How To Expand Hematopoietic Stem Cells
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1 Purification and Expansion of Hematopoietic Stem Cells Based on Proteins Expressed by a Novel Stromal Cell Population Our bodies are constantly killing old, nonfunctional, and unneeded cells and making new cells. Without many different types of stem cells in our bodies we could not live! Prof. Harvey F. Lodish Whitehead Institute for Biomedical Research, Cambridge, MA, and Departments of Biology and Bioengineering, Massachusetts Institute of Technology, Cambridge, MA
2 What are stem cells, and what is the difference between adult and embryonic stem cells? What are hematopoietic stem cells (HSCs) - the cells that form all red and white blood cells and all cells of the immune system? How do we analyze them? How did we identify a population of supportive, or stromal cells that supports HSC growth in culture? How did we use these supportive cells to identify new hormones that support HSC growth in culture? How are we using these findings to expand human cord blood hematopoietic stem cells in culture for clinical use?
3 What are stem cells, and what is the difference between adult and embryonic stem cells? What are hematopoietic stem cells (HSCs) - the cells that form all red and white blood cells and all cells of the immune system? How do we analyze them? How did we identify a population of supportive, or stromal cells that supports HSC growth in culture? How did we use these supportive cells to identify new hormones that support HSC growth in culture? How are we using these findings to expand human cord blood hematopoietic stem cells in culture for clinical use?
4 What is a stem cell? Immortal When a stem cells divides, one or both of its daughters must also be a stem cell. Thus the number of stem cells of a particular type either increases or remains constant over time. Undifferentiated Exhibits few if any of the traits that are distinguishable as cells acquire the ability to form specialized, functional tissues and organs. Able to generate multiple types of body cells A stem cell divides such that one or both of its daughters are able to give rise (after yet more cell divisions) to a few defined types of differentiated, functional body cells (Multipotent).
5 What is a stem cell? Embryonic stem cell (ES cell) Grown in the laboratory from embryos Not clear whether these normally exist in adult animals/ people Can give rise to all types of cells in the body (Totipotent) Adult- type stem cell Normally exist in adult animals and people but are extremely rare Each type of adult stem cell can give rise to a few specific types of cells in the body (Multipotent)
6 Embryonic stem cells are totipotent and are derived from the inner cell mass in a blastocyst Sperm Fertilized Egg/Zygote Unfertilized Egg Blastocyst (5 days) Morula (3/4 days)
7 Adult stem cells generate replacement cells for those that are normally subject to destruction Intestine Stem Cells Muscle Bone Marrow Stem Cells Muscle Stem Cells Blood Cells
8 The mesenchymal stem cell Found normally in bone marrow Can be grown in petri plates in the laboratory (cell culture) When treated with specific hormones, can differentiate in cell culture to form one of three types of cells: Bone- forming cells Cartilage- forming cells Fat cells
9 What are stem cells, and what is the difference between adult and embryonic stem cells? What are hematopoietic stem cells (HSCs) - the cells that form all red and white blood cells and all cells of the immune system? How do we analyze them? How did we identify a population of supportive, or stromal cells that supports HSC growth in culture? How did we use these supportive cells to identify new hormones that support HSC growth in culture? How are we using these findings to expand human cord blood hematopoietic stem cells in culture for clinical use?
10 The hematopoietic (blood- forming) stem cell Extremely rare - about one cell in ten thousand in adult bone marrow In embryos found in the yolk sac and fetal liver The basis of bone marrow transplantation Gives rise to: All cells of the immune system All red blood cells All types of white blood cells
11 Red Blood Cells Comprise about 45-47% of the volume of blood. Filled with the red oxygen- binding protein hemoglobin. Transports oxygen from the lungs to all tissues in the body. Transports carbon dioxide from tissues to the lungs. Survive for 120 days; ~0.8% replaced each day
12 Hematopoietic stem cells: 1:10,000 cells in the bone marrow
13 Clinical Applications of HSCs Allogeneic (HLA match of donor and recipient) transplantation: leukemia, lymphoma, and other inherited blood disorders Autologous (self) transplantation: solid cancers, autoimmune diseases Potentially: gene therapy
14 Challenges in the application of hematopoietic stem cells (HSC) for therapy Bone Marrow Transplantation for Cancer Patients Improve separation and purification of HSCs from cancer cells Grow HSC in Culture Bone Marrow or Cord Blood Transplantation Generation of sufficient numbers of stem cells for bone marrow transplants Gene Therapy Selection of cells with appropriate genetic alteration - gene therapy treatment of: Sickle cell anemia, thalassemia Immune deficiencies, cancers
15 Hematopoietic stem cells are found in a niche interspersed among many other types of cells in the bone marrow
16 Multiple fates of hematopoietic stem cells: Each HSC integrates multiple extracellular signals and HSC self renewal requires multiple growth factors Self-renewal HSC Migration Protein Factors ECM Niche Cells Apoptosis (programmed cell death) Differentiation
17 Bone marrow transplant experiments in mice are used to identify and quantify both human and mousehematopoietic stem cells
18 To identify and enumerate hematopoietic stem cells we do a bone marrow transplantation experiment Donor bone marrow cells are removed from a CD45.1 (Ly 5.1) mouse, and usually fractionated to remove most of the non- stem cells. A recipient CD45.2 (Ly 5.2) mouse is irradiated, to kill off its own stem cells. Defined numbers of donor CD45.1 cells are injected into a vein of the irradiated recipient mice. One to nine months after transplantation blood is removed from the recipient mouse. Blood cells are reacted with a monoclonal antibody that fluoresces green light and that binds only to CD45.2 recipient cells, and with another monoclonal antibody that fluoresces red light and that binds only to CD45.1 donor cells A Fluorescence- Activated Cell Sorter (FACS) allows us to examine cells one- at- a- time to determine whether they fluoresce red or green light and thus are CD45.1 (donor derived) or recipient CD45.2 cells.
19 Bone marrow transplant experiments are used to identify and quantify hematopoietic stem cells
20 Cell separation through Fluorescenceactivated cell sorting (FACS)
21 Transplantation of enriched hematopoietic stem cells
22 What are stem cells, and what is the difference between adult and embryonic stem cells? What are hematopoietic stem cells (HSCs) - the cells that form all red and white blood cells and all cells of the immune system? How do we analyze them? How did we identify a population of supportive, or stromal cells that supports HSC growth in culture? How did we use these supportive cells to identify new hormones that support HSC growth in culture? How are we using these findings to expand human cord blood hematopoietic stem cells in culture for clinical use?
23 Ex vivo expansion of HSCs has been difficult Input Hematopoietic cells Known hormones or stromal cells After culture Proliferation, differentiation and death predominate The key: Identify new growth factors
24 Expansion of mouse fetal liver hematopoietic stem cells during in vitro culture Natural expansion of hematopoietic stem cells occurs in the fetal liver between embryonic days 15 and 21 (birth) Fetal liver hematopoietic stem cells will repopulate all blood cell lineages in irradiated adult recipient mice The properties of fetal liver and adult bone marrow hematopoietic stem cells are similar. Expansion of both fetal liver and adult bone marrow HSCs in culture is enhanced by a novel population of fetal liver CD3 + cells.
25 Growing hematopoietic stem cells in culture We have developed a simple culture system for expansion of bone marrow hematopoietic stem cells. The medium contains no serum and low levels of five hormones: SCF (Stem cell factor) TPO (Thrombopoietin) IGF-2 (Insulin- like growth factor- 2) FGF-1 (Fibroblast growth factor- 1) Angptl-2 (Angiopoietin- like protein 2) After a 10 day culture there is a 30- fold increase in the number of hematopoietic stem cells. This may be sufficient for most therapeutic purposes.
26 Identification of a novel HSC supportive population: fetal liver CD3 + cells Fetal liver Coculture: HSCs with different non-hsc subpopulations Competitive reconstitution analysis HSC supportive cell population? Zhang CC and Lodish HF Blood (7): DNA array: identification of candidate growth factors
27 Coculture with fetal liver CD3 + cells promotes a three fold expansion of HSCs Medium alone with 50 fetal liver CD3 + cells Fifty freshly isolated Day 15 CD45.2 fetal liver Lin - Sca-1 + Kit + (LSK) cells, an enriched population of Hematopoietic Stem Cells ( ~ 0.07% of fetal liver cells) were cultured 3 days in 30 µl medium containing fetal calf serum supplemented with SCF, FL and IL-6.
28 IGF-2 is specifically expressed in fetal liver CD3 + cells Gene description Fetal liver CD3 + cells (normalized) Adult spleen CD3 + cells (normalized) Fetal liver Gr cells (normalized) IGF Angiopoietin-like protein 2 Plasminogen Dlk1 - like homolog Angiopoietin-like protein 3 CD p glycoprotein CD24a CD98 light chain Prion protein
29 Coculture with fetal liver CD3 + Ter119 - cells promotes ex vivo expansion of HSCs by producing IGF-2 Fifty freshly isolated day 15 CD45.2 fetal liver LSK cells were cultured 3 days in 30 µl medium containing fetal calf serum supplemented with SCF, FL and IL-6. Medium alone with 50 fetal liver CD3 + cells with 50 fetal liver CD3 + cells and anti-igf-2 antibody
30 All HSCs bind to IGF-2-hFc fusion protein: IGF-2 acts directly on HSCs 60 3 weeks 6 months 50 %Repopulation Binding to IGF2-hFc
31 Culture with IGF - 2 increases in vivo repopulating activity of cultured HSCs % Repopulation Neg 0-4 H SCF, FL and IL-6 H H H SCF and TPO H H H H SCF, FL, IL-6, and 500 ng/ml IGF-2 H H H H SCF and TPO and 500 ng/ml IGF-2 Fifty freshly isolated day 15 CD45.2 fetal liver Lin - Sca Kit + cells were cultured for 3 days in medium containing FBS and supplemented as indicated
32 Multilineage contribution of cultured cells at 4 months post-transplant (n = 6) %Repopulation T-lymphoid B-lymphoid myeloid
33 ? 1. Fetal liver CD3 + cells are a novel HSC supportive population 2. IGF-2 is a new HSC growth factor produced by fetal liver CD3 + cells 3. HSCs can be expanded 8 -fold by using IGF-2 and other factors in serum- free medium 4. Can we identify other novel HSC growth factors and further improve ex vivo expansion of HSCs? Zhang CC and Lodish HF. Blood Apr 1;103(7): Zhang CC and Lodish HF. Blood Jun 1;105(11):
34 Angiopoietin- like proteins 2 and 3 are specifically expressed in fetal liver CD3 + cells Gene description Fetal liver CD3 + cells (normalized) Adult spleen CD3 + cells (normalized) Fetal liver Gr cells (normalized) IGF Angiopoietin-like protein 2 Plasminogen Dlk1 - like homolog Angiopoietin-like protein 3 CD p glycoprotein CD24a CD98 light chain Prion protein
35 Angptl2 supports expansion of highly enriched bone marrow SP Sca-1 + CD45 + long- term repopulating HSCs in serum-free medium 5 day culture containing only SCF ± Angptl2 10 day culture containing SCF, TPO, FGF-1, and IGF-2 ± Angptl2 %Repopulation weeks 4 months 8 months ** * * Day 0 Day 5 SCF Day 5 SCF+Angptl2 Day 10 S+T+I+F Day 10 S+T+I+F+A Day 0 Day 5 SCF Day 5 SCF+Angptl2 Day 10 S+T+I+F Day 10 S+T+I+F+A Day 0 Day 5 SCF Day 5 SCF+Angptl2 Day 10 S+T+I+F Day 10 S+T+I+F+A **
36 Growing hematopoietic stem cells in culture We have developed a simple culture system for expansion of bone marrow hematopoietic stem cells. The medium contains no serum and low levels of five hormones: SCF (Stem cell factor) TPO (Thrombopoietin) IGF-2 (Insulin- like growth factor- 2) FGF-1 (Fibroblast growth factor- 1) Angptl-2 (Angiopoietin- like protein 2) After a 10 day culture there is a 30- fold increase in the number of hematopoietic stem cells. This may be sufficient for most therapeutic purposes.
37 Important Questions Are CD3 + cells bona fide stromal cells in the fetal liver? In the bone marrow? What is the function of each of these individual growth factors on hematopoietic stem cells? Stimulate division? Prevent cell death? Prevent differentiation of daughter cells? What signaling pathways are induced in hematopoietic stem cells by each of these growth factors and how do these signaling pathways interact? How do hematopoietic stem cells interpret these signals and decide whether to divide, differentiate, undergo death, or simply remain quiescent? Does a similar culture system support expansion ex vivo of human umbilical cord blood hematopoietic stem cells?
38 What are stem cells, and what is the difference between adult and embryonic stem cells? What are hematopoietic stem cells (HSCs) - the cells that form all red and white blood cells and all cells of the immune system? How do we analyze them? How did we identify a population of supportive, or stromal cells that supports HSC growth in culture? How did we use these supportive cells to identify new hormones that support HSC growth in culture? How are we using these findings to expand human cord blood hematopoietic stem cells in culture for clinical use?
39 Culture of total human cord blood cells in the presence of Angptl5 stimulates ex vivo expansion of HSCs. Cultures contain SCF, TPO, Flt3-L, IGF-2, and Angptl 5 Extent of human chimerism in the bone marrow of NOD/SCID mice two months posttransplantation with 8,000 or 15,000 uncultured human cord blood CD133 + cells, or the progeny from 8,000 initial CD133 + cells cultured for 11 days. * Significantly different from lanes 1-3; Student s t-test, p < 0.05.
40 Culture of CD133 + human cord blood cells in the presence of Angptl5 stimulates ex vivo expansion of HSCs. Cultures contain SCF, TPO, Flt3-L, IGF-2, and Angptl 5 Myeloid B Primitive Myeloid B Primitive Summary of multilineage reconstitution from mice 2 months post-transplant with freshly isolated human cord blood CD133 + cells, or the progeny from 8,000 initial CD133 + cells cultured for 11 days.
41 The stem cell team: Cheng Cheng Zhang, Megan Kaba, Guangtao Ge, Kathleen Xie, Wei Tong, and Christopher Hug
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