Summary hematopoietic stem cells and disease
2nd year Bachelor course Biology 2021
Lecture 1: Stem cells and hematopoiesis Schuringa May 6th
This course gives an overview of the development of mature components of the blood (leukocytes like
granulocytes, macrophages, dendritic cells, NK cells, T cells and B cells, erythrocytes, platelets) from
hematopoietic stem cells (HSCs), and everything that can go wrong within this process.
Hematopoietic stem cells are found in the bone marrow.
These cells can self-renew and differentiate into mature
blood cells (pluripotency). It starts as LT-HSCs (long term
HSC), then goes to ST-HSC (short term HSC), which can
differentiate into LMPP (lymphoid myeloid progenitor) that
can mature into CLP (forms lymphocytes, NK cells and DCs),
GMP (forms macrophages and granulocytes) or CMP (forms
platelets and erythrocytes). ST-HSC can also differentiate to
MkEP, which can also form CMP (and therefore platelets and
erythrocytes).
Using biomarkers in flow cytometry gives an idea about the amounts of stem cells. Another way is
transplantation into mice. Stem cells are relatively quiescent. Older stem cells reproduce more but are of
less quality.
Division of a stem cell leads to one self-renewed cell and one differentiated cell: asymmetric division.
Proteins, RNA, metabolites, and DNA (immortal strand ends up in self-renewed cell, so no mutations here)
are distributed asymmetrically. Mutations are both wanted (evolution) and unwanted (cancer).
• How is this asymmetry achieved? à stochastic, cell intrinsic,
extrinsic/niche dependent. Example: stem cell bound to
microenvironment with integrins. This sends signals to the
closest centrosome. Based on the position of the centrosome,
the cell self-renews or differentiates or both. Too much HSC
production causes leukemia, too little HSC production causes
exhaustion (ageing).
o Also in Drosophila: germ stem cells (GSCs) are bound to a
niche (Hub). A dividing cell has centromere close to the Hub and one opposed of it. GSCs
with misoriented centrosomes occur with age and are delayed in cell cycle.
Embryonic stem cells (ESCs) are obtained from the inner mass blastocyst and are totipotent. They have
unlimited self-renewal but come with plenty ethical and political issues (might be solved with iPS).
Adult stem cells (like HSCs) are multipotent and can form cells into certain directions (blood). They have
limited self-renewal but less ethical and political issues.
• A lot of cells are dying and being produced: tissue homeostasis is maintained by adult stem cells
(rather than embryonic stem cells.
Embryonic stem cell generation
- ESCs can be derived from the blastocyst using a pipet and putting them in a dish (e.g. patients who have
completed IVF treatment, or fresh embryos unsuitable for clinical use. à problems: ethics, non-self.
- ESCs can also be obtained via nuclear transfer: putting nucleus of a patient into enucleated egg,
developing an embryo and isolating the inner cell mass (like sheep Dolly) à 3 mothers and no father, and
patient specific so no rejection problems when using the stem cells for therapeutic purposes.
,- ESC like cells can be formed through production of induced pluripotent stem cells (iPS): reprogramming
differentiated cells using 4 factors: SOX2, Nanog, OCT3/4 and Klf4. à very useful, can be used for tissue
replacement, gene therapy (by correcting the mutation in the cells themselves, was also done with
mutated CCR5 gene to prevent HIV), cell-based models of human disease and drug discovery.
Blood production from ESC/iPS cells
It is not easy to produce blood from these stem cells. Embryoid body formation using hanging drops
(mimic embryogenesis) or coculture conditions are standard protocols to produce HSCs from stem cells.
Generation lacks definitive engraftments done by overexpression of HOXP4 or STAT5 (blood-specific TFs).
- It is hard because normally, the stem cells are moved between different stromal systems during
embryogenesis before going to the bone marrow: first in AGM, then to the liver, then to the bone
marrow. This ‘education’ must also be incorporated in the production of HSCs to produce definitive
hematopoietic stem cells.
Hematopoiesis
For hematopoiesis, HSCs, growth factors and microenvironment are needed. This microenvironment
consists of blood vessels, bone, and other cells like MSCs. The centrosome closest to the bone niche
becomes a new stem cell, and the one closest to the vasculature differentiates (so high self-renewal close
to the bone). Adult somatic cells are hard to grow because of different niches. Bone marrow niche has low
oxygen (important for metabolism), low ROS, high HIFa and high calcium. In the vascular niche this is the
opposite. Id the cell is activated, it goes into the vasculature to be transported to other parts of the body.
The cells are kept at the bone-marrow niche surface (and kept quiescent) by:
• Physical linkage to niche (through SNO cells): cadherins, integrins, binding to ECM
• Growth factor/cytokine mediated signaling for quiescence
• Gene expression based on certain transcription factors
• Cell intrinsic mechanisms like epigenetics and polycomb mediated repression
Different differentiation programs: HSCs (large nucleus, small cytoplasm) can go into lymphoid
differentiation, myeloid differentiation, megakaryocyte differentiation, erythroid differentiation.
• Erythrocyte (red blood cells) are very flexible. During differentiation, the nucleus is kicked out. They
have a lifespan of about 3 months. Erythrocytes contain hemoglobin (2 alpha chains, 2 beta chains,
but in fetal hemoglobin, the beta chains are absent and gamma chains are present) to transport
oxygen. The expression of the different chains is regulated by the LCR region. Increased
hemoglobin leads to polycythemia, decreased hemoglobin leads to anemia. Hematocrit is the
percentage of the concentration of the red blood cells in the blood (females about 40%, males
about 47%).
o Glutamate to valine mutation in the beta chain of hemoglobin leads to sickle cell anemia.
• Megakaryopoiesis is the formation of platelets (thrombocytes). These are small, irregularly shaped
cell fragments that do not have a nucleus. Cells become bigger but do not divide: they explode and
platelets are formed. Platelets can form a clot to heal a wound.
• White blood cells fight infections. Increased white blood cells lead to leukocytosis, decreased white
blood cells lead to leukopenia. There are innate immune cells (neutrophils, eosinophils, basophils,
macrophages) and adaptive immune cells (B cells and T cells).
o Neutrophils are the primary defense against bacterial infection: they can phagocytose and
digest microorganisms. They are granulocytes with neutral staining and have nuclei with 3
to 5 lobes. The cytoplasm contains granules. Increased neutrophils leads to neutrophilia
(caused by pyogenic bacterial infection), decreased neutrophils leads to neutropenia,
caused by B12 & folate deficiency or blood cancer.
o Eosinophils are important for the response in allergies and parasitic infections. They are
granular leukocytes with a nucleus with two lobes. Cytoplasm contains granules that can be
stained by eosin. Increased: eosinophilia (caused by asthma, hypersensitivity reaction) and
decreased: eosinopenia (caused by alcohol intoxication, overproduction of steroids).
, o Basophils play a role in parasitic infections and allergies. They contain large cytoplasmic
granules that obscure the cell nucleus. When unstained, the nucleus is visible and has two
lobes. Increased: basophilia (caused by CML) and decreased: basopenia (caused by
hyperthyroidism, pregnancy and irradiation).
o Monocytes/macrophages can phagocytose dead or damaged cells (without granulocytes):
monocytes move into tissues and become macrophages. They have one kidney shaped
nucleus and are slightly larger than a lymphocyte. Increased: monocytosis (caused by
tuberculosis, malaria and typhoid & Kala azar). Decreased: monocytopenia (caused by
aplastic anemia, lymphocytic anemia and glucocorticoids)
§ Phagocytosis is done by
recognition of certain
bacteria or damage (also
chemotaxis involved).
Actin is rearranged to
allow engulfment: put
into phagosome which
fuses with lysosome and
breaks down the
bacteria)
o Lymphocytes are the primary
source of viral defense and
antibodies. They are
mononuclear and small, do not
have a lot of cytoplasm.
Increased: lymphcytosis (caused by viral infection, leukemia, bone marrow, cancer) and
decreased: lymphocytopenia (caused by acute stages of infection, excess irradiation)
B cells develop with recombination in the genes to form specific antibodies, which occurs in the bone
marrow. B cells that recognize self-antigens are removed. Specialization happens in secondary lymphoid
tissue (lymph nodes, spleen, Peyer’s patches). B cells then circulate the lymph and blood. At sight of
pathogens, they come back to the secondary lymphoid tissue to proliferate. Plasma cells (secrete
antibodies) and memory B cells are produced. In hematopoietic stem cells, VDJ loci cannot be accessed.
When becoming a B cell, PAX5 can bind the promoter regions to open the chromatin and allow RAGS to
cause rearrangements (first D-J, then V).
- The different B cell tumors reflect different developmental stages of B cells.
T cells can by either helper (CD4), regulatory, or cytotoxic (CD8). TCR genes are rearranged to cause the
different TCRs to be present on the T cells and to recognize different antigens presented on MHC. Cells
that won’t bind MHC or cells that bind self-antigens are killed.
- Also in this case: the different T cell tumors reflect different developmental stages of T cells.
Innate vs adaptive immunity:
• Innate is quick, adaptive is slow
• Innate is non-specific, adaptive is specific
• Innate consists of macrophages, neutrophils, NK cells, DCs, basophils eosinophils, adaptive consists
of B cells and T cells
• Innate secretes antimicrobial peptides and proteins, adaptive secretes antibodies
• Innate is based on self vs non self-discrimination, adaptive is less good at distinguishing this
• Innate has limited memory (priming, tolerance, epigenetics?), adaptive has lots of memory
• Innate diversity is limited (standard receptors), adaptive is highly diverse (customized in genetic
recombination)
2nd year Bachelor course Biology 2021
Lecture 1: Stem cells and hematopoiesis Schuringa May 6th
This course gives an overview of the development of mature components of the blood (leukocytes like
granulocytes, macrophages, dendritic cells, NK cells, T cells and B cells, erythrocytes, platelets) from
hematopoietic stem cells (HSCs), and everything that can go wrong within this process.
Hematopoietic stem cells are found in the bone marrow.
These cells can self-renew and differentiate into mature
blood cells (pluripotency). It starts as LT-HSCs (long term
HSC), then goes to ST-HSC (short term HSC), which can
differentiate into LMPP (lymphoid myeloid progenitor) that
can mature into CLP (forms lymphocytes, NK cells and DCs),
GMP (forms macrophages and granulocytes) or CMP (forms
platelets and erythrocytes). ST-HSC can also differentiate to
MkEP, which can also form CMP (and therefore platelets and
erythrocytes).
Using biomarkers in flow cytometry gives an idea about the amounts of stem cells. Another way is
transplantation into mice. Stem cells are relatively quiescent. Older stem cells reproduce more but are of
less quality.
Division of a stem cell leads to one self-renewed cell and one differentiated cell: asymmetric division.
Proteins, RNA, metabolites, and DNA (immortal strand ends up in self-renewed cell, so no mutations here)
are distributed asymmetrically. Mutations are both wanted (evolution) and unwanted (cancer).
• How is this asymmetry achieved? à stochastic, cell intrinsic,
extrinsic/niche dependent. Example: stem cell bound to
microenvironment with integrins. This sends signals to the
closest centrosome. Based on the position of the centrosome,
the cell self-renews or differentiates or both. Too much HSC
production causes leukemia, too little HSC production causes
exhaustion (ageing).
o Also in Drosophila: germ stem cells (GSCs) are bound to a
niche (Hub). A dividing cell has centromere close to the Hub and one opposed of it. GSCs
with misoriented centrosomes occur with age and are delayed in cell cycle.
Embryonic stem cells (ESCs) are obtained from the inner mass blastocyst and are totipotent. They have
unlimited self-renewal but come with plenty ethical and political issues (might be solved with iPS).
Adult stem cells (like HSCs) are multipotent and can form cells into certain directions (blood). They have
limited self-renewal but less ethical and political issues.
• A lot of cells are dying and being produced: tissue homeostasis is maintained by adult stem cells
(rather than embryonic stem cells.
Embryonic stem cell generation
- ESCs can be derived from the blastocyst using a pipet and putting them in a dish (e.g. patients who have
completed IVF treatment, or fresh embryos unsuitable for clinical use. à problems: ethics, non-self.
- ESCs can also be obtained via nuclear transfer: putting nucleus of a patient into enucleated egg,
developing an embryo and isolating the inner cell mass (like sheep Dolly) à 3 mothers and no father, and
patient specific so no rejection problems when using the stem cells for therapeutic purposes.
,- ESC like cells can be formed through production of induced pluripotent stem cells (iPS): reprogramming
differentiated cells using 4 factors: SOX2, Nanog, OCT3/4 and Klf4. à very useful, can be used for tissue
replacement, gene therapy (by correcting the mutation in the cells themselves, was also done with
mutated CCR5 gene to prevent HIV), cell-based models of human disease and drug discovery.
Blood production from ESC/iPS cells
It is not easy to produce blood from these stem cells. Embryoid body formation using hanging drops
(mimic embryogenesis) or coculture conditions are standard protocols to produce HSCs from stem cells.
Generation lacks definitive engraftments done by overexpression of HOXP4 or STAT5 (blood-specific TFs).
- It is hard because normally, the stem cells are moved between different stromal systems during
embryogenesis before going to the bone marrow: first in AGM, then to the liver, then to the bone
marrow. This ‘education’ must also be incorporated in the production of HSCs to produce definitive
hematopoietic stem cells.
Hematopoiesis
For hematopoiesis, HSCs, growth factors and microenvironment are needed. This microenvironment
consists of blood vessels, bone, and other cells like MSCs. The centrosome closest to the bone niche
becomes a new stem cell, and the one closest to the vasculature differentiates (so high self-renewal close
to the bone). Adult somatic cells are hard to grow because of different niches. Bone marrow niche has low
oxygen (important for metabolism), low ROS, high HIFa and high calcium. In the vascular niche this is the
opposite. Id the cell is activated, it goes into the vasculature to be transported to other parts of the body.
The cells are kept at the bone-marrow niche surface (and kept quiescent) by:
• Physical linkage to niche (through SNO cells): cadherins, integrins, binding to ECM
• Growth factor/cytokine mediated signaling for quiescence
• Gene expression based on certain transcription factors
• Cell intrinsic mechanisms like epigenetics and polycomb mediated repression
Different differentiation programs: HSCs (large nucleus, small cytoplasm) can go into lymphoid
differentiation, myeloid differentiation, megakaryocyte differentiation, erythroid differentiation.
• Erythrocyte (red blood cells) are very flexible. During differentiation, the nucleus is kicked out. They
have a lifespan of about 3 months. Erythrocytes contain hemoglobin (2 alpha chains, 2 beta chains,
but in fetal hemoglobin, the beta chains are absent and gamma chains are present) to transport
oxygen. The expression of the different chains is regulated by the LCR region. Increased
hemoglobin leads to polycythemia, decreased hemoglobin leads to anemia. Hematocrit is the
percentage of the concentration of the red blood cells in the blood (females about 40%, males
about 47%).
o Glutamate to valine mutation in the beta chain of hemoglobin leads to sickle cell anemia.
• Megakaryopoiesis is the formation of platelets (thrombocytes). These are small, irregularly shaped
cell fragments that do not have a nucleus. Cells become bigger but do not divide: they explode and
platelets are formed. Platelets can form a clot to heal a wound.
• White blood cells fight infections. Increased white blood cells lead to leukocytosis, decreased white
blood cells lead to leukopenia. There are innate immune cells (neutrophils, eosinophils, basophils,
macrophages) and adaptive immune cells (B cells and T cells).
o Neutrophils are the primary defense against bacterial infection: they can phagocytose and
digest microorganisms. They are granulocytes with neutral staining and have nuclei with 3
to 5 lobes. The cytoplasm contains granules. Increased neutrophils leads to neutrophilia
(caused by pyogenic bacterial infection), decreased neutrophils leads to neutropenia,
caused by B12 & folate deficiency or blood cancer.
o Eosinophils are important for the response in allergies and parasitic infections. They are
granular leukocytes with a nucleus with two lobes. Cytoplasm contains granules that can be
stained by eosin. Increased: eosinophilia (caused by asthma, hypersensitivity reaction) and
decreased: eosinopenia (caused by alcohol intoxication, overproduction of steroids).
, o Basophils play a role in parasitic infections and allergies. They contain large cytoplasmic
granules that obscure the cell nucleus. When unstained, the nucleus is visible and has two
lobes. Increased: basophilia (caused by CML) and decreased: basopenia (caused by
hyperthyroidism, pregnancy and irradiation).
o Monocytes/macrophages can phagocytose dead or damaged cells (without granulocytes):
monocytes move into tissues and become macrophages. They have one kidney shaped
nucleus and are slightly larger than a lymphocyte. Increased: monocytosis (caused by
tuberculosis, malaria and typhoid & Kala azar). Decreased: monocytopenia (caused by
aplastic anemia, lymphocytic anemia and glucocorticoids)
§ Phagocytosis is done by
recognition of certain
bacteria or damage (also
chemotaxis involved).
Actin is rearranged to
allow engulfment: put
into phagosome which
fuses with lysosome and
breaks down the
bacteria)
o Lymphocytes are the primary
source of viral defense and
antibodies. They are
mononuclear and small, do not
have a lot of cytoplasm.
Increased: lymphcytosis (caused by viral infection, leukemia, bone marrow, cancer) and
decreased: lymphocytopenia (caused by acute stages of infection, excess irradiation)
B cells develop with recombination in the genes to form specific antibodies, which occurs in the bone
marrow. B cells that recognize self-antigens are removed. Specialization happens in secondary lymphoid
tissue (lymph nodes, spleen, Peyer’s patches). B cells then circulate the lymph and blood. At sight of
pathogens, they come back to the secondary lymphoid tissue to proliferate. Plasma cells (secrete
antibodies) and memory B cells are produced. In hematopoietic stem cells, VDJ loci cannot be accessed.
When becoming a B cell, PAX5 can bind the promoter regions to open the chromatin and allow RAGS to
cause rearrangements (first D-J, then V).
- The different B cell tumors reflect different developmental stages of B cells.
T cells can by either helper (CD4), regulatory, or cytotoxic (CD8). TCR genes are rearranged to cause the
different TCRs to be present on the T cells and to recognize different antigens presented on MHC. Cells
that won’t bind MHC or cells that bind self-antigens are killed.
- Also in this case: the different T cell tumors reflect different developmental stages of T cells.
Innate vs adaptive immunity:
• Innate is quick, adaptive is slow
• Innate is non-specific, adaptive is specific
• Innate consists of macrophages, neutrophils, NK cells, DCs, basophils eosinophils, adaptive consists
of B cells and T cells
• Innate secretes antimicrobial peptides and proteins, adaptive secretes antibodies
• Innate is based on self vs non self-discrimination, adaptive is less good at distinguishing this
• Innate has limited memory (priming, tolerance, epigenetics?), adaptive has lots of memory
• Innate diversity is limited (standard receptors), adaptive is highly diverse (customized in genetic
recombination)
