Application protocol

Isolation, expansion, and analysis of human hematopoietic stem cells (HSC)

This application protocol describes how human hematopoietic stem cells (HSCs) can be isolated from various sources, such as bone marrow or cord blood, expanded in highly defined cell culture conditions, and analyzed by flow cytometry or cell culture assays.

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Protocol

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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

1. Things to prepare in advance

Preparation of buffers for cell isolation

PE buffer for isolation of mononuclear cells

Prepare a solution with phosphate buffered saline (PBS), pH 7.2, and 2 mM EDTA. Keep buffer cold (2–8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).

PBE buffer for isolation of CD34⁺ cells

Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution. Keep buffer cold (2−8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as respective serum albumin, respective serum, or fetal bovine serum (FBS). Buffers or media containing Ca²⁺ or Mg²⁺
are not recommended for use.

2. Isolation of mononuclear cells

Isolate mononuclear cells by density gradient centrifugation using Ficoll-Paque™. Follow the Ficoll-Paque protocol from the special protocol corresponding to the source tissue.

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Isolation of mononuclear cells from peripheral blood

Isolation of mononuclear cells from bone marrow aspirates

Isolation of mononuclear cells from cord blood

3. Isolation of CD34⁺ cells

Isolate CD34⁺ cells among the mononuclear cells using the CD34 MicroBead Kit UltraPure, human and LS or MS columns. Follow the protocol of the kit data sheet.

We recommend filtering the magnetically labeled cell suspension before separation on the column to guarantee a single-cell suspension.

Place a Pre-Separation filter (30 µm) on the separation column.
Rinse the column three times with PBE buffer, pre-wetting the filter.
Apply the cell suspension and washing buffer to the filter on the column.

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CD34 MicroBead Kit UltraPure, human

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Figure 1: High purity CD34⁺ cells isolated from a PBMC sample using the CD34 MicroBead Kit UltraPure. CD34⁺ cells were isolated from a debris-rich PBMC sample using the CD34 MicroBead Kit UltraPure, two MS Columns, and a MiniMACS™ Separator. Cells were stained with CD34-PE, CD45-FITC and propidium iodide. Cells were analyzed after gating on propidium iodide^–, lymphoid cells.

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Figure 2: Reduced debris carry-over during CD34⁺ cell isolation. A comparison of the ungated forward scatter versus side scatter profile of the same sample after separation with the CD34 MicroBead Kit or the CD34 MicroBead Kit UltraPure demonstrates reduced debris carry-over with the CD34 MicroBead Kit UltraPure.

4. Expansion of CD34⁺ cells

For expansion, culture the isolated CD34⁺ cells in StemMACS™ HSC Expansion Media XF supplemented with StemMACS HSC Expansion Cocktail. Follow the protocol of the media data sheet.

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StemMACS HSC Expansion Media XF, human

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Figure 3: High levels of CD34⁺ cells. Cryopreserved CD34⁺ cells separated from cord blood were expanded for 7 days in the indicated media supplemented with StemMACS HSC Expansion Cocktail (n=4; mean +/– SD). Values were normalized to StemMACS HSC Expansion Media XF. StemMACS HSC Expansion Media XF consistently maintains high levels of CD34⁺ cells during expansion.

5. Flow cytometric analysis of expanded CD34⁺ cells

Analyze the expanded CD34⁺ cells for HSC-specific surface markers via flow cytometry using REAfinity™ antibodies, or perform CFU-GEMM analysis to assess their myeloid differentiation potential.

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Figure 4: Preserved HSC surface phenotype. CD34⁺ cord blood cells were expanded for 7 days in StemMACS™ HSC Expansion Media XF (red) or alternative media (blue) supplemented with StemMACS HSC Expansion Cocktail. The cell surface phenotype was assessed by flow cytometry. Cells expanded in StemMACS HSC Expansion Media XF maintain the characteristic surface phenotype of early
hematopoietic progenitors and typically show a higher proportion of phenotypically primitive cells.

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Figure 5: Preserved broad differentiation potential. Isolated CD34⁺ cord blood cells were expanded in the indicated media plus StemMACS HSC Expansion Cocktail. After 7 days, cells were analyzed for their CFU potential. Cells cultured in StemMACS HSC Expansion Media XF maintained broad hematopoietic differentiation potential, giving rise to all major CFU types, including primitive CFU-GEMMs.

Resources

The following is a list of relevant resources that might be of interest:

Material

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

Reagents and instrumentation requirements

For mononuclear cell isolation

PE buffer: Phosphate buffered saline (PBS), pH 7.2, and 2 mM EDTA. Keep buffer cold (2–8 °C).
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD).
15 mL of Ficoll-Paque™ (ρ = 1.077 g/mL)
MACS® SmartStrainers (100 µm) (# 130-098-463)

For CD34⁺ cell isolation

CD34 MicroBead Kit UltraPure, human (# 130-100-453)
CD34 Stem Cell Analysis Cocktail, anti-human (# 130-093-427) for flow cytometric analysis of separated cells.
(Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., CD34-FITC, CD34-PE, CD34-APC, CD133 (293C3)-PE, CD45-FITC, CD45-PE, or CD45-APC. For more information about antibodies refer to www.miltenyibiotec.com/antibodies.
(Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD Staining Solution (# 130-111-568) for flow cytometric exclusion of dead cells.
(Optional) Dead Cell Removal Kit (# 130-090-101) for the depletion of dead cells.
(Optional) Pre-Separation Filters (30 µm) (# 130-041-407) to remove cell clumps.
PBE buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution (# 130-091-376) 1:20 with autoMACS® Rinsing Solution (# 130-091-222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubble could block the column.
▲ Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca²⁺ or Mg²⁺ are not recommended for use.
MACS Columns and MACS Separators: CD34⁺ cells can be enriched using MS, LS, or XS Columns (positive selection). Cells that express the CD34 antigen strongly can also be depleted using MS, LS, or XS Columns.
▲ Note: Positive selection or depletion can be automated using the autoMACS Pro or the autoMACS Separator.

Column	Max. number of labeled cells	Max. number of total cells	Separator
Positive or negative selection
MS	1×10⁷	2×10⁸	MiniMACS™, OctoMACS™, SuperMACS II
LS	1×10⁸	2×10⁹	MidiMACS™, QuadroMACS™, SuperMACS II
XS	1×10⁹	2×10¹⁰	SuperMACS II
Positive or negative selection
autoMACS	2×10⁸	4×10⁹	autoMACS Pro, autoMACS

▲ Note: Column adapters are required to insert certain columns into SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.

For CD34⁺ cell expansion

StemMACS™ HSC Expansion Media XF, human (# 130-100-463)
StemMACS HSC Expansion Cocktail (# 130-100-843)

Cytokines included in the StemMACS HSC Expansion Cocktail:

Human SCF (# 130-096-693)
Human FLT3-Ligand (# 130-096-476)
Human Thrombopoietin (TPO) (# 130-094-011)

For flow cytometry analysis

StemMACS HSC-CFU complete with Epo, human (# 130-091-280)
CD45 Antibody, anti-human, VioBlue
CD45RA Antibody, anti-human, FITC
CD133/2 (293C3) Antibody, anti-human, PE
CD34 Antibody, anti-human, APC

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