Ferrari: Lecture IX Flashcards
miRNA: Regulation in Hematopoiesis
Where does miRNA regulate and what characterizes its presence?
it is regulated in many different body systems and characterized by the presence of stem cells
What are the 4 model linages of stem and progenitor cells?
muscles
skin
nervous system
hematopoietic
Describe the muscle lineage differentiation
muscles contain muscle progenitors and differentiated cells
the main feature of the stem cells is the capacity of self-renewal
in this case the muscle progenitor is able to differentiate into different cells like satellite and myoblasts
Describe skin differentiation
embryo: embryonic epidermal skin cells
adult: epidermal skin cells which are responsible for skin renewal as we change them every 3-4 weeks and are localized in the derma
Describe nervous system differentiation
neuroepithelial stem cells give rise to differentiated cells
Describe hematopoietic differentiation
Hematopoietic stem cells are found in the peripheral blood and the bone marrow and give rise to rbc, wbc, etc.
What advantages for study do hematopoetic stem cells offer?
easier to isolate from bone marow (this is why HPSC are the most well studied and known)
What are some TF expressed only in the muscular system that regulate master genes?
MyoD
Myogenin
MYF5
PAX3
PAX7
Describe the hematopoietic hierarchy:
hematopoietic stem cells
↓
committed progenitors (not able to renew like HPSC) but they can differentiate in more than 1 lineage
↓
common myeloid progenitor (can give rise to both granulocytes and megakaryocytes and erythrocytes) so they are not lineage restricted, just committed
↓
common lymphoid progenitor (gives rise to B cells, T cells, and the NK compartment)
What characterizes the differential states of the hematopoietic hierarchy?
specific factors (cytokines and growth factors) and the expression of specific miRNA
Why are the factors that make the hematopoietic hierarchy important?
they allow for cell cultures to be synthesized and placed into specific lineages
How would we make a cell line to study erythropoiesis?
with stem cell factors IL3, and erythropoietin
How would we make a cell culture to study granulocytes?
with granulocyte stem factors IL3, IL6, and so on
When and who discovered the hematopoiesis was organized in a cellular hierarchy?
Maximow in the 19th century
Why was the organization of hematopoiesis important?
it was sustained by the consequences of the atomic war
survivors had cancers and neoplasia of the lymhoid/hematopoietic system, so they needed transplants from donors
Stem Cells
self-renew, generate daughter stem cells, and can contibute to a pool of differentiated stem cells
these features CANNOT be found in progenitors and in differentiated cells
How were HPSC found and defined?
TIll and McCulloch performed a clonal in-vivo repopulating assay in a mouse in 1961
The pool of hematopoietic stem cells, progenitors, or the differentiated cells express different _______
surface markers
Why is it important to note that hematopoietic stem cells, progenitors, or the differentiated cells express different surface markers?
we can use antibodies to have cell sorting
What are the 2 essential factors that induce the proliferation of differentiation of stem cells in vivo?
intrinsic factors and extrinsic factors
Give examples of intrinsic factors
TF
miRNA
Give examples of extrinsic factors
cytokines
networks
Where are HPSC located in embryo?
fetal liver
Where are HPSC located in adults?
bone marrow
What is the microenvironment of the bone marrow?
bone marrow niche
Why are the cells that pertain to the stromal component, the vascular component, and the osteoblastic component of the bone marrow important?
they are the ones able to release factors and do cell-cell interactions
What is required for the formation and function of hematopoietic stem cells?
critical transcription factors
What defines the stem cells?
miRNAs
How are HPSC from the primitive compartment isolated?
bone marrow cells fro mouse are extracted using a syringe → surface markers of primitive compartment are exploited using antibodies → cells are sorted using flow cytometry
What was a result that was being sought out in this paper in regards to HPSC?
isolate the HPSC-specific miRNAs
Gain of Function Approach
expression of a gene is induced at a supraphysiological level to see the effects of overexpression
Loss of Function
abrogate the expression of a gene
Why are vectors derived from viruses used?
because a virus’ job is to transfer their genome into the host cell and using the machinery for their cell cycle
What are the most powerful viral systems?
vector derived from Mo-MLV: retrovirus (Moloney leukemia virus that infects mice, carries oncogenes, and causes leukemia)
vector derived from HIV-1: human virus (Lentivirus class of the retroviral family)
*the genome for both is RNA
Mo-MLV
small piece of RNA
at the ends of the terminal are located long terminal repeats, where the strong promoter is located
fundamental genes (form the viral particles) code for: capsid proteins, reverse transcriptase, envelope proteins
What are the 2 type of elements that make HIV complex?
cis-acting and trans-acting elements
trans-acting elements
sequences required for the formation of the viral particle:
gag
pol
env
cis-acting sequences
required for the assembly of the viral genome into the viral particles:
PBS (primer binding site)
DIS (genome dimerization signal)
Ѱ (packaging signal)
PPT (polypurine track, downstream env)
polyA (poly adenylation sequence, in R)
U & R (unique and redundant sequence)
SD & SA (splice donor and acceptor , scattered)
Which one of the cis-acting sequences is of particular importance?
Ѱ signal: in the RNA genome and allows this genome to be included in the viral particle
What happens if you use technology to remove almost all of the genes of a virus and only keep the LTR and Ѱ signal?
the retroviral vector is cloned and the expression of the construct of interest (gene, miRNA, protein coding gene) will be driven by the strong viral promoter
What does it mean if miRNA are expressed at a lower level?
no leukemia
What does it mean if the same miRNA is expressed at a high level?
leukemia
Why is the field of miRNA in literature full of paper regarding cancer?
the expression of specific miRNAs are completely dysregulated in cancers, so much so that they are almost markers for neoplastic growth, useful for diagnosis and prediction
What are the 3 sources of HPSC in humans?
cord blood
bone marrow
peripheral blood
What is a surface marker for stem cells in humans in cord blood, peripheral blood, and bone marrow?
CD34 (this is the marker used for transplantation)
What happens after CD34 is extracted from the core blood for a transplant?
qPCR is performed (not microarray because the mouse model shows us miRNAs are present) after the miRNA has been designed
What is a human mouse chimera?
mouse that can accept transplantation of human cells and can have human blood circulating
How can a mouse have human blood circulating?
it is immunodeficient in B, T, and NK murine cells
*they are extremely immunodeficient
If miR-125b1 is added to immunodeficient mice, what occurs?
human cells demonstrate the expression of this miRNA and enable better engraftment, homing, and differentiation
*it is also observed in humans
there is an evolutionary conserved subset of miRNAs, which are particularly important in terms of expression in the __________
hematopoietic primitive compartment
miRNAs in the hematopoietic primitive compartment must be _____ because _____ means cancer
tightly regulated
dysregulation
