Transcript Hematopoietic Stem Cells

Immune cells
by
Soohyun Kim, Ph.D
Contact Info
E-mail: [email protected]
02-457-0868
Immune cells (Leukocytes; white
blood cells)
1. Hematopoietic stem cells (HSCs)
2. Cytokine in immune cell differentiation
(T- and B-cells developments)
•
Immune cell and cytokine related diseases.
Hematopoietic stem cells (HSCs)
• Hematopoietic stem cells (HSCs) are
pluripotent stem cells that give rise to all
the blood cell types.
– Two major immune cell types
• Myeloid lineages
• Lymphoid lineages
Immune cell development
proliferation
Self renewal
Differentiation
proliferation
proliferation
Self renewal
proliferation
HSCs self-renewal, differentiation,
and proliferation
Common Lymphoid Progenitor (CLP)
Common Myeloid Progenitor (CMP)
Hematopoietic stem cells (HSCs)
• Myeloid linages
• Monocytes
• Macrophages
• Dendritic cells
• Neutrophils,
• Basophils
• Eosinophils
• Erythrocytes
• Megakaryocytes/platelets
Hematopoietic stem cells (HSCs)
• Lymphoid lineages
– T-cells
– B-cells
– NK-cells
Components of blood
Red blood cells
ABO Blood Types
1). Antigens & Antibodies
Red blood cells have proteins called antigens on the membranes
Percentage of the population with each blood type
Rh+
Rh-
O
38.5%
6.5%
A
34.3%
5.7%
B
8.6%
1.4%
AB
4.3%
0.7%
Three types of immune cells according to
a classical staining method
Neutrophils
contains fine granules
a 3-6 lobed nucleus
Eosinophils
large course granules
bi-lobed nucleus
Basophils
stains very dark
large histamine granules
Monocytes
2). Monocytes
Largest leukocytes
U-shaped nucleus
Lymphocytes
1). Lymphocytes
Large dark spherical nucleus
i). T-lymphocytes (T-cells)
ii). B-lymphocytes (B-cells)
Platelets
Platelets (Thrombocytes)
Platelets development
Immune cell function
• Red blood cells (erythrocytes) carry
oxygen to the tissues.
• Platelets (derived from megakaryocytes)
help prevent bleeding.
Immune cell function
• Granulocytes (neutrophils, basophils
and eosinophils) and macrophages
(collectively known as myeloid cells)
fight infections from bacteria, fungi,
and other parasites such as nematodes
(ubiquitous small worms).
• Some of these cells are also involved in
tissue and bone remodeling and
removal of dead cells.
Immune cell function
• B-lymphocytes produce antibodies.
• T-lymphocytes can directly kill or
isolate by inflammation cells recognized
as foreign to the body, including many
virus-infected cells and cancer cells.
Immune cell function
• Many blood cells are short-lived and need to
be replenished continuously; the average
human requires approximately one hundred
billion new hematopoietic cells each day.
• The continued production of these cells
depends directly on the presence of
Hematopoietic Stem Cells (HSCs), source of
all these cells.
Myeloblasts/ Monoblasts/ Lymphoblasts
•Myeloblasts become all of the granular leukocytes
•Monoblasts become monocytes
•Lymphoblasts become lymphocytes
Historical Overview
• The search for stem cells began in the after
math of the bombings in Hiroshima and
Nagasaki in 1945.
• Those who died over a prolonged period from
lower doses of radiation had compromised
hematopoietic systems that could not
regenerate either sufficient white blood cells
to protect against otherwise nonpathogenic
infections or enough platelets to clot their
blood.
Historical Overview
• Higher doses of radiation also killed the stem
cells of the intestinal tract, resulting in more
rapid death.
• Later, it was demonstrated that mice that
were given doses of whole body X-irradiation
developed the same radiation syndromes; at
the minimal lethal dose, the mice died from
hematopoietic failure approximately two
weeks after radiation.
Historical Overview
• Soon thereafter, using inbred strains of
mice, scientists showed that wholebody-irradiated mice could be rescued
from otherwise fatal hematopoietic
failure by injection of cells from bloodforming organs such as the bone
marrow.
Historical Overview
• In 1956, three laboratories
demonstrated that the injected bone
marrow cells directly regenerated the
blood-forming system in human, rather
than releasing factors that caused the
recipients' cells to repair irradiation
damage.
Historical Overview
• To date, the only known treatment for
hematopoietic failure following whole
body irradiation is transplantation of
bone marrow cells or HSCs to
regenerate the blood-forming system in
the host organisms.
• Where this method can be used?
The isolation of HSCs in mouse
and human
• The development of Fluorescence Activated
Cell Sorting (FACS) has been crucial for this
field.
• This technique enables the recognition and
quantification of small numbers of cells in
large mixed populations.
• More importantly, FACS-based cell sorting
allows these rare cells (1 in 2000 to less than
1 in 10,000) to be purified, resulting in
preparations of near 100% purity.
Magnectic bead sorting and Flow cytometry
(FACS)
Enrichment and purification methods for hematopoietic stem cells.
illustrate column-based magnetic enrichment.
Magnectic bead sorting and Flow cytometry
(FACS)
Lower panels illustrate Fluorescence Activated Cell Sorting (FACS).
In this setting, the cell mixture is labeled with fluorescent markers that
emit light of different colors after being activated by light from a laser.
Fluorescence Activated Cell Sorting (FACS)
• Modern FACS machines use three
different lasers (that can activate
different set of fluorochromes), to
distinguish up to 8 to 12 different
fluorescence colors and sort 4 separate
populations, all simultaneously.
Functional Characteristics
• Multipotency and self-renewal
– As stem cells, they are defined by their
ability to form multiple cell types
(multipotency) and their ability to selfrenew.
Functional Characteristics
• It is known that a small number of HSCs can
expand to generate a very large number of
progeny HSCs.
• This phenomenon is used in bone marrow
transplant when a small number of HSCs
reconstitute the hematopoietic system.
• This indicates that, at least during bone
marrow transplant, symmetrical cell divisions
that give two progeny HSCs must occur.
Functional Characteristics
• Stem cell self-renewal is thought to
occur in the stem cell niche in the bone
marrow, and it is reasonable to assume
that key signals present in this niche will
be important in self-renewal.
stem cell niche
Functional Characteristics
• There is much interest in the
environmental and molecular
requirements for HSC self-renewal, as
understanding the ability of HSC to
replenish themselves will eventually
allow the generation of expanded
populations of HSC ex vivo that can be
used therapeutically.
Hematopoietic stem cell marker
• In reference to phenotype, hematopoeitic
stem cells are identified by their small size,
lack of lineage (lin) markers, low staining with
vital dyes such as rhodamine 123
(rhodamineDULL, also called rholo) or Hoechst
33342, and presence of various antigenic
markers on their surface, like:
– CD34, CD38, CD90, CD133, CD105, CD45
and also c-kit- the receptor for stem cell
factor.
Hematopoietic stem cell marker
• The hematopoietic stem cells are
negative for the markers that are used
for detection of lineage commitment,
and are, thus, called Lin-; and, during
their purification by FACS.
Hematopoietic stem cell marker
• A bunch of up to 14 different mature
blood-lineage marker, e.g.,
• For human
–
–
–
–
CD13
CD71
CD19
CD61
& CD33 for myeloid,
for erythroid,
for B cells,
for megakaryocytic, etc.
Hematopoietic stem cell marker
• A bunch of up to 14 different mature
blood-lineage marker, e.g.,
• For mouse
–
–
–
–
–
B220 (murine CD45) for B cells,
Mac-1 (CD11b/CD18) for monocytes,
Gr-1 for Granulocytes,
Ter119 for erythroid cells,
Il7Ra, CD3, CD4, CD5, CD8 for T cells, etc.)
Hematopoietic stem cell marker
• There are many differences between the
human and mice hematopoietic cell
markers for the commonly-accepted
type of hematopoietic stem cells.
Proposed cell-surface markers of
undifferentiated hematopoietic stem cells
Mouse
Human
CD34low/-
CD 34+
SCA-1+
CD59+*
Thy1+/low
Thy1+
CD38+
CD38low/-
C-kit+
C-kit-/low
lin-*
lin-**
Physical characteristics
• With regard to morphology,
hematopoietic stem cells resemble
lymphocytes.
• They are (i)non-adherent, and
(ii)rounded, with (iii)a rounded nucleus
and (iv)low cytoplasm-to-nucleus ratio.
Physical characteristics
• Since Physical characteristics cannot
be isolated as a pure population, it is
not possible to identify them in a
microscope.
• The above description is based on the
morphological characteristics of a
heterogeneous population.
HSCs Mobility
• HSCs have a higher potential than other
immature blood cells to pass the bone
marrow barrier, and thus may travel in
the blood from the bone marrow in one
bone to another bone.
HSCs Mobility
• If they settle in the thymus they'll
develop into T cells.
• In the case of fetuses and other
extramedullary hematopoiesis HSCs
may also settle in the liver or spleen and
develop.
• This ability is the reason why HSCs may
be harvested directly from the blood.
HSCs Source
• Sketch of bone marrow and its cells
HSCs are found in the bone marrow of
adults, which includes femurs, hip, ribs,
sternum, and other bones.
HSCs Source
• Cells can be obtained directly by
removal from the hip using a needle and
syringe, or from the blood following
pre-treatment with cytokines, such as
G-CSF (granulocyte colony-stimulating
factors), that induce cells to be
released from the bone marrow
compartment.
• What Are the Sources of Hematopoietic Stem Cells?
• Bone Marrow
• The classic source of hematopoietic stem cells
(HSCs) is bone marrow. For more than 40 years,
doctors performed bone marrow transplants by
anesthetizing the stem cell donor, puncturing a
bone—typically a hipbone—and drawing out the bone
marrow cells with a syringe. About 1 in every 100,000
cells in the marrow is a long-term, blood-forming
stem cell; other cells present include stromal cells,
stromal stem cells, blood progenitor cells, and
mature and maturing white and red blood cells.
HSCs Source
• Other sources for clinical and scientific
use include umbilical cord blood,
placenta, mobilized peripheral blood.
• For experimental purposes, fetal liver,
fetal spleen, and AGM (Aorta-gonadmesonephros) of animals are also
useful sources of HSCs.
Hematopoietic stem cell marker
• Mouse HSC : CD34lo/-, SCA-1+ ,
Thy1.1+/lo, CD38+, C-kit+, lin-
• Human HSC : CD34+, CD59+,
Thy1/CD90+,CD38lo/-, C-kit/CD117+,
lin-
The Manhattan Project
• The Manhattan Project was a top-secret
operation carried out by the United
States, the United Kingdom, and
Canada during the Second World War.
The purpose of the project was to
create the first nuclear weapon.
The Manhattan Project
• Created as a response to the fear that
Nazi Germany was developing nuclear
weapons, the Manhattan Project
eventually employed more than 200,000
people and cost nearly $2 billion.
The Manhattan Project
• In 1942, physicist Enrico Fermi
successfully controlled a nuclear
reaction in his reactor called CP-1
(Chicago Pile 1). On July 6, 1945, the
project exploded its first atomic bomb
at Los Alamos.
The Manhattan Project
and Preparations for Dropping the Bomb
Victim of Atomic Bomb