Hematopoietic stem cells (HSC) CFU-E BFU-E CFU-M CFU-GM CFU-GEMM 2006 Terese Winslow
26 Long-Term Repopulation p Assay 1. It involves transplantation of test cells into irradiated hosts. 2. Donors and hosts are distinguished by different alleles of the hematopoietic cell marker CD45 such as CD45.1 and CD Blood samples are collected at various intervals, and donor vs. host cells are analyzed by FACS for their potential presence and contribution ti to major hematopoietic ti lineages, typically including myeloid as well as lymphoid lineages. 4. A test population is considered to contain LT-HSC when a recipient contains donor-derived cells of all these lineages even after at least 16 weeks following transplantation.
27 Competitive Repopulation Assay 1. A specific number of whole bone marrow cells, a standard source of HSCs, typically derived from mice congenic with the host, are included with the test cells. 2. This allows for the quality of the test cells to be assessed relative to the standard. However, it does not allow for rigorous quantification of HSC frequency and number in the test population.
28 Limiting Dilution Assay 1. It utilizes a titration of varying doses of test cells. The percentage of hosts which fail to engraft donor cells is determined for each dose. 2. With a minimum of three different doses in which both positive and negative recipients are present, a best-fit line is generated by Poisson statistics. This is utilized to calculate the frequency of competitive repopulating units (CRUs) and thus the number of HSCs by H IBELGAUFTS. ENTRY LAST MODIFIED:
29 Serial Transplantation Assay 1. The serial transplantation t ti assay is the most stringent t test t for HSC stemness. 2. It can utilize any of the previous assays. Here, test cells are transplanted into primary recipients as previously mentioned but then harvested from these primary recipients and transplanted into secondary recipients and further from secondary into tertiary hosts. Only the most primitive HSC can yield long-term, multilineage repopulation in this assay. Tertiary Transplant rcipient
30 Heterogeneity of HSCs
31 et al
32 Single cell gene expression to study heterogeneity and hierarchy of HSCs
33
34 Guo et al
35 Guo et al
36 Genome-wide approach to identify new markers of HSCs?
37 Bone marrow transplantation
38 The Nobel Prize in Physiology or Medicine 1990 Joseph E. Murray E. Donnall Thomas "for their discoveries concerning organ and cell transplantation for their discoveries concerning organ and cell transplantation in the treatment of human disease"
39 World-wide application of Bone Marrow Transplantation
40 Mesenchymal Stem Cells/ Bone Marrow Stromal Cells (MSC)
41 Discovery of MSC 1. In 1970s, Friedenstein and colleagues demonstrated that seeding of Bone Marrow cells at clonal density results in establishment of discrete fibroblastic colonies initiated by single cells called the colony-forming unit fibroblastic, CFU- Fs. 2. Multiple skeletal tissues including bone, cartilage and adipose tissues can be generated in vivo from a single BM stromal cells. Image from StemCell Technologies
42 MSC Stemness assays A single MSC is able to generate multiple skeletal lineages both in vitro and in vivo!! In vitro differentiation Mesenchymal stem cells (MSC) Self-renewal & in vivo differentiation Osteoblast Adipocyte Chondrocyte
43 CD146 as a surface marker of MSCs
44 Antibody Screening
45
46 CD146+ cells form hematopoiesis associated Stromal cells in vivo
47 Self-renewal of CD146+ cells in vivo
48 Self-renewal of CD146+ cells in vivo
49 Nestin as marker of MSCs
50
51 Cell origin of MSCs
52
53
54
55 Application of MSCs Djouad, F. et al. (2009) Mesenchymal stem cells: innovative therapeutic tools for rheumatic diseases Nat. Rev. Rheumatol. doi: /nrrheum
56 Immunological function of MSCs Front. Immunol., 23 July 2013
57 Mechanisms of the therapeutic effect of MSCs Treatment Stem Cell Res Ther 2011 May 11;2(3):23.
58 Lineage tracing to study adult stem cell biology
59 Different approaches to lineage tracing A: Direct observation B-C: Dye D: DNA label E: LacZ retroviral vector F: Speman and Mangold s organizer s experiment G-H: Adult mouse chimeras from GFP+ and GFP- mice.
60 Adult stem cell in vivo: DNA label-retaining cells (LRC)
61 Development 130,
62 Lineage tracing through genetic recombination
63 1. Genetic recombination has been used for lineage tracing since the early 1990s and is now the preferred approach in most situations. 2. A recombinase enzyme is expressed in a cell- or tissue-specific manner to activate the expression of a conditional reporter gene, and thus permanent genetic labeling of all progeny of the marked cells is achieved. 3. Two site-specific recombination systems adapted from bacteriophage P1 (CreloxP) and Saccharomyces cerevisiae (FLP-FRT) have been widely employed, in combination with different conditional reporter genes in a range of organisms.
64
65
66 A Label-Retaining Cell Population Expressing SC Markers Is Specified Early in HF Morphogenesis and Gives Rise to the Adult Bulge SC Niche Cell Stem Cell 3, 33 43, July 2008
67 Sox9 Is Expressed Early in Developing HFs, and Sox9-Derived Progeny Can Contribute to All Skin Epithelial Lineages Cell Stem Cell 3, 33 43, July 2008
68 Conditional Knockout of Sox9 affects the formation of Quiescent Early-Bulge SCs Cell Stem Cell 3, 33 43, July 2008
69 CreER, an inducible Genetic Recombination System
70
71 NATURE Vol October 2007
72 NATURE Vol October 2007
73 Lineage tracing of Lgr5+ cells NATURE Vol October 2007
74 Dual and multicolor reporter systems
75 Choosing an appropriate lineage tracing strategy
76 Embryonic stem cells
77 Mario R. Capecchi Sir Martin J. Evans Oliver Smithies "f th i di i f i i l f i t d i "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells".
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80 2006 Terese Winslow
81 2006 Terese Winslow
82 PSC Stemness assays Pluripotent stem cells (PSC) Ectoderm Mesoderm Endoderm Neural rosette Pluripotent stem cells (PSC) Immunodeficient NOD/SCID mice Cartilage Columnar epithelium
83 Chimera formation Author:-Priya Pi Rathore
84 Tetraploid complementation assay 1. The assay starts with taking an embryo at the two-cell stage and fusing the two cells by applying an electrical current. The resulting tetraploid cell will continue to divide, and all daughter cells will also be tetraploid. t 2. A tetraploid embryo can develop normally to the blastocyst stage and will implant in the wall of the uterus. The tetraploid cells can form the extra-embryonic tissue (placenta etc.), however a proper fetus will rarely develop. 3. Tetraploid complementation assay is the most stringent assay to evaluate developmental potential of pluripotent stem cells. In this assay, a tetraploid embryo (either at the morula or blastocyst stage) combines with normal diploid pluripotent stem cells from a different organism. The embryo will then develop normally; the fetus is exclusively derived from the pluripotent stem cells, while the extra-embryonic tissues are exclusively derived from the tetraploid cells.
85 Tetraploid complementation assay Modified from Kang and Gao Journal of Animal Science and Biotechnology :5
86
87 References Reading 1. Hematopoietic stem cell heterogeneity takes center stage. Copley MR, Beer PA, Eaves CJ. Cell Stem Cell. 2012;10(6): Mesenchymal stem cells: revisiting history, concepts, and assays. Bianco P, Robey PG, Simmons PJ. Cell Stem Cell. 2008; 2(4): Lineage Tracing. Kretzschmar K and Watt FM. Cell. 2012; 148(1-2):33-45.
88 Assignment 1. Glycolysis-mediated changes in acetyl-coa and histone acetylation control the early differentiation of embryonic stem cells. Cell Metab (3): Presenter: 刘洋 2 T 10 C di t E i ti C t l f S E h A ti it i 2. Tex10 Coordinates Epigenetic Control of Super-Enhancer Activity in Pluripotency and Reprogramming. Cell Stem Cell pii: S (15) Presenter: 眭伟浩
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