Ex- vivo expansion of hematopoietic stem cells (HSCs) derived from Acute
Myeloid Leukemia (AML) bone marrow patients.
OR
Ex-vivo culturing of Hematopoietic Stem Cells.
Instructor: Tal Gabay, Department of Medicine
Abstract
Stem cells of the blood system, termed hematopoietic stem cells (HSCs), are
generated in the bone marrow (BM) niche and represent 0.01% of all cells in the BM.
A stem cell can either divide in a self-renewal manner to form another stem cell or
alternatively, to become differentiated and form the hematopoietic system.
Mature hematopoietic cells are continuously generated from a small self-renewing
pool of pluripotent HSC.
Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy resulting
in accumulation of primitive and partially differentiated clonal cells (so-called blasts)
in bone marrow (BM), blood and/or extra medullary organs .Thus, AML is
characterized by disruption of HSC and progenitor cell differentiation.
There is evidence from cell purification studies that a subset of HSC, termed leukemic
stem cell (LSC) or cancer-initiating cells, are responsible for the initiation and
progression of the disease. Therefore, researchers focus their studies on HSC and lots
of efforts are directed to establish the right conditions for HSC expansion.
Although HSC self-renewal division in-vivo (mice models) clearly occur, induction of
self-renewal in vitro (outside a live model) is still difficult. Normal HSCs cannot be
cultured for more than a few days without losing their stem cell properties (i.e. self –
renewal and ability to reconstitute hematopoiesis after transplantation) and entering
into the pathway of lineage commitment and terminal differentiation.
In this respect, hematopoietic stem cells differ from other types of stem cells, such as
embryonic stem cells, which can undergo multiple cell divisions in culture while still
retaining their stem cell properties.
The aim of this study is to define the appropriate conditions and culture which will
support an un-limited ex-vivo expansion of HSCs from AML BM source. Culture
condition will include screening of several experimentals models including different
cytokines, growth factors and small molecules that are known to be critical for cells
expansion. Moreover, in order to give preferential conditions for stem cells expansion,
we will try to mimic the bone marrow niche by co-culturing the HSCs on a supportive
feeder layer of cells derived from BM source.
AML patient cells (CD34+)
MS5 Feeder layer
Figure 1: Microscopic view of AML CD34+ cells co-culture with mouse stromal cell-line
(MS-5) as a feeder layer.
Student mission/Objective:
Our main goal is to reveal the conditions that will enable growing HSC and mainly
leukemic HSC in un-limited ex-vivo manner. Such a study will shed light on AML
stem cells features and will enable studying other properties of these cells such as
genetic analyses and functional assays. Moreover, the results of this research project
will pave the way to better understanding of AML heterogeneity and thus
personalized therapy.
Our laboratory is located in the Cancer Research Institution in Rambam hospital. BM
or peripheral blood samples from AML patients are supplied from the hematology
department in the hospital.
In this study, we will purify HSCs and progenitors cells from AML patients and
healthy donors. Three models of culturing HSC will be tasted: 1) co-culture with a
supportive feeder layer of mouse stromal cell line (MS-5) (figure 1) 2) culturing in a
serum-free medium contain combination of cytokines and other required small
molecules 3) culturing in a semi-solid medium. In each culture model, proliferation of
HSC and progenitor cells will be monitored by the time.
To achieve the above goal we will use cutting the edge techniques that are used in
biology research:
1. Ficoll gradient: separation of white blood cells from human bone
marrow/peripheral blood.
2. MACS technology: magnetic beads conjugated to an antibody against CD34+
hematopoietic progenitor and stem cells.
3. Fluorescence Activated Cell Sorting (FACS): A technology that uses lasers
and antibodies conjugated to fluorophores to separate cells based on their
surface markers. We will use the FACSaria device for separate the tumor into
HSC sub-populations.
4. Colony Forming Cell (CFC) assay: also known as methylcellulose assay. We
will use that in vitro assay to study of hematopoietic stem cells characteristics.
5. Cell proliferation assay: measure the number of cells, or the change in the
proportion of cells, that is dividing.
Additionally, we will practice how to plan an experiments, how to make results
analysis and how to make a scientific conclusions interpretations.
Requirements:
1. 5 units or college level in biology/ life science.
2. Willing to work with human bone marrow and peripheral blood.
4. Curiosity and enthusiasm for science work
Paper name:
Marta A.W. et al. Hematopoietic stem cell expansion: challenges and opportunities.
Ann. N.Y. Academy of Science, 138–150 (2012).
Questions about the paper:
1. What is the main purpose of ex-vivo expansion of HSCs?
2. What is the role of small molecule SR1 (StemRegenin1) in the expansion of
HSCs?
We will discuss the answers when we meet at the dinner in the opening ceremony.
Please fill free to contact me with questions regarding the project at:
[email protected]
Recommended reading material:
Pabst.C. et al. Identification of small molecules that support human leukemia stem
cell activity ex vivo. Nature Methods 11,436-42 (2014).