Stem Cells: Intro, Properties & Characteristics

Introduction to Stem Cells

• Stem cells exist in the brain, blood vessels, skeletal muscle, peripheral blood, bone marrow, teeth, skin, heart, liver, and gut.
• Instructor of this information is Assoc. Prof. Nihal Karakaş.

What is a Stem Cell?

• Stem cells from an embryo, fetus or adult can reproduce themselves under certain conditions for long periods. For adult stem cells, this occurs throughout the organism's life.
• Stem cells produce specialized cells making up the body's tissues and organs.

Special Properties of Stem Cells

• Stem cells self-renew through division and create copies of themselves.
• Stem cells differentiate, giving rise to mature cell types that make up organs and tissues.

Characteristics Distinguishing Stem Cells

• Stem cells are unspecialized. They renew themselves for long periods via cell division.
• Stem cells can be induced under certain physiological or experimental conditions to become cells with special functions. These include heart muscle cells or insulin-producing cells of the pancreas.

Stem Cell Composition

• The body comprises about 200 kinds of specialized cells, including muscle, nerve, fat, and skin cells.
• All cells come from stem cells.
• A stem cell is not yet specialized.
• Specialization is called differentiation.
• Once a stem cell's differentiation pathway is determined, it cannot typically become another cell type on its own.

Types of Stem Cells

• Embryonic Stem Cells:
  • Early Embryonic Stem Cells
  • Blastocyst Embryonic Stem Cells
• Tissue Specific Stem Cells (Adult Stem Cells):
  • Hematopoietic stem cells
  • Mesenchymal stem cells
  • Fetal Stem Cells
  • Umbilical Cord Stem Cells
  • Inducible Pluripotent Stem Cells (IPSCs)

Classification According to Differentiation Potency

• Totipotent: Each cell can develop into a new individual. Examples are cells from early (1-3 days) embryos.
• Pluripotent: Cells can form any (over 200) cell types. Examples are some cells of blastocyst (5 to 14 days).
• Multipotent: Cells are differentiated but can form a number of other tissues, such as fetal tissue, cord blood, and adult stem cells.

Detailed Classification of Stem Cells

• Totipotent stem cells can generate a viable embryo (including the placenta) under the right conditions; they exist until about four days after fertilization, before the blastocyst stage.
• Pluripotent stem cells can become all cell types in an implanted embryo, fetus, or developed organism. Embryonic stem cells are pluripotent.
• Multipotent stem cells give rise to different types of specialized cells but are restricted to certain organ or tissue types. Blood stem cells are a multipotent example, producing all blood cell types, but not cells of the liver/brain.

Definitions and General Concepts

• Pluripotent stem cell: A single pluripotent stem cell can give rise to cells that develop from the three germ layers (mesoderm, endoderm, and ectoderm) from which all body cells arise. Known sources are isolated and cultured from early human embryos and fetal tissue destined to be part of the gonads.
• Embryonic stem cell: It is derived from a group called the inner cell mass, part of the early (4- to 5-day) embryo called the blastocyst. Once moved from the blastocyst, inner cell mass cells can be cultured into embryonic stem cells.

Term Definitions

• Embryo: Early developing organism, the period between the fertilized egg and the fetal stage.
• Blastocyst: A very early embryo with a ball shape, consists of about 150-200 cells. Has an inner cell mass, from which embryonic stem cells derive, and an outer trophoblast layer which forms the placenta.
• Self-renewal: Cell division process generating another cell with the same potential.

Additional Term Definitions

• Embryonic germ cell: It is derived from fetal tissue specifically isolated from primordial germ cells of the gonadal ridge of the 5- to 10-week fetus.
• Differentiation: Unspecialized cells (like stem cells) specialize into the body's diverse cells. Genes activate/inactivate in a regulated manner during differentiation. This results in differentiated cells that develop specific structures.
• Directed differentiation: Stem cells are manipulated in the laboratory to become specialized or partially specialized cell types i.e. heart muscle, nerve, or pancreatic cells

More Term Definitions

• Adult stem cell: It is an undifferentiated cell in a differentiated tissue. It renews itself and specializes to yield all cell types of its tissue of origin.
• Adult stem cells can make identical copies of themselves for the life of the organism; this is self-renewal. Adult stem cells divide to generate progenitor/precursor cells that then differentiate into "mature" cell types with specialized functions.
• Sources of adult stem cells are bone marrow, blood, cornea/retina of eye, brain, skeletal muscle, dental pulp, liver, skin, lining of gastrointestinal tract, and pancreas.

Additional Stem Cell Characteristics

• The most abundant information about stem cells comes from studies of hematopoietic (blood-forming) stem cells derived from bone marrow/blood. They've been studied and therapeutically applied for diseases.
• Adult stem cells are rare, difficult to identify/isolate/purify. Insufficient numbers are available for transplantation, and adult stem cells do not replicate indefinitely in culture.
• Plasticity: An adult stem cell's ability from one tissue to generate specialized cells for another tissue, or under specific experimental conditions, bone marrow adult stem cells can generate neuron-resembling cells. Given the appropriate environment, some adult stem cells can be genetically reprogrammed to generate cells characteristic of different tissues.

Clonality

• Clonality/clonally derived stem cell: Cell generated from a division of a single cell and is genetically identical; concept important in stem cell research. Understanding identities/capabilities/qualities of stem cells is important to generating replacement cells/tissues. Human pluripotent stem cells from embryos and fetal tissue are by nature clonally derived.
• Very few studies have shown clonal properties of the cells developed from adult stem cells.
• Knowing if a single cell can develop an array of cell types, or if multiple stem cell types grown together can form multiple cell types is crucial. Recent studies show mixtures of fat tissue/umbilical cord blood cells can develop into blood/bone cells.

Precursor Cells

• Progenitor/precursor cell: A precursor occurs in fetal or adult tissues and is partially specialized; divides/gives rise to differentiated cells. When a stem cell divides, one of the new cells is a similar stem cell capable of replicating itself; in contrast, when a precursor cell divides, it forms more precursor cells or two specialized cells incapable of replicating.
• Progenitor/precursor cells can replace damaged or dead cells, maintaining tissue integrity/function (liver/brain). They give rise to lymphocytes like T cells and B cells, but do not generate a variety of cell types.

Types of Stem Cells - Early Embryonic Stem Cells

• The first human development step occurs when a newly fertilized egg, or zygote, begins to divide, producing a group of stem cells (embryo). These early stem cells are totipotent, so they can become any kind of cell in the body.

Types of Stem Cells - Blastocyst Embryonic Stem Cells

• Five days after fertilization, the hollow-like structure is called a blastocyst. Embryos in the blastocyst stage contain two types of cells.
• Embryonic stem cells form the inner cell mass. This ultimately develops into the fetus.
• Trophoblast cells make up the outside of the ball and eventually become the placenta, supplying nutrients to the fetus.
• Embryonic stem cells in the blastocyst are pluripotent, meaning they can become almost any cell type. An embryo in blastocyst stage lacks features characteristic of the human body.

Development of Blastocyst In Vitro

• Human oocyte, post-fertilization, the following timeline is observed:
  • Day 1: 18-24 hours post in vitro fertilization of oocyte.
  • Day 2: (24-25 hours) the zygote (fertilized egg) has its first cleavage producing a 2-cell embryo.
  • Day 3: (72 hours) the 8-cell stage is called a morula. The embryo controls its development.
  • Day 4: the embryo's cells compress tightly.
  • Day 5: the blastocyst's cavity is complete. Inner cell mass separates from outer cells.

Day-5 Blastocysts for ES Cell Cultures

• A normal day-5 human embryo in vitro consists of 200-250 cells, and most constitute the trophectoderm.
• In deriving ES cell cultures, the trophectoderm is removed through microsurgery or immunosurgery, thus freeing the inner cell mass.
• At this stage, the inner cell mass is composed of only 30–34 cells. Growth techniques are similar when inner cell mass is from mouse/human blastocysts.

Embryonic Stem Cells

• The derivation of human embryonic stem cells from human blastocysts first occurred in 1994.
• An embryonic stem cell derives from the inner cell mass, part of the early (4-5 day) embryo (the blastocyst).

Embryonic Stem Cell Origins

• These cells are derived from pre-implantation/peri-implantation embryos. Has self-renewal allowing dividing to make copies of itself for long periods of time without differentiating. Give rise to cells from three embryonic germ layers.

Stem Cells: Totipotent and Pluripotent

• The fertilized egg and cells arising in those first divisions are totipotent. They can generate a viable embryo (includes support tissues). These cells transition days later to being pluripotent.

Pluripotent Stem Cells Characteristics

• Embryonic stem cells are immortal.
• Months of growth in culture dishes produces any body cell type, making it pluripotent. Human ES cells has a proliferative/developmental potential promising essentially unlimited supply of specific cell types that treat heart disease to leukemia.

Embryonic Stem Cells

• As long as maintained in certain culture conditions, they can stay undifferentiated, or unspecialized.
• If grown in cell cultures that allow clumping together to form embryoid bodies, they will begin to differentiate and form muscle cells and nerve cells.

Identifying Embryonic Stem Cells

• What tests exists for identifying embryonic stem cells?
  • Subculturing stem cells for many months to ensure long-term self-renewal.
  • Inspecting cultures via microscope, ensuring cells look healthy/undifferentiated.
  • The presence of surface markers indicates undifferentiated cells.
  • Oct-4 protein presence test. It helps turn genes on/off at right time, impacting differentiation/embryonic development.
  • Chromosome examination using a microscope. Checking chromosome damage or a changed number of chromosomes.
  • Determines the ability of subculturing post-freezing, thawing and replating.
• Pluripotency tests exist:
  • Allowing cells differentiate spontaneously in culture.
  • Manipulating cells into forming any specific cell type.
  • Injecting cells into immunosuppressed mice to test for benign tumor (teratoma) formation.

Embryonic Stem Cell Stimulation for Differentiation

• Undifferentiated culture allows for differentiation to begin only if cells form embryoid bodies.
• Spontaneous differentiation is a good indication the culture is healthy, but not an efficient method to creating specific cell type cultures.

Potential Use of Human Stem Cells

• Transplant therapy: replacing/restoring tissue damaged by disease/injury.
• Human ES-derived applications exist for Parkinson's, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne's muscular dystrophy, heart failure, and osteogenesis imperfecta.
• Advantages; it has unlimited in vitro proliferation and generates broad range of cells thru directed differentiation. However disadvantages exist:
  • They may cause undifferentiated tumors to form.
  • Immunological rejection may occur. Avoid using genetically engineered ES cells to express MHC antigens of transplant recipients.

Other stem-cell uses

• Studying early events in human development is possible. It can identify genetic/molecular/cellular events leading to problems such as birth defects/placental abnormalities.
• Test therapeutic drugs (preclinical tests). Screen for potential toxins.
• They're used to develop new genetic engineering methods, and can be used to direct differentiation to a specific cell type.

Fetal Stem Cells

• After the eighth week of development, an embryo is a fetus.
• Stem cells in the fetus are responsible for the development of all tissues before birth.
• Fetal stem cells, like embryonic stem cells, are pluripotent.

Umbilical Cord Stem Cells

• Recognized in the late 1980s as an important human stem cell source.
• The cord transports nutrients and oxygenated blood from the placenta to the fetus.
• Hematopoietic stem cells are genetically identical to the newborn child.
• They are multipotent and differentiate into a limited range of cell types.

Umbilical Stem Cells

• UCB is harvested and frozen, stored in cord blood banks as either donor-specific or general resource.
• Cord blood effectively transplants children, but less so in adults. Specific limitations includes:
  • Limited quantity of stem cells harvested.
  • Delayed immune reconstitution following transplant, leaving patients vulnerable to infections.
• However, advantages exist:
  • Availability and ease of obtaining
  • Lowered risk of graft-versus-host disease.
  • Greater proliferative capacity than in adult HSCs.

Adult Stem Cells

• Infants and children have these, residing in already-developed tissues and directing tissues growth.
• Adult = Multipotent.
• Sometimes referred to as tissue stem cells. They reside in most body tissues and are involved in repair/replacement. It's hard to isolate.

Adult Stem Cells (Tissue Stem Cells)

• Adult stem cells can make identical copies of themselves for long periods of time (self-renewal).
• They can give rise to mature cell types that have the morphologies/specialized functions that are characteristic to each type.
• Typically, stem cells first generate an intermediate cell type (precursor or progenitor).
• Adult stem cells maintain homeostasis and replace cells that die (injury/disease).
• Unlike embryonic stem cells, the origin of adult stem cells is often unknown in mature tissue.

Locations and Functions

• Adult stem and progenitor cells reside throughout the body.
• Reside in specific areas of tissues; the stem cell niche.
• Niche is a microenvironment promoting resident stem cells.
• Mutations, signals, microenvironment changes can activate these adult stem cells.

Adult Tissues Containing Stem Cells

• Bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelia of the skin and digestive system, cornea, retina, liver, and pancreas.

Plasticity of Adult Stem Cells

• Plasticity is the ability of a stem cell from one adult tissue to generate the differentiated cell types of another tissue. It is referred to as "plasticity”, “unorthodox differentiation" or "transdifferentiation".

Additional Notes on Plasticity

• Hematopoietic stem cells may differentiate into brain cells, skeletal muscle cells, cardiac muscle cells, and liver cells.
• Bone marrow stromal cells may differentiate into cardiac/skeletal muscle cells.
• Brain stem cells may differentiate into blood/skeletal muscle cells.

Hematopoietic Stem Cells

• These (HSCs) give rise to all blood cell types. There are myeloid and lymphoid subtypes. HSCs are found in bone marrow from very early development, plus umbilical cord blood/placental tissue.
  • Myeloid: monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • Lymphoid: T-cells, B-cells, NK-cells.

Mesenchymal Stem Cells

• They're multipotent (non-blood cells) are derived from blood organs like bone marrow/fetal liver, and support blood cell growth in vitro. hMSC Sources come from bone marrow and mobilized peripheral blood.
• MSCs can differentiate in vitro/in vivo to osteoblasts, chondrocytes, myocytes, adipocytess, neural cells, and beta-pancreatic islet cells.
• Easy to isolate/culture with high ex vivo expansive potential that allows for regenerative medicine/tissue engineering.

Neural Stem Cells

• Located in the subventricular zone lining lateral ventricles and subgranular zone of hippocampus.
• Give rise to neurons / oligodendrocytes / astrocytes.

Epithelial Stem Cells

• Give rise to epithelial cells. These make up 60% of the differentiated body cells.
• Responsible for covering internal (intestinal) and external surfaces (skin). Found in bulge region of the hair follicle and thelining of vessels and some glands.

Final Clarification on Adult Cells

• Not all new adult cells arise from stem cells. Most by mitosis of differentiated cells.

Inducible Pluripotent Stem Cells - History

• John Gurdon in 1962 eliminated the frog egg cell nucleus. Then, he replaced it with the nucleus of a specialized tadpole cell. This created a normal tadpole, and subsequent nuclear transfers generated cloned mammals. Injected genetic info was contained. Later, he found mammals can also be cloned.
• Shinya Yamanaka won the Nobel Prize when he and Kazutoshi Takahashi demonstrated in 2006 that four transcription factors are capable of turning embryonic/adult fibroblasts into pluripotent cells. These cells, in vivo, product teratomas.

Modern Inducible Stem Cells

• iPS cells derived here display pluripotency for ES cells, to form teratomas that have all germ layers and can be injected into mouse embryos and can form those chimeric mice. Myc, Oct4, Sox2 and Klf4 are Yamanaka's factors for transmission.iPS cells generated from humans/disease patients. Nerve, heart, and liver cells can derive from these. and study disease/new therapies.

Use of Adult Stem Cells

• Reprogrammed from the patient's cells. Can be made from fat, skin, and fibroblasts.

Potential Uses of all Types of Stem Cells

• Cellular Immunotherapy (tumor vaccines, immune system with differentiated or undifferentiated stem cells) and for Tissue engineering (bone, cartilage, heart, etc)
• Stem Cell Transplantation: allogeneic (treatment of acute lymphoblastic leukemia and acute, chronic myelogenous/lymphoid leukemia, multiple myeloma, several forms of anemia/diseases), autologous (anti-aging, cancer treatment, cosmetics). Lastly Gene therapies (for treatment for genetic, neurodegenerative and cancer diseases)