V6: direct cell reprogramming

1. V6: direct cell reprogramming Canadian researchers transform skin into blood, National Post. When scientists at Hamilton’s McMaster University noticed that a petri dish full of long, thin skin cells now also had a quite different occupant — distinctive, round blood cells — they suspected they were on to something big. ... The team under Dr. Mick Bhatia ... devised a process that transforms skin cells directly into blood, a mind-bending breakthrough that one expert suggests may turn cell biology “upside down.”The discovery opens the door to creating healthy blood from a mere patch of skin. For leukemia sufferers unable to find a bone-marrow donor — and other patients — the innovation could one day prove life saving. Mickie Bhatia, McMasters Univ. Ontario/Canada The discovery began as Dr. Bhatia’s team were trying to create iPS cells from skin. Eva Szabo, a post-doctoral student, noticed one day what looked like blood cells in the dish of skin cells, an observation soon borne out by testing. The team then manipulated 3 different types of “factors” – proteins that turn on or off genes within cells – until they could intentionally convert the skin into blood “progenitors” or precursors, which in turn become blood cells. Eva Szabo, Born in Transylvania National Post WS 2010 – lecture 6 Cellular Programs 1

2. Direct reprogramming Dr. Bhatia’s discovery appears to go a major step beyond indirect cellular reprogramming involving a middle stage of creating induced pluripotent stem (iPS) cells Directly reprogramming skin into other types of cell has a number of advantages: (1) Just deriving stem cells from skin can take 5 or 6 months; reprogramming them into other types of cell is another laborious task beyond that. In contrast, Dr. Bhatia’s lab can get from skin to blood within a few weeks. (2) The produced adult blood cells are much more useful for clinical applications like transfusing into a cancer patient. Stem cells tend to produce fetal blood cells. “One of the most important things is, you’re actually making the right cells,” said Dr. Bhatia. More fundamentally, the findings suggest that even cells that have differentiated into a particular type — be it skin, heart or liver — are not frozen in that state and can be directly reprogrammed to do other things, ... And the process was not overly complex. It just required a single transcription factor. Modified after National Post WS 2010 – lecture 6 Cellular Programs 2

3. Medical perspective: fighting leukemia Dr. Bhatia’s lab is now working on converting skin into cells other than blood, as well. The blood process, though, could eventually prove crucial to the many leukemia patients who linger on waiting lists for bone marrow transplants, where stem cells from the marrow are given to the patient to rebuild a blood system decimated by cancer. Compatible donors are often few and far between. Instead, patients would be transfused with blood derived from healthy skin cells, bearing the person’s own genetic make-up. Big hurdles remain, such as “scaling up” the system to produce enough blood, conducting trials and winning regulatory approval. Dr. Bhatia said he hopes that this could be achieved in a couple of years. Dr. Dunbar predicted it would be 5 years at the earliest, and more likely 10 to 15, assuming the lab’s early results are borne out under more testing. National Post WS 2010 – lecture 6 Cellular Programs 3

4. Fibroblasts A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of connective tissue in animals. The main function of fibroblasts is to maintain the structural integrity of connective tissues by continuously secreting precursors of the extracellular matrix. NIH/3T3 Fibroblasts in cell culture www.wikipedia.org Cellular Programs

5. Haematopoiesis Haematopoiesis (from Ancient Greek: αἷμα, "blood"; ποιεῖν "to make") is the formation of blood cellular components. All cellular blood components are derived from haematopoietic stem cells. In a healthy adult person, approximately 1011–1012 new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation. Development of different blood cells from haematopoietic stem cell to mature cells www.wikipedia.org Cellular Programs

6. Experimental strategy To make blood progenitor cells, Bhatia and his team collected skin fibroblasts from several volunteers. They infected the cells with a virus that inserted the gene OCT4, and then grew them in a soup of immune-stimulating proteins called cytokines. Cytokines (Greek cyto-, cell; and -kinos, movement) are small cell-signaling protein molecules. They are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication. Why cytokines? www.nature.com WS 2010 – lecture 6 Cellular Programs 6

7. CD45 The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. This PTP contains an extracellular domain, a single transmembrane segment and two tandem intracytoplasmic catalytic domains, and thus belongs to receptor type PTP. Protein tyrosine phosphatase, receptor type, C also known as PTPRC is an enzyme that, in humans, is encoded by the PTPRC gene. PTPRC is also known as CD45 antigen (CD stands for cluster of differentiation). www.wikipedia.org Cellular Programs

8. CD45 CD45 is specifically expressed in hematopoietic cells and is an essential regulator of T- and B-cell antigen receptor signaling. It functions either through direct interaction with components of the antigen receptor complexes or by activating various Src family kinases required for the antigen receptor signaling. CD45 also suppresses JAK kinases, and, thus, functions as a regulator of cytokine receptor signaling. Four alternatively spliced transcripts variants of this gene, which encode distinct isoforms, have been reported. CD45 is a type I transmembrane protein that is in various forms present on all differentiated hematopoietic cells except erythrocytes and plasma cells that assists in the activation of those cells (a form of co-stimulation). www.wikipedia.org Cellular Programs

9. Cytokine cocktail Cytokines can be classified as proteins, peptides, or glycoproteins. The term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin. The term "cytokine" is sometimes used to refer only to the immunomodulating agents, such as interleukins and interferons. Biochemists disagree as to which molecules should be termed cytokines and which hormones. As we learn more about each, anatomic and structural distinctions between the two are fading. www.wikipedia.org Cellular Programs

10. Cytokines vs. hormones Classic protein hormones circulate in nanomolar (10-9 M/l) concentrations that usually vary by less than one order of magnitude. In contrast, some cytokines (such as IL-6) circulate in picomolar (10-12 M/l) concentrations that can increase up to 1,000-fold during trauma or infection. The widespread distribution of cellular sources for cytokines may be a feature that differentiates them from hormones. Virtually all nucleated cells, but especially endo/epithelial cells and resident macrophages (many near the interface with the external environment) are potent producers of IL-1, IL-6, and TNF-α. In contrast, classic hormones, such as insulin, are secreted from discrete glands (e.g., the pancreas). www.wikipedia.org Cellular Programs

11. Response to cytokines • Each cytokine has a matching cell-surface receptor. • Subsequent cascades of intracellular signalling then alter cell functions. • This may include • the upregulation and/or downregulation of several genes and their TFs, resulting in the production of other cytokines, • an increase in the number of surface receptors for other molecules, • or the suppression of their own effect by feedback inhibition. www.wikipedia.org Cellular Programs

12. Erythrocyte differentiation pathway Stem cells in the bone marrow produce a variety of hematopoietic cell types from common progenitor cells under the influence of cytokines and growth factors. CFU-GEMM cells are a key intermediate in the differentiation of granulo–cytes, erythrocytes, monocytes and megakaryocytes. Erythropoietin (EPO) is a cytokine produced in the kidneys that, along with other cytokines, induces red blood cell (erythrocyte) differentiation in the bone marrow from CFU-GEMM cells. As the erythrocyte lineage progresses, cells lose their nuclei, and move out of the bone marrow into circulation. The ability of EPO to selectively induce red blood cell differentiation has allowed extensive therapeutic use of the recombinant form of this cytokine to treat anemias. EPO is also used for doping! www.biocarta.com Cellular Programs

13. IGF-2 Insulin-like growth factor 2 (IGF-2) is one of three protein hormones that share structural similarity to insulin. In human and mouse, Igf2 is imprinted, with expression resulting favourably from the paternally inherited allele. IGF-2 exerts its effects by binding to the IGF-1 receptor. IGF2 may also bind to the IGF-2 receptor (also called the cation-independent mannose 6-phosphate receptor). IGF2R functions to clear IGF2 from the cell surface to attenuate signalling The major role of IGF2 is as a growth promoting hormone during gestation. IGF2, 1igl.pdb Insulin, 2g4m.pdb www.wikipedia.org Cellular Programs

14. IGF-1 receptor Two alpha subunits and two beta subunits make up the IGF-1 receptor. In response to ligand binding, the α chains induce the tyrosine autophos–phorylation of the β chains. This triggers a cascade of intracellular signaling that, while somewhat cell type specific, often promotes cell survival and cell proliferation. www.wikipedia.org Cellular Programs

15. IGF-1 signalling pathway Insulin like growth factor 1 (IGF-1) and its receptor (IGF-1R) provide a potent proliferative signaling system that stimulates growth in many different cell types and blocks apoptosis. In vivo IGF-1 acts as an intermediate of many growth hormone responses, and may stimulate the growth of some types of cancer. IGF-1 also provides a mitogenic signal to act as a growth factor for many tissue culture cells. One component of IGF-1 mitogenic signaling is association of the receptor tyrosine kinase with Shc, Grb2, and Sos-1 to activate ras and the Map kinase cascade (raf, Mek, Erk). An endpoint of the Map kinase pathway is modification of transcription factor activity, such as activation of ELK transcription factors. Serum response factor (SRF) and AP-1 contribute to mitogenic signaling by many factors. Phosphorylation of IRS-1 and PI3 kinase activation are also involved in IGF-1 signaling, similar to insulin signaling. www.biocarta.com Cellular Programs

16. Stem cell factor Stem Cell Factor (also known as SCF, kit-ligand, KL, or steel factor) is a cytokine that binds to the c-Kit receptor (CD117). SCF can exist both as a transmembrane protein and a soluble protein. The soluble and transmembrane forms of the protein are formed by alternative splicing of the same RNA transcript. This cytokine plays an important role in hematopoiesis (formation of blood cells), spermatogenesis, and melanogenesis. www.wikipedia.org Cellular Programs

17. Stem cell factor In particular, SCF plays an important role in the hematopoiesis during embryonic development. Sites where hematopoiesis takes place, such as the fetal liver and bone marrow, all express SCF. Mice that do not express SCF die in utero from severe anemia. Mice that do not express the receptor for SCF (c-Kit) also die from anemia. Non-lethal point mutants on the c-Kit receptor can cause anemia, decreased fertility, and decreased pigmentation SCF may serve as guidance cues that direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which a stem cell resides), and it plays an important role in HSC maintenance. www.wikipedia.org Cellular Programs

18. Stem cell niche SCF plays a role in the regulation of HSCs in the stem cell niche in the bone marrow. SCF increases the survival of HSCs in vitro and contributes to the self renewal and maintenance of HSCs in-vivo. HSCs at all stages of development express the same levels of the receptor for SCF (c-Kit). The stromal cells that surround HSCs are a component of the stem cell niche, and they release a number of ligands, including SCF. In the bone marrow, HSCs and hematopoietic progenitor cells are adjacent to stromal cells, such as fibroblasts and osteoblasts, see figure. Diagram of a hematopoietic stem cell (HSC) inside its niche. It is adjacent to stromal cells that secrete ligands, such as stem cell factor (SCF). www.wikipedia.org Cellular Programs

19. Summary Direct reprogramming of somatic cells avoids complications of iPS cell formation. Cytokines involved in intracellular communication have an important role in cell reprogramming. Oct4 appears to be the major „reset“ button of differentiation. New medical applications? To treat leukemia? Mickie Bhatia: red blood cells created from stem cells do not make the adult form of haemoglobin. „Those cells, because they think they are embryonic, make embryonic and fetal blood.“ Ian Wilmut: „Because the progenitor cells bypass pluripotency, there is little risk of them forming tumours when implanted into patients.“ www.nature.com Cellular Programs