Chapter 22 - Stem Cells

A stem cell can become any of the 220 cell types

development
1. begins life as a single cell - a zygote
2. divides into the 3 embryonic germ layers
  1. endoderm
    1. gives rise to the epithelial lining of the gut (associated organs: lungs, esophagus, and intestine)
  2. mesoderm
    1. connective tissue ( cartilage, bone, muscles, and vascular system)
  3. ectoderm
    1. epidermis, nervous system, sensory organs
Early stages of development
1. The zygote undergoes cell division and cleavage events and forms a hollow ball called a blastula
2. blastula cells will differentiate into the 3 germ layers
Cell potency
1. Through development, individual cells become more restricted in their developmental potential
2. Totipotent - a cell with the potential to give rise to all cell types of the body
  1. zygote and blastula
3. pluripotent - a cell with the potential to give rise to almost all cell types
  1. late blastula
4. Multipotent - a cell with the potential to give rise to multiple cell types
  1. ectodermic, endodermic, mesodermic cells
Regulatory DNA defines gene expression patterns in development
1. differentiation - a process where the cell becomes specialized to perform a specific function
  1. Beta-islet cells are the only cells in the body that produce insulin
  2. Differentiation
    1. zygote → blastomere
    2. blastomere → endoderm cell
    3. endoderm cell → pancreatic bud cell
    4. pancreatic bud cell → endocrine pancreas cell
    5. endocrine pancreas cell → beta-islet cell
2. All cells have the same genome, but what transcription factors and transcription regulators are present determines what the function of the cell is
Cell signaling pathways coordinate spatial patterning
1. Cells become different according to their position in the embryo
  1. inductive signal (local)
    1. releasing signal molecules, and nearby cells are directed to a new developmental pathway
  2. Cells become different according to their position in the embryo
2. Morphagen signaling
  1. a cell that tells other cells to differentiate or not
Lateral inhibition - cell interactions force neighboring cells to be different
1. An example of this is seen in the notch pathway
2. Transient Bias: Amplified by feedback pathways. Creates a slight asymmetry to start differentiation between the 2 cells. Positive feedback: asymmetry is self-amplifying
Asymmetric cell division generates diversity
1. symmetric sister cells become different as a result of influences acting on them after birth
2. Asymmetric divisions: sister cells are born different
  1. Usually, transcription factors are all given to one daughter and not the other
3. Both of these can happen in the body
Stem Cells
1. Development occurs throughout the lifetime for tissue renewal and repair
2. Stem cells are specialized cells that provide a fresh supply of differentiated cells
  1. defining properties:
    1. not terminally differentiated
    2. can divide without limit (immortal)
    3. have the choice to differentiate or remain a stem cell
Stem Cell Origins
1. Not all stem cells come from human embryos
Epithelial tissue renewal in the small intestine
1. Not all tissues have stem cells
2. Anything you eat will wear down your gut, so it is constantly renewed
3. pathway
  1. Villi fingers are present to increase the surface area of the gut
  2. between each villi is a crypt
  3. Intestinal stem cells are buried at the bottom of crypts
  4. As cells in the crypt divide, they are pushed upward into the villi portion, and worn-out cells undergo apoptosis
4. Intestinal stem cells become 1 of the 4 main types of differentiated cells
  1. absorptive cells - they take up nutrients
  2. goblet cells - they secrete mucus into the gut lining to lubricate and protect
  3. paneth cells - innate immunity, stop the absorption of bacteria. Helps maintain the system population. Only cell type that lives in the crypt.
  4. enteroendocrine cell - regulatory, secretes hormones to help regulate the digestive process
5. These are not potent; they will always become what they are
6. The stem cell is multipotent
Transient amplifying cells
1. undifferentiated cells that rapidly divide
2. Cell division is risky, so stem cells are restricted
Cells move up the crypt in a conveyor belt manner
1. The bottom of the crypts has stem cells
2. Stem cells don’t divide a lot. When they do, those become transient amplifying cells
3. Transient amplifying cells rapidly divide and move to the top of the villi, then die by apoptosis
Stem cell daughters do not always have to make different choices
1. asymmetric division: a parent cell divides into two daughter cells with different sizes, compositions, or fates
2. independent choice: regulated based on tissue needs, need more stem cells, or more differentiated cells
3. which they do is dependent on the need of the tissue
Can 1 intestinal crypt stem cell create all cell types in the gut
1. Lrg5 stem cell marker
  1. Added a dye to the stem cell, so when the cell replicates, it will also replicate the dye.
  2. It was grown and found all 4 cell types coming from the original stem cell
2. “minigut”
  1. When a single stem cell was separated onto a plate, it not only differentiated, but was able to form a mini gut structure
Paneth cells create the stem cell niche
1. The crypt is undifferentiated cells, and the villus is differentiated cells
  1. This is violated by Paneth cells
  2. Paneth cell is the only differentiated cell that lives in the crypt
2. Paneth cell uses Wnt to maintain the niche of stem cells
  1. Paneth cell at the bottom of the crypt secretes Wnt
  2. wnt signal is received by neighboring cells, the stem cells
  3. When stem cells receive Wnt, they also start producing Wnt
    1. They start engaging in an autocrine loop
    2. secreting and binding to its own Wnt
  4. Transient amplifying cells are next to stem cells, and they do not make their own wnt, but are kept undifferentiated by the presence of wnt
3. The crypt ends at the last cell receiving Wnt, and the first cell that is not
  1. It has a very firm and distinctive border
  2. 2 transmembrane proteins, EphB and EphrinB, keep it that way
    1. EphB is under the control of the Wnt pathway, and cells will produce EphB where Wnt is present
    2. If cells don’t get Wnt, they will express Ephrin B
    3. EphB and Ephrin B cells do not mix
  3. Paneth cells start off the Wnt pathway and make sure the crypt is a good environment for undifferentiated cells
Notch and Wnt signaling maintain and drive diversification
1. Notch is downstream of Wnt, so delta is also downstream of Wnt
  1. If cells are exposed to Wnt, they will produce either notch or delta, depending on their cell type
  2. If a cell is expressing notch and notch is activated, that cell is inhibited from differentiating
  3. pathway
    1. Paneth cells in the crypt are under the influence of Wnt
    2. When a Paneth cell receives Wnt, it produces delta
    3. Next to a Paneth cell is a stem cell, which is also under the influence of Wnt, so it produces notch
    4. Contact-dependent signaling between the stem cell and Paneth cell activates the Notch pathway
    5. When the notch pathway is activated, the notch cell can’t differentiate
    6. The stem cell (a notch cell) can and does still divide
    7. daughter cells get pushed up and aren’t next to Paneth cells anymore
      1. They are not being inhibited from differentiating anymore
    8. daughter cells undergo differentiation pathways
    9. They will undergo lateral inhibition with each other
      1. Some will become notch producers, others will become delta producers
    10. If notch and delta are next to each other, they will bind and activate the notch pathway and be inhibited from differentiating
    11. At this point, they are shoved out of the crypt, so there is no more Wnt, Notch, or Delta
    12. Cells that were notch-producing become absorptive cells, and cells that were delta-producing become secretory cells
Connective tissue fibroblasts
1. Connective tissue cells are related and often interconvertible
  1. Since cells are similar anyway, the differentiation process is easier
  2. fibroblasts, bone cells, cartilage cells, smooth muscle cells, and fat cells
2. Bone marrow stromal cells are fibroblasts that are multipotent, referred to as mesenchymal stem cells (precursor stem cells)
Bone vs. Cartilage
1. cells
  1. cartilage - chrondrocytes
  2. Bone - osteo(cyte, blast, clast)
    1. Osteoclast comes from a different stem cell
2. matrix
  1. cartilage - uniform and highly hydrated for a cushioning role
  2. bone - dense and rigid
3. growth
  1. cartilage - slow chondrocyte division
  2. bone - apposition (forming layers like a tree)
Growth of cartilage
1. doublet cells caused by chondrocyte division are surrounded by a highly hydrated matrix
2. Most of the compressive forces of the body are absorbed by the matrix
Deposition of bone matrix by osteoblasts
1. Osteoblasts are the precursor cells on the top layer of bone
  1. They sit on top of the bone and build new matrix, secreting all three forms of ECM proteins, and are responsible for the calcification of that
  2. Once they are completely surrounded by matrix, they finish differentiating and become osteocytes
2. Little holes in bone are called the lacuna, and inside the lacuna is the cell body
3. Osteocytes are connected to each other through little channels called canaliculi
  1. through the canaliculi, they reach out their cytoplasm and communicate and share nutrients with each other through gap junctions
Bone is remodelled by osteoclasts
1. Osteoclasts degrade the ECM of bone
  1. They are “bone chewers”.
  2. Osteoclasts have 5 nuclei and a ruffled border
    1. The ruffled border is a wavy part of the membrane that secretes acid and proteases
      1. secretes to get rid of calcium phosphate crystals in bone
Remodelling of complex (bone)
1. The cell will need to grow a new blood vessel
2. To grow a new blood vessel in bone, there needs to be a tunnel dug by osteoclasts
3. Osteoblasts will lay new matrix on the sides of the tunnel, and a blood vessel will sprout a new vessel through the tunnel
Blood cell formation - a hierarchical stem cell system
1. Blood contains many different cell types
2. All blood cell types are produced continually throughout the lifetime of the organism and have a limited life span
3. All come from the hematopoietic stem cell
  1. These also give rise to osteoclasts
4. Red blood cells (erythrocytes) are all alike
Leukocytes - white blood cells
1. Granulocytes
  1. neutrophils, eosinophils, basophils
2. monocytes
3. lymphocytes
  1. B cells, T cells, and natural killer cells
Chemical signal molecules recruit white blood cells to damaged tissues
1. white blood cells are usually in the circulatory system or more prominently in the lymphatic system
2. If you get a cut or infection, the white blood cells will squeeze out of the system and crawl to find the pathogen
Identifying hematopoietic stem cells
1. Radiated a mouse with X-ray radiation
2. Radiation killed all of the bone marrow
3. they took bone marrow from a healthy mouse and injected the marrow into the irradiated mouse
4. The healthy mouse cells filled in the bone marrow of the irradiated mouse and reconstituted the entire blood system
  1. All blood cells were then made from donor marrow in the irradiated mouse
5. Which stem cell caused this?
  1. They used FACS to sort the different cell types, then put those into an irradiated mouse one at a time
  2. found that the hematopoietic stem cells reconstituted the whole system and saved the mouse
Commitment is a stepwise process
1. A multipotent hematopoietic stem cell becomes either an osteoclast or a multipotent hematopoietic progenitor, which is a precursor for all other blood cells, which will then become either a myeloid or lymphoid precursor
Factors that regulate hematopoiesis: stem cell niche
1. steel, or stem cell factor (SCF), regulates hematopoiesis
2. It is expressed in bone marrow stroma, helping to form the niche
3. along the migration pathway, it is bound and secreted
  1. If a stem cell divides, 1 daughter will stay attached to the stromal cell and remain stemness, the other loses contact-dependent signaling, so it will either complete a differentiation pathway or die
4. How the population is maintained
  1. If a stromal cell in the bone marrow with the kit ligand (SCF) engages in contact-dependent signaling with the kit receptor on a stem cell, it will keep the stem cell from differentiating
Factors that regulate hematopoiesis: Erythropoietin
1. boosts the production of red blood cells in response to low oxygen conditions or an erythrocyte shortage
  1. The kidney will release erythropoietin
  2. Erythropoietin goes through the bloodstream and stimulates hematopoietic stem cells to become red blood cells
    1. In differentiation, the cell will discard everything that is useless, including the nucleus, mitochondria, and pretty much everything. Red blood cells need hemoglobin
Colony-stimulating factors (CSFs)
1. CSFs promote the growth of colonies of differentiated blood cells
  1. An example is erythropoietin
  2. All steps in the stem cell life can be regulated by a CSF
Stem cell transplants for leukemia
1. Autologous
  1. Take the bone marrow from the patient themselves
    1. have to guess if the mutation is in the cells in the marrow or the transient amplifying cells
2. Allogenic
  1. Take marrow from a donor
    1. unlikely to relapse
    2. Immune system could attack the body
3. Stem cells in the clinic: Adult stem cells used in bone marrow transplants for leukemia
  1. Cells from the bone marrow of the donor are frozen down, and the patient is treated with high-dose chemotherapy to wipe out their bone marrow
  2. donor cells are then transplanted into the patient
Additional example - skeletal muscle
1. Skeletal muscle cells have multiple nuclei because they form by the fusion of multiple cells
2. A satellite cell is the stem cell for a muscle fiber
  1. If a muscle fiber is damaged, the satellite cell can come off and proliferate, and then fuse back into the muscle fiber
regeneration and repair
1. Many tissues of the body are self-renewing and self-repairing as we have seen
2. largely thanks to stem cells
3. Regeneration can also occur in the case of the liver
4. Some animals can do much better
Planarian worms can regenerate a whole new body
1. is radiate it will die, but if it is injected with a healthy neoblast (embryonic planarian cell), it will regenerate all parts of its body
Newts can regenerate whole limbs
1. If you amputate a limb, it will grow back
2. bud where the limb was will reform the 3 embryonic germ layers and form the limb as it did in development
Some tissue renewal does not depend on stem cells
1. A few types of fully differentiated cells can divide
2. beta cells of the pancreas
  1. attacked by the immune system in Type 1 diabetes
  2. They are replenished by simple duplication
Some tissues are not renewable
1. auditory epithelium
2. retinal epithelium
3. some nerve cells in the brain
Cell reprogramming
1. transplanted cells remain largely faithful to their origins (memory)
2. nuclei can be programmed
3. Embryonic stem (ES) cells can generate any part of the body
4. Fibroblasts can be programmed to be pluripotent
  1. Pluripotent cells can become anything
Nuclei can be reprogrammed by transplantation into foreign cytoplasm
1. Transcription factors are in the cytoplasm and control what genes are expressed in the nucleus
2. Dolly the sheep was the first fully cloned animal
  1. She was cloned by taking a cell from an adult sheep and a fertilized egg, and then they sucked the nucleus out of both
  2. They put the nucleus of the adult sheep into the cytoplasm of the fertilized egg
  3. The fertilized egg cytoplasm stripped all of the differentiation from the adult cell and turned it into a totipotent cell
3. Dolly died young of old age because her telomeres were already shortened because she was born an adult
Embryonic stem cells
1. come from the inner cell mass of the fertilized egg
2. pluripotent (cannot form a placenta)
  1. took a fertilized egg and collected cells of the inner cell mass, and grew them on a plate
  2. took a second embryo and injected some of the embryonic stem cells from the first embryo that were fluorescently labelled pink, and the cells merged right in
  3. resulted in a mouse that had random tissues that were pink. They ran the experiment multiple times and got mice with all kinds of pink tissues
  4. All cells eventually came from embryonic stem cells
  5. This made what is known as a chimeric mouse
3. ES cells - fertilize an egg cell in vitro, harvest the inner cell mass, causing the destruction of embryo
Adult fibroblasts can be programmed to create induced pluripotent stem (iPS) cells
1. made by adding 4 transcription factors
2. positive ways
  1. They divide indefinitely in culture (immortal)
  2. perfect chimeras
  3. can be derived from adult human cells
  4. self-sustaining
3. negatives
  1. inefficient and slow
4 essential factors to create iPS cells
1. OCT4, Sox2, Klf4, Myc
  1. these genes:
    1. downregulated differentiation genes
    2. caused the upregulation of embryonic stem cell genes
    3. upregulated cell proliferation
    4. caused loosening of chromatin structure
Major events during iPS reprogramming
1. One of the cells, after being activated with OSKM, has to lose its differentiating specific genes
  1. Many cells just do step one and then do not continue
2. The cell then needs to gain embryonic genes
3. The cell needs to turn in its own OCT4 gene in its genome
  1. If it fails to turn on its own OCT4, it does not progress all the way
4. If all three steps are completed, you will have a successful iPS cell
In culture, one can differentiate ES and iPS cells into certain cell types
1. The cells can become almost everything. How do we make sure they become the cells we want them to
  1. Stem cells are influenced by their niche; the environment tells them what they should be
    1. If they are treated with retinoic acid, they will become neurons
    2. If treated with retinoic acid, then insulin and thyroid hormones, they will become fat cells
  2. If the cell is confused, it can become a teratoma mass
ES cells can form whole, small organs
1. If you encourage ES cells to become eye cells, they will become the cell types in the retina and start to form the tissue structure
2. Whether pluripotent cells are embryonic or natural, they have an idea of what their tissue architecture should be
Uses of iPS cells for patient care and drug discovery
1. Take skin cells, treat them with OSKM, turning them into iPS cells, then fix the mutation in plates, differentiate the cells into healthy kidney cells, and put them back
2. Take skin cells, turn them into iPS cells, turn them into kidney cells, and treat with drugs to see what will work for the patient
  1. Since iPS cells can become any cell type, you could make some of the cells into other tissues and test for side effects
ES cells vs. iPS cells
1. iPS
  1. positives
    1. avoids tissue rejection by the immune system
    2. allows for a study of diseases ranging from many cell types
    3. avoids moral debate and regulation
  2. negative
    1. The product is inefficient
    2. cell memory
    3. variation because we make them
  3. both are immortal