Lesson 9 Flashcards
where are adult stem cells located?
somatic stem cells - only in defined tissues such as haematopoiesis, myogenesis, osteogenesis. etc
what are the four model lineages of stem cells?
in muscles, in the skin, in the nervous system, and in hematopoietic stem cells
what are the two parts of the muscle cell lineage?
muscle progenitors and the differentiated cells → muscle progenitor is able to differentiate into different cells
what is the main feature of muscle stem cells?
the capacity for self-renewal
what are the two levels of stem cells in the skin?
at the embryonic level = embryonic epidermal cells and in adults there are the epidermal stem cells (responsible for skin renewal)
where are epidermal stem cells located?
in the profound layer of the derma
describe stem cells in the nervous system:
neuroepithelial stem cells that give rise to the differentiated cells
why is it easier to study hematopoietic stem cells as opposed to the other three categories?
easiest to harvest → the most well known and studied
name some transcription factors of the muscular system important for the expression of genes in the muscular system (do not occur during development and define ms):
MyoD, Myogenin, MYF5, PAX3 and PAX7
what are committed progenitors?
cells that are not able to self-renew like HSC but have the possibility to differentiate into more than one lineage
what three cell lines can common myeloid progenitors give rise to?
mature granulocytes, megakaryocytes, and erythrocytes
what is each differential stem cell state characterized by?
using specific factors (not transcription factors) such as cytokines and growth factors which are able to sustain the differentiation and proliferation of the specific lineages
what specific characteristics can be found in stem cells, but not progenitor cells or differentiated cells?
stem cells can durably self-renew, they generate daughter stem cells and they can contribute to the pool of differentiated cells
how can you experimentally tell the difference between hematopoietic stem cells, differentiated cells, and progenitors?
the use of antibodies → they all express different surface markers
what two ways are differentiated cells and progenitor cells induced in vivo?
through intrinsic and extrinsic factors
what are intrinsic factors?
present in the cell → regulate master genes of hematopoietic self-renewal or differentiation (transcription factors and miRNA)
what are some examples of extrinsic factors?
growth factors, cytokines, or a complex network of cell-cell interactions
what is the microenvironment of HSC in adult bone marrow called?
bone marrow niche
what do HSC serve as in the bone marrow?
the stromal component, the vascular component, and the osteoblastic compotent
in the molecular regulation of hematopoiesis, what are the critical transcription factor required for?
the formation and function of hematopoietic stem cells
in the molecular regulation of hematopoiesis, what are the other classes of transcription factors necessary for?
lineage specific differentiation
what percentage of the bone marrow do hematopoietic stem cells make up?
1-2%
describe a gain of function approach:
the expression of a gene is induced at supra physiological level in order to see the effects of the over expression
describe a loss of function approach:
to abrogate the expression that particular gene
why are vectors derived from viruses used in research?
a virus’ job is to transfer their host genome into a host cell
what are some of the most powerful viral systems we can use for hematopoietic research?
Mo-MLV and HIV-1
what are vectors derived from Mo-MLV?
derived from a family of retroviruses, particularly from the Moloney leukemia virus (virus affecting mice that carries oncogenes and causes leukemia)
describe the HIV-1 vector:
a human virus belonging to the Lentivirus class which is part of the retroviral family
which is more complex, Mo-MLV or HIV-1?
HIV-1
what are the two components of HIV-1?
cis-acting sequences and trans-acting sequences
what are trans-acting sequences?
sequences required for the formation of the viral particle gag-pol-env
what are cis-acting sequences in HIV-1?
viral sequences required for the assembly of the viral genome into the viral particles
what are the viral particles of the cis-acting sequences?
-PBS (primer binding site)
-DIS (genome dimerization signal)
-Ψ (packaging signal)
-PPT (polypurine track, downstream env)
-polyA (poly adenilation sequence, in R)
-U & R (unique and redundant regions)
-SD and SA (splice donor and acceptor, scattered)
what is the most important cis-acting sequence signal?
Ψ signal → signal in the RNA genome that allow this genome to be included into the viral particle
what are trans-acting sequences?
gag, pol and env are genes that you can remove from the original genome and insert in another construct ( a plasmid) in order to complement the function
the expression of your construct of interest will be driven by what?
the strong viral promotor
what is the first step in the gain of function approach:
clone the sequence
in order to test the function of a stem cell, what must you look at?
the self-renewal and the differentiation
what is the only way to test the self-renewal of stem cells?
transfer them into an animal model
what is used to to reconstitute the haematopoiesis starting from the transplanted cells and begining with the elimination of the endogenous marrow, in order to prevent/avoid the competition among the transplanted cells and the wt cells of the recipient?
chemotherapy or irradiations
how can you detect the transplanted cells in the recipient mice?
you have different markers and GFP
when doing experiments with miRNA what is important to remember?
not only the expression matters but the level of expression as well → ex: miRNA expressed at low levels can be okay, but when expressed in higher levels induced leukemia
why is the level of expression of miRNA important?
important to confer advantages at the hematopoietic stem cells, but if the level is dysregulated the advantages can turn into neoplastic growth
where are the three sources of hematopoietic stem cells in humans?
core blood, bone marrow, and peripheral blood
what is cord blood?
in the cord blood we have circulating hematopoietic stem cells, reason why, once the baby is born, cord blood can be conserved. Cord blood is a good source of stem cells even if a unit of cord blood is not sufficient to transplant an adult
what is the specific surface marker found in cord blood, bone marrow, and peripheral blood?
CD34 - expressed mostly in stem cells
why is CD34 useful?
you can isolate a population of cells particularly enriched in hematopoietic stem cells
what can occur after labeling cells with CD34?
you can extract primitive cells from core blood
what is the best technique you can use to analyze the expression of a gene once you have already known the gene of interest?
qPCR
when performing qPCR with the gene of interest, what is the aim?
to target CD34 positive cells → detect which are the miRNAs expressed only in the positive fraction
what is a human mouse chimera?
a mouse that can accept the transplantation of human cells
how do human mouse chimera work?
this strain of mice is immunodeficient for B, T, and NK murine cells
