Stem Cells and Regenerative Medicine

Stem Cells and Regenerative Medicine 纪俊峰 Email: jijunfeng@zju.edu.cn May 07, 2015

Definition of a Stem Cell

Potency of Stem Cells

Totipotent Pluripotent Multipotent /mucosa /enamel /cartilage/denti n

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

Derivation of Pluripotent Stem Cells ipsc

Fetal Stem Cells

Adult Stem Cells

Discovery of Adult Stem Cells

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

Colony Forming Unit-Spleen (CFU-S) assay

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.

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):115-125. Till, J.E., McCulloch, E.A. (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research 14:213-22. 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 197:452-4 4. Siminovitch, L., McCulloch, E.A., Till, J.E. (1963) The distribution of colony- forming cells among spleen colonies. Journal of Cellular and Comparative Physiology 62:327-36. 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.

Purification of Hematopoietic stem cells (HSCs) with surface marker Lin-Thy-1.1lowSca-1+ 1998

1998

Identification of c-kit as HSC marker

Discovery of new Hematopoietic stem cells surface markers

HSCs Isolation 2006 Terese Winslow

HSC Stemness assays Hematopoietic stem cells (HSC) CFU-E BFU-E CFU-M CFU-GM CFU-GEMM 2006 Terese Winslow

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 CD45.2. 3. 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.

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.

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. 2012 by H IBELGAUFTS. ENTRY LAST MODIFIED:

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

Heterogeneity of HSCs

et al

Single cell gene expression to study heterogeneity and hierarchy of HSCs

Guo et al

Genome-wide approach to identify new markers of HSCs?

Bone marrow transplantation www.scripps.org

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"

World-wide application of Bone Marrow Transplantation

Mesenchymal Stem Cells/ Bone Marrow Stromal Cells (MSC)

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

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

CD146 as a surface marker of MSCs

Antibody Screening

CD146+ cells form hematopoiesis associated Stromal cells in vivo

Self-renewal of CD146+ cells in vivo

Nestin as marker of MSCs

Cell origin of MSCs

Application of MSCs Djouad, F. et al. (2009) Mesenchymal stem cells: innovative therapeutic tools for rheumatic diseases Nat. Rev. Rheumatol. doi:10.1038/nrrheum.2009.104

Immunological function of MSCs Front. Immunol., 23 July 2013

Mechanisms of the therapeutic effect of MSCs Treatment Stem Cell Res Ther 2011 May 11;2(3):23.

Lineage tracing to study adult stem cell biology

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.

Adult stem cell in vivo: DNA label-retaining cells (LRC)

Development 130, 5241-5255

Lineage tracing through genetic recombination

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.

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

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

Conditional Knockout of Sox9 affects the formation of Quiescent Early-Bulge SCs Cell Stem Cell 3, 33 43, July 2008

CreER, an inducible Genetic Recombination System

NATURE Vol 449 25 October 2007

Lineage tracing of Lgr5+ cells NATURE Vol 449 25 October 2007

Dual and multicolor reporter systems

Choosing an appropriate lineage tracing strategy

Embryonic stem cells

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".

2006 Terese Winslow

PSC Stemness assays Pluripotent stem cells (PSC) Ectoderm Mesoderm Endoderm Neural rosette Pluripotent stem cells (PSC) Immunodeficient NOD/SCID mice Cartilage Columnar epithelium

Chimera formation Author:-Priya Pi Rathore

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.

Tetraploid complementation assay Modified from Kang and Gao Journal of Animal Science and Biotechnology 2012 3:5

References Reading 1. Hematopoietic stem cell heterogeneity takes center stage. Copley MR, Beer PA, Eaves CJ. Cell Stem Cell. 2012;10(6):690-7. 2. Mesenchymal stem cells: revisiting history, concepts, and assays. Bianco P, Robey PG, Simmons PJ. Cell Stem Cell. 2008; 2(4):313-9. 3. Lineage Tracing. Kretzschmar K and Watt FM. Cell. 2012; 148(1-2):33-45.

Assignment 1. Glycolysis-mediated changes in acetyl-coa and histone acetylation control the early differentiation of embryonic stem cells. Cell Metab. 2015. 21(3):392-402. 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 2015. pii: S1934-5909 (15)00159-9. Presenter: 眭伟浩