Properties and Culturing of HSC

Properties and Culturing of Hematopoietic Stem Cells (HSCs)
Key properties of HSCs:

1. Self-renewal: ability of HSC to divide maintaining their biological properties
2. Multipotency: ability of HSC to generate all mature hematopoietic cell types (B, T and
myeloid lineage)
3. Quiescence: ability of HSC to remain inactive and unresponsive to external stimuli, but
capable of rapid proliferation in response to stress.
In normal, steady-state conditions, HSCs exist in a quiescent state, which serves to conserve their
long-term regenerative potential, minimize the risk of DNA damage that can arise from frequent cell
division, and prevent depletion of the stem cell pool to ensure availability for future needs.
Several signals play a crucial role in maintaining this quiescence, such as
 Notch signaling is essential for keeping HSCs quiescent by regulating gene expression.
 Transforming growth factor-beta (TGF-β) suppresses HSC proliferation and promotes a
quiescent state
 Thrombopoietin (TPO) interacts with the MPL receptor on HSCs, aiding their maintenance in
this state.
 CXCL12, a chemokine secreted by stromal cells in the niche, binds to CXCR4 receptors on
HSCs, anchoring them in a quiescent state within the bone marrow.
Signals for entry into active cell cycling:
 From BM niche: G-CSF, IL-6, SCF push HSCs into the cell cycle. E.g. G-CSF can
downregulate CXCL12 expression in the bone marrow, reducing HSC anchorage and
promoting mobilization and activation.
 Sympathetic Nervous System: During stress, norepinephrine from the SNS signals to the
BM niche, decreasing CXCL12 secretion, which releases HSCs from their quiescent state.
 Wnt/β-Catenin: Increased stress signals β-catenin to translocate to nucleus and activate
genes that promote cell division.
 mTOR pathway: Increased protein synthesis, energy metabolism, and entry into the cell
cycle, shifting HSCs to an active state.
 FOXO TFs: FOXO activity is reduced, which permits the cell to enter the cell cycle and
proliferate.
 Cell cycle regulators like cyclin-D and CDKs initiate HSC proliferation.
 Metabolic shift from glycolysis to OxPhos, which provides more ATP needed for cell
division but increases ROS. The increase in ROS can act as a signal for cell cycle entry.
Culturing of HSCs
I. Isolation of HSCs
 Primary sources of HSCs are:
1. BM: A direct source of HSCs, but the collection process (bone marrow aspiration) is invasive.
2. Peripheral blood (PB): HSCs can be mobilized into the bloodstream by administering G-CSF,
after which they can be collected through a process called leukapheresis.
3. Umbilical cord blood (CB): Contains a high concentration of HSCs and is non-invasive to
collect, commonly used for clinical and research purposes.
II. Sample Preparation:

1. The blood or bone marrow sample is diluted 1:1 in PBS without Mg²⁺ or Ca²⁺
2. Ficoll Layering: 20 ml Ficoll is taken into a 50-ml tube and slowly layered with 25 mL of the
diluted blood sample on top by tilting the tube and gently running down the side to avoid
mixing.
3. The tube are centrifuged at 1100g for 20 minutes at RT (25-27℃).
4. The upper half of the top layer containing plasma is carefully discarded.
5. Carefully collect the "cloudy" interface layer (approximately 10 ml, containing mononuclear
cells) and transferred to a clean 50-ml tube.
6. The cells are washed by adding 50ml PBS without Mg²⁺ and Ca²⁺.
7. The washed cells are resuspend in a medium containing serum or protein (such as D-PBS or
Hank’s with 2–6% FBS or HSA).
8. RBS are lysed in cold NH₄Cl solution by keeping on ice for 10 minutes.
9. The cells are washed twice with PBS without Mg²⁺ and Ca²⁺
10. The cells are resuspended in Hank’s + FBS at a concentration of 10⁷ cells/ml.
III. Cell Sorting
1. To the isolated cells, appropriate antibodies are added for the chosen surface markers (e.g.,
anti-CD34, anti-CD45, anti-CD38, and anti-CD71).
2. Samples are incubated for 30 minutes at 4°C.
3. After incubation, cells are washed twice with Hank’s + FBS and resuspended the cells in
Hank’s + FBS containing 2 µg/mL PI to label dead cells.
4. The cells are now ready for sorting by FACS or MACS to purify HSCs based on specific
surface markers.
• For sorting murine HSCs: Lin− (Lineage−) Sca-1+ c-Kit+ (LSK), and more specific
markers like CD150+, CD48−.
• For sorting human HSCs: CD34+, CD38−, CD133+, and CD90+ are used to identify the
primitive stem cell population.
5. FACS: In FACS, a stream of single cells is passed through a laser beam in a sequential
manner, allowing for the measurement of light scattered by the cell and the fluorescence
emitted from the bound antibodies. As cells flow past the laser, data on forward scatter (size)
and side scatter (granularity) are collected.
6. Gating: Gating involves setting specific criteria (gates) to isolate populations of interest from
the total cell population based on the acquired data. This is typically visualized using scatter
or dot plots. Common gating strategies include:
 Forward Scatter (FSC): Indicates the size of the cells. Larger cells scatter more
light forward.
 Side Scatter (SSC): Reflects the granularity or internal complexity of the cells. More
granular cells scatter light at wider angles.
7. PI staining to exclude non-viable cells (PI-negative cells are viable).
8. HSCs express high levels of CD34 and low levels of CD45, CD38, and CD71.
9. Representation: A dot plot (or scatter plot) shows individual cells as dots based on their
fluorescence intensity for two different markers. Different cell populations form distinct
clusters, each representing cells that express certain markers.
IV. Culturing of HSCs

The following media and supplements are used for culturing HSCs in vitro:
1. Culture media
 DMEM: Vitamins, amino acids, glucose, FBS and specific supplements like IL-6 and SCF.
Example: DMEM with valproic acid and 10% FBS for culturing CD133+lin−CD45− cells
from CB samples.
• RPMI 1640: Bicarbonate-buffered, with glutathione as a reducing agent, supplemented with
L-glutamine, 2-beta mercaptoethanol, gentamicin, FBS, and growth factors like GM-CSF, IL-
4, TNF-alpha, IL-3 for culturing PBMNCs.
• Iscove’s Modified Dulbecco’s Medium (IMDM): Contains additional amino acids,
vitamins, selenium, and transferrin. Used in combination with IL-3, EPO, SCF, insulin, and
human serum for CD45+ cell proliferation.
• Serum-Free Media (SFM): Serum poses risks for human stem cell culture, particularly for
therapeutic purposes, promoting the use of serum-free media. E.g. StemPro-34 SFM, which
is a specialized serum-free medium designed for the culture, expansion, and differentiation of
BM, PB, and CB-HSCs. Often supplemented with cytokine cocktails for optimal results.
2. Cytokine Cocktails
These include SCF, TPO, IL-3, IL-6, and G-CSF are used in combination for efficient HSC self-
renewal and differentiation. E.g. SCF alone supports 25% self-renewal but for a short period. TPO
and SCF together induce around 90% self-renewal. IL-3, IL-6, and G-CSF with SCF significantly
increase division potential (50-60%).
V. Expansion of HSCs
Expansion of HSCs in vitro is crucial for various applications, including stem cell transplantation,
gene therapy, and the study of blood-related diseases. Expansion media are used for expanding HSC
lineages is given in Table 1. Each media type, such as SFEM, Stem Pro 34SFM, XVIVO 10, and
IMDM, is paired with specific cell sources, including umbilical cord blood (UCB), peripheral blood
mononuclear cells (PBMC), and BM. For each media type, specific cytokine and supplement
combinations are listed to enhance stem cell expansion. For instance, SFEM with UCB includes
antibiotics (100 U/ml penicillin-streptomycin), 2 mM L-glutamine, and cytokines like IL-6, TPO, and
FLT3-ligand, while IMDM with UCB includes 20% FBS, selenium, insulin, and additional factors
like SCF, FLT3-ligand, and folic acid.
Protocol for expansion of HSCs

1. Prepare the Expansion Medium: Combine the Basal Medium (based on the chart) and the
Supplement along with appropriate amount of Cytokine Mix.
2. Pre-equilibrate the required amount of the supplemented medium in the incubator at 37°C and
5% CO₂ for 30 minutes before use.
3. Plate the Cells:
• For freshly isolated HSCs: Plate the cells at a density of 5 x 10³ cells/mL in the pre-
equilibrated medium.
• For cryopreserved HSCs: Thaw the cells in a 37°C water bath for 2 minutes.
Immediately transfer the thawed cells into the pre-equilibrated medium at a density of
5,000 cells/ml.
• Incubate the plated cells in the incubator at 37°C and 5% CO₂ for 4 days.
4. Partial Medium Change on Day 4:
 Remove the cells from the incubator, pipette up and down several times to create a single cell
suspension and transfer the contents into a 50 mL conical tube.
 Centrifuge the cells at 24g for 10 minutes.
 Carefully aspirate and discard the upper two-thirds of the medium, leaving the cells in the
bottom third.
 Gently resuspend the cells in the remaining medium and replenish the medium back to the
original volume using fresh media.
 Place the cells back in the incubator and continue incubating for another 6–8 days.
 Every 3 days, replace two-thirds of the medium as described in the partial medium change
step above.

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Properties and Culturing of HSC

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Properties and Culturing of Hematopoietic Stem Cells (HSCs)

Key properties of HSCs:

II. Sample Preparation:

IV. Culturing of HSCs

Protocol for expansion of HSCs

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