Stem Cells and Cancer PDF

Stem Cells and Cancer Day 2 Day 9 Day 13 Katie Sawai INSERM U1312 BRIC Team #11 October 5, 2022 WHAT IS A STEM CELL?  WHAT DEFINES CELL IDENTITY? The ability to self-renew, to regenerate themselves, but also to differentiate...

Stem Cells and Cancer Day 2 Day 9 Day 13 Katie Sawai INSERM U1312 BRIC Team #11 October 5, 2022 WHAT IS A STEM CELL?  WHAT DEFINES CELL IDENTITY? The ability to self-renew, to regenerate themselves, but also to differentiate and give rise to whatever more specialized cells would make up that tissue.  “Reprogramming” of somatic cells The idea is that your cells contain all the DNA. From one cell to another, that DNA should be within an organism. That DNA should largely be the same. That DNA is what encodes that information. It can be reprogrammed from a somatic cell, where the cell is already differentiated, and you can reprogram it to become like a stem cell. That shows that there are factors in the recipient cell, the oocyte, that can induce this stem cell state. Campbell, Nature 1996  Inducing pluripotency: mature cells can be reprogrammed to become pluripotent Takahashi, Cell 2006  Functional studies of stem cells Technological advances Read outs of stem cell function Flow cytometry & sorting Colony assays In vivo gene targeting Transplantation Microarray/next generation Lineage tracing sequencing These are white blood cells that we talk about when we talk about immune cell function. We have megakaryocytes and Hematopoietic cell types erythroid cells. Erythroid cells are the red blood cells. They're important for transporting oxygen as well as transporting all kinds of things and removing waste from different tissues. Megakaryocytes give rise to platelets, which are really important in wound and maintaining correct blood flow. Myeloid Lymphoid Mega/Erythroid O. Naegeli, 1908  A hematopoietic stem cell (HSC) Artur Pappenheim (1870-1916) Alexander Maximov (1874-1928) (Pappenheim, 1905) …a Common Stem Cell of the Different Blood Elements During Embryonic Development and in the Post-Fetal Life of Mammals (Maximov, 1909) Stem cell pioneers (1960s) Spleen colony assay (1961) Ernest McCulloch James Till 1926-2011 * Single cell-derived Clonogenicity * Multi-lineage (erythroid/myeloid) Pluripotency * Transplantable Self-renewal CAN THESE PRIMITIVE CELLS BE GROWN IN VITRO?  Development of in vitro culture conditions Semi-solid media review Metcalf, Science 1985  Caveats Early assays primarily demonstrate myeloid lineage potential Lymphoid lineage rarely read out, true multilineage potential? No distinction between self-renewing stem cell vs non-self- renewing progenitor How to ensure stemness? WHAT PHENOTYPE BEST CHARACTERIZES HSCs?  Technological advance: flow cytometry What we do is we stain our pool of cells with a combination of flexibly tied antibodies. Based on the positive or negative expression of each antibody, a cell will basically travel through the flow cell and pass different lasers that will illuminate. If the antibody that the cell expresses will fluoresce or glow in a way, that light will be captured and detected through a set of filters. That information of whether there is light or not will be processed and then spit out and give you the phenotypic profile of each of your cells. When we look at the flow cytometry information, each dot represents a cell and its level of expression of different markers. MPP3/4 MPP2 57.7 3.7 CD48 14.4 9.3 ST-HSC HSC CD150 Cell Signaling Technology  Transplantation: “gold standard” assay of HSC function HSC BM cells One is CD45.1 and the other one is CD45.2. Based on this difference here at this locus, Donor 1º Recipient 2º Recipient you can distinguish cells from a donor mice that is CD45.2+ CD45.1+ CD45.1+ CD45.2 compared to the cells that are in the recipient mice that are CD45.1. You can see that on flow cytometry. You can identify donor your donor cells from the cells that were originally in the mouse. You transplant these into your mouse and you can track their activity. You can take blood from the mouse over time. You can CD45.2 take bone marrow, a little sampling of bone marrow as well to look at what's going recipient on in the bone marrow. To really show that these donor cells are true stem cells, they have to be able to engross in CD45.1 a second recipient.  Prospective identification of HSCs Positive/ negative selection Spangrude, Heimfeld & Weissman Science 1988  Identification of progenitors downstream of HSCs Population defined as HSCs (Lin- Thy1.1 lo Sca1+) consists of cells with long- term reconstitution potential as well as differentiation potential that can be separated (Morrison & Weissman Immunity 1994) Identification of common lymphoid progenitor (Kondo et al, Cell 1996) Identification of common myeloid progenitor (Akashi et al, Nature 2000)  Classical view of hematopoiesis Bifurcation myeloid vs lymphoid lineages Adapted from Passegué et al PNAS 2003 review Laurenti & Göttgens Nature 2018 WHAT REGULATES HSC FUNCTION? Basically, a lot of candidate genes have been identified for regulating HSC functions. These can be transcription factors that will determine the differentiation programs and are required for driving specific lineages. They can be factors that regulate cell cycle or metabolism or other basic cell functions. There are epigenetic and epitranscriptome factors that would also regulate stem cell functions. Basically, any aspect of a cell's biology that you can think of, most likely there has been at least one, if not multiple, genes that have been identified that would regulate and be important for hematopoietic stem cell functions  Technological advance: in vivo genome modification Knock-in/knock-out Transgene ATG 5’ UTR 3’ UTR ATG Wild type gene exon 1 2 3 4 genomic DNA Transgene GFP pA Targeted gene GFP pA genomic DNA Random transgene integration Homologous targeting recombination vector Injection into Embryo Transgenic fertilized egg transfer ES cell ES cell Blastocyst Chimera electroporation screening injection/transfer We can make knock-in or knock-out animals, and we can make transgenic models, where we would have, for instance, we could have a reporter that would be expressed, so a GFP reporter that would be expressed anytime that your gene of interest is turned on. With knock-in or knock-out, we can basically remove exons from a specific gene that we want to basically inactivate. With knock-ins, we can test the idea of replacing one gene by something else. This happened because we found ways that we could basically incorporate whatever sequence of DNA into the genome. We can do that in a very precise way, where it will go to the specific locus that we want to target, or we could do it in a more random way, which is what happens here when we make transgenes, where you put a piece of DNA and it gets randomly incorporated into the genome. You don't know exactly where it is, but there are certain spots that seem more open and allow this kind of insertion and incorporation of your transgene. review Upadhaya, Reizis & Sawai Exp Hem 2018  New markers for prospective identification of HSCs Kiel, Cell 2005  Regulation of HSCs review Haas, Trumpp & Milsom, Cell Stem Cell 2018 WHAT HAPPENS AT THE SINGLE CELL LEVEL?  Single cell transplantation Nakauchi Science 1996 Mueller-Sieburg Blood 2002, 2004, 2006 Eaves Cell Stem Cell 2007 Goodell Cell Stem Cell 2010 Iscove Cell Stem Cell 2010 review Eaves, Blood 2015  Some HSCs are better than others (in retrospect) The different reconstitution patterns are shown by the different colors here in this bar. You can see the different colors here indicate the different types of lineages that they were tracking. Here, this first primary secondary part represents what happened in the primary transplantation, and then here, this is what happens in the secondary transplantation. You can see that some cells seem to do really good, giving all the different lineages in both the primary and the secondary transplantation. Then you can see that some did all five lineages really well in the primary transplantation, but then in the secondary, you have some lineages dropping out. Then you have some that didn't even do all five lineages in the first transplantation, and only one lineage is maintained in the secondary transplantation. But this shows us that actually all of those cells with the same phenotype don't have the same function Yamamoto et al., Cell 2013  HSCs are heterogeneous in transplantation Single cell, phenotypically-defined HSCs show different types of lineage output upon transplantation Deficiency in production of one or more lineages (myeloid vs lymphoid bias) So transplantation tells you what a cell can do, is capable of doing, in kind of extreme conditions, but it doesn't show what cells are doing at the steady state. WHAT HAPPENS AT THE STEADY STATE?  Finding an HSC-specific gene Tal1 Slamf1 HSC Prog Pdzk1ip1 Slamf1 (Map17) (CD150) M/E My B NK T And these boxes represent different cells, intermediates, starting from the hematopoietic stem cell at the top left, and then becoming more restricted progenitors, and giving more mature cells here at the bottom. And pink means high expression, and blue means low expression. Gene Expression Commons  HSC-specific reporters Vwf Fgd5 Pdzk1ip1 HSC Prog Ctnna1-GFP Fgd5-mCherry Gprc5c-GFP Hoxb5-mCherry Tie2-GFP Vwf-GFP M/E My B NK T Gene Expression Commons  In vivo lineage tracing: fluorescent reporters So the idea with lineage tracing is that you use your reporter to drive either an expression of a fluorescent cassette, or what you can do is to induce the activity of CRE. So you can use CRE locks to basically have your stem cell-specific gene of interest driving the CRE, and there is a form that can be induced with a dose of tamoxifen. So this tamoxifen allows the CRE to enter the nucleus, and then it's active. And so this would turn on the CRE-inducible fluorescent reporter. So what that means is that you can temporarily control the expression of your reporter at any given moment. And once you have fluorescent labeling, it's irreversible, it doesn't get turned off, and then it's stable, right? So it gets passed on to all the daughter cells. ATG Genetic 5’ UTR CreERT2 reporter Rosa26 Tamoxifen Time CAG STOP tdTomato pA gene trap CreERT2 Tamoxifen Upadhaya, Reizis & Sawai Exp Hem 2018  In vivo lineage tracing of HSCs: Tie2 system