HMB204 Midterm Notes
- Lecture 1: What are Stem Cells?
Stem cells: can develop multiple cell types (development, during life)
Somatic stem cells: undifferentiated, multipotent, responsible for maintaining-repairing tissues.
Embryonic stem cells: NOT multipotent
SCs can bypass normal limits on cell division to replenish other cell types.
SC: 1) Self-renewal 2) Differentiation
Self-renewal
Symmetric: A = Two undifferentiated cells (A + A)
Asymmetric: A (intestinal)= One undifferentiated (A-intestinal) + one committed (B-goblet cell)
Depletion (not maintain cell pool): A = B + B
1) Developmental potency:
Toti: can produce an entire viable organism including extra-embryonic cells = zygote, morula, post-fertilization, 2-cell stage, 4-
cell stage, 8 cell-stage
Pluri: derivatives from each 3 primary embryonic lineages (germ layer) = blastocyst, germ cells (endoderm, mesoderm,
ectoderm), octodermal stem cell, inner cell mass, epiblast
Multi: multiple cell types within a single germ layer (tissue-specific function) = placenta, HSC, NSC, mesenchymal NSC,
trophectoderm, primitive endoderm (hypoblast), extraembryonic mesoderm, yolk sac
Uni: only a single cell type (specialized cell type) = blood cell, cells of the nervous system, connective tissue, bone, cartilage, fat
Differentiation has many steps: morphology, molecular profile, and function.
Progenitor: undifferentiated cell with a limited ability to self-renew
Challenges in stem cell therapies: right stimuli for induction, purification of correct cell type, expand it to a therapeutically relevant
dose, ensure the survival and functionality.
Developmental Hourglass Theory: middle stages exhibit greater divergence and specialization.
BM contained single cells that could give rise to colonies in the spleen. Given enough time, the donor cells can repopulate the BM
and protect the lethally irradiated host mouse (which constitutes the entire organ blood system)
Potential SCs can be located within the niche using bromodeoxyuridine.
Short-term exposure (pulse) of living tissues to Brdu will label cells actively undergoing cell division for detection later (chase) with
anti-Brdu antibodies.
Gastrulation results in 3 embryonic lineages: endoderm (in), mesoderm (mid), ectoderm (out) = each layer contributes to specific
tissues in the body
Teratomas (benign) & teratomas (malignant) are germ cell tumors (ovarian or testicular) composed of tissues from all 3 germ layers.
Individual pluripotent cells called embryonal carcinoma (EC) can divide indefinitely and differentiate into teratomas when ejected
into mice.
EC cells were used to create the first pluripotency assays and to improve cell culture conditions for embryonic cells by adding a layer
of mouse embryonic fibroblasts (MEFs) which supports the growth and maintenance of the undifferentiated state of mouse
embryonic stem cells.
Chimera assay with GFP-labelled donor ESCs
Chimera: a single organism composed of genetically distinct cells
Discovery of induced pluripotent stem cells (iPSCs)
Created by engineering or “reprogramming” (adult) tissue-specific cells into cells that behave like ESCs.
Yamanaka factors: Oct 4, SOX 2, KLF 4, C-Myc*
Assay stringency (robustness) increases from 1 to 4.
1) Undifferentiated under certain conditions (least informative) = colony or cell morphology, marker abundance (nuclear, cell
surface)
2) Self-renewal = single-cell cloning (clonogenicity), serial-transplantation
3) Developmental potency = spontaneous or directed differentiation, tissue integration
Observation, if candidate stem cell differential potential, is stronger evidence of an undifferentiated phenotype than
morphology.
A teratoma produced by an injection of embryonic stem cells or embryonal carcinoma cells must contain these lineages:
endoderm, ectoderm, and mesoderm.
A balance between self-renewal and differentiation in vivo (in the body) is supported by interactions with a specific anatomical
location called the niche.
This study source was downloaded by 100000899194722 from CourseHero.com on 05-29-2025 09:26:39 GMT -05:00
https://www.coursehero.com/file/246864313/HMB204-Midterm-Notesdocx/
, - Lecture 2: Tissue Microenvironments and Organoid Systems
The embryonic niche rapidly changes throughout development.
Trophectoderm, epiblast, extra-embryonic endoderm: separate tissues but actively communicating (multi-way: direct, soluble
factors)
ECM: biophysical, mechanical, and biochemical properties specific to each tissue
Blood vessels (provide nutrients and carry away wastes) / supportive cells / neural inputs (both in adult and fetal tissues) /
acellular components (proteins, polysaccharides, lipids)
Bone, connective, cartilage: stiff, elastic ECM vs CNS: less stiff, elastic
Intestine: villus (barrier), lamina propria (dense ECM, support), Transit amplifying zone (differentiating progenitor cells), crypt base
(niche for intestinal stem cells)
Enterocyte cells (barrier for absorption), enteroendocrine cells (hormone-producing that influence nearby cells), goblet cells
(secrete gel-forming proteins for mucous layer), Paneth cells (secrete protein factors that control ISCs), Intestinal stem cells
(active {aISC}: always undergoing self-renewal, found at crypt/reserve type {+4 – relative to crypt base - rISC}: dormant-reserve
stem cell, only activated when exposed to a chemical or something that Is a risk to damaging active type, can directly replace
them)
LGR5 (marker of intestinal stem cells) reporter gene with in-frame insertion of B-galactosidase (encoded by lacZ) the enzyme that
produces a blue metabolic product.
Intestinal stem cells (tagged by YFP) are interspersed within supportive Paneth cells in the crypt base (Lysozyme: a marker of Paneth
cells)
Lineage-tracing experiments: better than using a reporter gene, as it can be turned off and you can lose track of stem cells.
The label must be distributed to daughter cells over a few or many cells (e.g. BrdU)
When a SC is labeled, all its progeny will also carry the label.
Low cell density labeling at the start is important for a good lineage-tracing experiment.
LGR5 supports the Wnt signaling pathway by blocking the endocytosis and degradation of the Frizzled/LRP receptor complex.
In the presence of RSPO, Frizzled/LRP remains at the cell membrane and disrupts the beta-catenin destruction complex.
When both LGR and RSPO ligands are high, B-catenin can translocate to the nucleus, and the beta-catenin signaling pathway is
promoted.
Extrinsic signaling factors in the intestinal SC niche.
BMP: secreted by peri cryptal fibroblasts that are found in the TA zone, promotes differentiation if intestinal SC
TA cell: transit amplifying cells that are no longer capable of prolonged self-renewal.
Myofibroblast: secretes Wnt towards the bottom of crypt => activates beta-catenin signaling
Closer crypt base: BMP decrease / BMP Antagonist (Noggin)increase
Up through the TA zone and ultimately into villus: Wnt signaling.
The base of the crypt: Wnt signaling increase
Undifferentiated self-renewing intestinal stem cell: Wnt sig inc / BMP sig dec
Mesenchymal components of lamina propria: physically support villus and crypt architecture.
Compartmentalization of villi and crypts:
Properly space villi and crypts for increased intestinal surface area, store a large population of transit-amplifying cells for
continuous cell turnover and protect ISCs in the crypt base from pro-differentiation signals.
Culture types: organ, explant, slice, dissociated.
In vitro cell growth conditions: culture medium, essential nutrients/supplements, growth factors, temperature, gasses, antibiotics, pH
In vitro cell growth systems
Adherent culture (anchorage-dependent, monolayer): some cells grow well on the plastic of culture dishes/flasks, while other
cells require an ECM component to grow (collagen, fibronectin, laminin) – cells stuck to the bottom of the culture dish,
preferred for cellular differentiation.
Non-adherent culture (anchorage-independent, suspension): cells are free-floating in the culture medium.
Holoclar is a graft or corneal epithelial cells grown on a fibrin scaffold from one type of eye stem cell (limb stem cells) for patients
with eye burns.
Scaffold: a 3D environment that promotes cell attachment, differentiation, and function
Hydrogels (polymers) are highly absorbent. Interconnected networks of polymer chains are often used as 3D scaffolds for tissue
engineering.
Popular naturally occurring (Matrigel) scaffolds are basal lamina extracts.
2D cell culture: soluble gradients absent, forced apical-basal polarity, continuous layer of matrix, high stiffness, adhesion
restricted to one plane, unconstrained spreading, and migration in one plane.
3D cell culture: soluble gradients present, no polarity, discrete matrix fibrils, variable stiffness, adhesion in all three dimensions,
spreading and migration hindered.
Increasing stiffness and elastic modulus (Pa) from 1 to 3
1) Epithelia, blood, CNS, Matrigel (hydrogel properties) / 2) connective tissues / 3) Bone, skin (cultured on plastics, stiff)
3D liquid culture systems: static suspension, hanging drop, spinner, rotation, microfluidic, gel embedding.
Bioreactor: a manufactured instrument intended to biologically support cells/tissues
Limitations of existing model systems for human biology
Advantages: Animal models (closest to recapitulating body functions and cellular interactions in human tissues), 2D cell
monolayers (simple and efficient, low cost, high reproducibility), 3D cell aggregates (better mimic cell-cell and cell-matrix
interactions)
Disadvantages: Animal models (differences in human and animal biology, limited usability in imaging and high-throughput
studies, high cost of maintenance), 2D cell monolayers (cells lose their phenotype, lack cell-cell & cell-matrix interactions, fail to
This study source was downloaded by 100000899194722 from CourseHero.com on 05-29-2025 09:26:39 GMT -05:00
https://www.coursehero.com/file/246864313/HMB204-Midterm-Notesdocx/
- Lecture 1: What are Stem Cells?
Stem cells: can develop multiple cell types (development, during life)
Somatic stem cells: undifferentiated, multipotent, responsible for maintaining-repairing tissues.
Embryonic stem cells: NOT multipotent
SCs can bypass normal limits on cell division to replenish other cell types.
SC: 1) Self-renewal 2) Differentiation
Self-renewal
Symmetric: A = Two undifferentiated cells (A + A)
Asymmetric: A (intestinal)= One undifferentiated (A-intestinal) + one committed (B-goblet cell)
Depletion (not maintain cell pool): A = B + B
1) Developmental potency:
Toti: can produce an entire viable organism including extra-embryonic cells = zygote, morula, post-fertilization, 2-cell stage, 4-
cell stage, 8 cell-stage
Pluri: derivatives from each 3 primary embryonic lineages (germ layer) = blastocyst, germ cells (endoderm, mesoderm,
ectoderm), octodermal stem cell, inner cell mass, epiblast
Multi: multiple cell types within a single germ layer (tissue-specific function) = placenta, HSC, NSC, mesenchymal NSC,
trophectoderm, primitive endoderm (hypoblast), extraembryonic mesoderm, yolk sac
Uni: only a single cell type (specialized cell type) = blood cell, cells of the nervous system, connective tissue, bone, cartilage, fat
Differentiation has many steps: morphology, molecular profile, and function.
Progenitor: undifferentiated cell with a limited ability to self-renew
Challenges in stem cell therapies: right stimuli for induction, purification of correct cell type, expand it to a therapeutically relevant
dose, ensure the survival and functionality.
Developmental Hourglass Theory: middle stages exhibit greater divergence and specialization.
BM contained single cells that could give rise to colonies in the spleen. Given enough time, the donor cells can repopulate the BM
and protect the lethally irradiated host mouse (which constitutes the entire organ blood system)
Potential SCs can be located within the niche using bromodeoxyuridine.
Short-term exposure (pulse) of living tissues to Brdu will label cells actively undergoing cell division for detection later (chase) with
anti-Brdu antibodies.
Gastrulation results in 3 embryonic lineages: endoderm (in), mesoderm (mid), ectoderm (out) = each layer contributes to specific
tissues in the body
Teratomas (benign) & teratomas (malignant) are germ cell tumors (ovarian or testicular) composed of tissues from all 3 germ layers.
Individual pluripotent cells called embryonal carcinoma (EC) can divide indefinitely and differentiate into teratomas when ejected
into mice.
EC cells were used to create the first pluripotency assays and to improve cell culture conditions for embryonic cells by adding a layer
of mouse embryonic fibroblasts (MEFs) which supports the growth and maintenance of the undifferentiated state of mouse
embryonic stem cells.
Chimera assay with GFP-labelled donor ESCs
Chimera: a single organism composed of genetically distinct cells
Discovery of induced pluripotent stem cells (iPSCs)
Created by engineering or “reprogramming” (adult) tissue-specific cells into cells that behave like ESCs.
Yamanaka factors: Oct 4, SOX 2, KLF 4, C-Myc*
Assay stringency (robustness) increases from 1 to 4.
1) Undifferentiated under certain conditions (least informative) = colony or cell morphology, marker abundance (nuclear, cell
surface)
2) Self-renewal = single-cell cloning (clonogenicity), serial-transplantation
3) Developmental potency = spontaneous or directed differentiation, tissue integration
Observation, if candidate stem cell differential potential, is stronger evidence of an undifferentiated phenotype than
morphology.
A teratoma produced by an injection of embryonic stem cells or embryonal carcinoma cells must contain these lineages:
endoderm, ectoderm, and mesoderm.
A balance between self-renewal and differentiation in vivo (in the body) is supported by interactions with a specific anatomical
location called the niche.
This study source was downloaded by 100000899194722 from CourseHero.com on 05-29-2025 09:26:39 GMT -05:00
https://www.coursehero.com/file/246864313/HMB204-Midterm-Notesdocx/
, - Lecture 2: Tissue Microenvironments and Organoid Systems
The embryonic niche rapidly changes throughout development.
Trophectoderm, epiblast, extra-embryonic endoderm: separate tissues but actively communicating (multi-way: direct, soluble
factors)
ECM: biophysical, mechanical, and biochemical properties specific to each tissue
Blood vessels (provide nutrients and carry away wastes) / supportive cells / neural inputs (both in adult and fetal tissues) /
acellular components (proteins, polysaccharides, lipids)
Bone, connective, cartilage: stiff, elastic ECM vs CNS: less stiff, elastic
Intestine: villus (barrier), lamina propria (dense ECM, support), Transit amplifying zone (differentiating progenitor cells), crypt base
(niche for intestinal stem cells)
Enterocyte cells (barrier for absorption), enteroendocrine cells (hormone-producing that influence nearby cells), goblet cells
(secrete gel-forming proteins for mucous layer), Paneth cells (secrete protein factors that control ISCs), Intestinal stem cells
(active {aISC}: always undergoing self-renewal, found at crypt/reserve type {+4 – relative to crypt base - rISC}: dormant-reserve
stem cell, only activated when exposed to a chemical or something that Is a risk to damaging active type, can directly replace
them)
LGR5 (marker of intestinal stem cells) reporter gene with in-frame insertion of B-galactosidase (encoded by lacZ) the enzyme that
produces a blue metabolic product.
Intestinal stem cells (tagged by YFP) are interspersed within supportive Paneth cells in the crypt base (Lysozyme: a marker of Paneth
cells)
Lineage-tracing experiments: better than using a reporter gene, as it can be turned off and you can lose track of stem cells.
The label must be distributed to daughter cells over a few or many cells (e.g. BrdU)
When a SC is labeled, all its progeny will also carry the label.
Low cell density labeling at the start is important for a good lineage-tracing experiment.
LGR5 supports the Wnt signaling pathway by blocking the endocytosis and degradation of the Frizzled/LRP receptor complex.
In the presence of RSPO, Frizzled/LRP remains at the cell membrane and disrupts the beta-catenin destruction complex.
When both LGR and RSPO ligands are high, B-catenin can translocate to the nucleus, and the beta-catenin signaling pathway is
promoted.
Extrinsic signaling factors in the intestinal SC niche.
BMP: secreted by peri cryptal fibroblasts that are found in the TA zone, promotes differentiation if intestinal SC
TA cell: transit amplifying cells that are no longer capable of prolonged self-renewal.
Myofibroblast: secretes Wnt towards the bottom of crypt => activates beta-catenin signaling
Closer crypt base: BMP decrease / BMP Antagonist (Noggin)increase
Up through the TA zone and ultimately into villus: Wnt signaling.
The base of the crypt: Wnt signaling increase
Undifferentiated self-renewing intestinal stem cell: Wnt sig inc / BMP sig dec
Mesenchymal components of lamina propria: physically support villus and crypt architecture.
Compartmentalization of villi and crypts:
Properly space villi and crypts for increased intestinal surface area, store a large population of transit-amplifying cells for
continuous cell turnover and protect ISCs in the crypt base from pro-differentiation signals.
Culture types: organ, explant, slice, dissociated.
In vitro cell growth conditions: culture medium, essential nutrients/supplements, growth factors, temperature, gasses, antibiotics, pH
In vitro cell growth systems
Adherent culture (anchorage-dependent, monolayer): some cells grow well on the plastic of culture dishes/flasks, while other
cells require an ECM component to grow (collagen, fibronectin, laminin) – cells stuck to the bottom of the culture dish,
preferred for cellular differentiation.
Non-adherent culture (anchorage-independent, suspension): cells are free-floating in the culture medium.
Holoclar is a graft or corneal epithelial cells grown on a fibrin scaffold from one type of eye stem cell (limb stem cells) for patients
with eye burns.
Scaffold: a 3D environment that promotes cell attachment, differentiation, and function
Hydrogels (polymers) are highly absorbent. Interconnected networks of polymer chains are often used as 3D scaffolds for tissue
engineering.
Popular naturally occurring (Matrigel) scaffolds are basal lamina extracts.
2D cell culture: soluble gradients absent, forced apical-basal polarity, continuous layer of matrix, high stiffness, adhesion
restricted to one plane, unconstrained spreading, and migration in one plane.
3D cell culture: soluble gradients present, no polarity, discrete matrix fibrils, variable stiffness, adhesion in all three dimensions,
spreading and migration hindered.
Increasing stiffness and elastic modulus (Pa) from 1 to 3
1) Epithelia, blood, CNS, Matrigel (hydrogel properties) / 2) connective tissues / 3) Bone, skin (cultured on plastics, stiff)
3D liquid culture systems: static suspension, hanging drop, spinner, rotation, microfluidic, gel embedding.
Bioreactor: a manufactured instrument intended to biologically support cells/tissues
Limitations of existing model systems for human biology
Advantages: Animal models (closest to recapitulating body functions and cellular interactions in human tissues), 2D cell
monolayers (simple and efficient, low cost, high reproducibility), 3D cell aggregates (better mimic cell-cell and cell-matrix
interactions)
Disadvantages: Animal models (differences in human and animal biology, limited usability in imaging and high-throughput
studies, high cost of maintenance), 2D cell monolayers (cells lose their phenotype, lack cell-cell & cell-matrix interactions, fail to
This study source was downloaded by 100000899194722 from CourseHero.com on 05-29-2025 09:26:39 GMT -05:00
https://www.coursehero.com/file/246864313/HMB204-Midterm-Notesdocx/
