Table of Content

Hematopoietic stem cells

1 Introduction

Hematopoietic stem cells (HSC) are tissue-specific adult stem cells capable of differentiating into all blood cell types to ensure homeostasis of blood throughout life.

Research on murine HSCs dates back to the late 1940s/early 1950s, when it was discovered that sub-lethally irradiated mice could be rescued from hematopoietic failure by injecting cells from blood-forming organs, such as the bone marrow. This finding led to the idea that cells in bone marrow with repopulation capacity could be further characterized and quantified in transplantation models. In early experiments, decreasing numbers of bone marrow cells were injected into recipient mice to determine the lowest cell number needed to fully reconstitute hematopoiesis. Further experiments transplanting sub-radioprotective doses of bone marrow cells into mice showed that these mice developed colonies of hematopoietic cells in their spleens. The number of colonies correlated to the amount of bone marrow cells originally injected. The advent of fluorescence-activated cell sorting technology and monoclonal antibodies then finally led to the identification of surface markers which are used to identify mouse HSCs.

Related PDFs:

Stem Cell Quick Guide (brochure)

2 HSCs in bone marrow

The main source to isolate mouse HSCs is bone marrow, where they represent approx. 0.01% of the total cells.

2.1 Cell subsets, frequencies, and marker expression

At a glance: HSCs in bone marrow

HSC source	Frequency	Marker expression	Function
Bone marrow	0.01%	Lin^–Sca-1⁺CD117⁺CD48^–CD150⁺	Hematopoiesis

Mouse HSCs are characterized and/or isolated using several surface markers. Firstly, mouse HSCs lack the expression of lineage-specific markers (meaning that they are are lin^–), and are positive for c-kit (CD117) and Sca-1. More recently, the HSC population was further narrowed down by including CD150 and CD48, resulting in the marker profile Lin^–c-Kit⁺Sca-1⁺CD48^–CD150⁺.

2.2 Miltenyi Biotec application protocols for HSCs from bone marrow

Miltenyi Biotec has created dedicated application protocols to work with and analyze HSCs.

2.3 Sample preparation of bone marrow

Murine HSCs are usually isolated from bone marrow prepared from the tibia of the hind leg.

2.4 Magnetic pre-enrichment of mouse HSCs from bone marrow

Miltenyi Biotec has developed products to pre-enrich lineage-negative cells containing HSCs from mouse bone marrow cells using the MACS® Cell Separation Technology.

For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation .

MACS Handbook:

Magnetic cell separation

2.4.1 Isolation of HSCs

At a glance: Kits and reagents for the separation of HSCs from bone marrow

Starting material	Isolation strategy	Comments	Automation	Product
Bone marrow	Positive selection of target cells			Anti-Sca-1 MicroBead Kit (FITC), mouse
Bone marrow	Positive selection of target cells			CD105 MultiSort Kit (PE), mouse
Bone marrow	Positive selection of target cells		No	CD117 MicroBeads, mouse
Bone marrow	Depletion of non-target cells	Direct pre-enrichment of lineage negative cells	Yes	Direct Lineage Cell Depletion Kit, mouse
Bone marrow	Depletion of non-target cells	Stringent two-step pre-enrichment of lineage negative cells	Yes	Lineage Cell Depletion Kit, mouse

The Lineage Cell Depletion Kit, mouse and the Direct Lineage Cell Depletion Kit, mouse enable fast pre-enrichment of mouse HSCs from bone marrow by depleting cells expressing lineage markers. Designed to magnetically label mature cells (T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, and erythroid cells) and their differentiation-committed precursors, the kits generate an enriched population of untouched HSCs and progenitor cells. The Direct Lineage Cell Depletion Kit, mouse is especially formulated for a quick one-step pre-enrichment process, whereas the Lineage Cell Depletion Kit, mouse enables the most stringent depletion of Lin⁺ cells from bone marrow.

Direct Lineage Cell Depletion Kit, mouse

Before separation

After separation

LSK staining

Before separation

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After separation

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LSK staining

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Quick, one-step separation of lineage-negative cells. Untouched lineage-negative cells were isolated from a single-cell suspension of mouse bone marrow using the Direct Lineage Cell Depletion Kit, mouse and a MidiMACS™ Separator with an LS Column. Cells were fluorescently stained with Lineage Cell Detection Cocktail-Biotin and Anti-Biotin-APC and analyzed by flow cytometry using the MACSQuant® Analyzer. To evaluate the LSK (Lin^–Sca-1⁺c-kit⁺) fraction, cells were further stained with CD117-PE (c-kit) and Anti-Sca-1-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

HSCs can be further separated from lineage negative (Lin^–) cells using specific MACS MicroBeads, like the CD117 MicroBeads, mouse or the Anti-Sca-1 MicroBead Kit.

CD117 MicroBeads isolate murine progenitor cells. CD117 (also known as c-kit, steel factor receptor, or stem cell factor receptor) encodes a 145 kDa cell surface glycoprotein belonging to the class III receptor tyrosine kinase family. It is expressed on the majority of hematopoietic progenitor cells, including multipotent HSCs and committed myeloid, erythroid, and lymphoid precursor cells. In addition to their hematopoietic cell differentiation potential, CD117⁺ stem cells from murine bone marrow are reportedly capable of differentiating into smooth muscle cells, myocytes, and endothelial cells in vivo. CD117 is also expressed on a few mature hematopoietic cells such as mast cells.

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Enriched murine hematopoietic progenitor cells. Lineage^–CD117⁺ cells were isolated from mouse bone marrow using the Lineage Cell Depletion Kit, CD117 MicroBeads, an LS Column, and a MidiMACS Separator. The cells were fluorescently stained with CD117-PE and Anti-Biotin-APC against the panel of biotinylated antibodies used for the Lineage Cell Depletion Kit, mouse. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Stem cell quick guide (brochure)

2.5 Characterization of HSCs by flow cytometry

At a glance: Dedicated antibody cocktail for flow cytometry analysis of HSCs

Markers in cocktail	Isolation kit or reagent used	Comment	Product
Biotin-conjugated monoclonal antibodies against CD5, CD11b, CD45R (B220), Anti-7-4, Anti-Gr-1 (Ly-6G/C), and Anti-Ter-119.	Lineage Cell Depletion Kit, mouse Direct Lineage Cell Depletion Kit, mouse	Requires the use of a secondary antibody such as Anti-Biotin-FITC, Anti-Biotin-PE, or Anti-Biotin-APC	Lineage Cell Detection Cocktail-Biotin, mouse

HSCs are routinely analyzed by flow cytometry based on various extracellular and intracellular markers. The Lineage Cell Detection Cocktail-Biotin, mouse simplifies the flow cytometry analysis of cells before and after cell separation using MACS Technology. All antibodies are optimally titrated to be simply added to an aliquot of a cell fraction before analysis.

Miltenyi Biotec also offers a wide range of unique and standard monoclonal antibodies for research of HSCs and progenitor cells, including CD117, Anti-SCA-1, CD150, and CD48. Fluorochrome-conjugated MACS Antibodies are perfectly suited for the identification, enumeration, and characterization of HSCs, which can be combined into panels tailored to specific research needs. An online tool to quickly build an appropriate multicolor flow cytometry panel for each research project is available through the Related resources panel to the right.

For details about Miltenyi Biotec antibodies and dyes for cell analysis, see chapter Cell analysis – reagents.

Related products:

Primary antibodies

Kits and support reagents

2.6 Cell culture of mouse HSCs

2.6.1 Expansion of HSCs

At a glance: Cytokines for the expansion of mouse HSCs

Use	Comment	Product
Expansion		Mouse SCF, premium grade
Expansion		Mouse FLT-3-Ligand, research grade
Expansion		Mouse IL-3 IS, premium grade
Expansion		Mouse IL-6, premium grade

Isolated mouse HSCs can be further expanded in cell culture with HSC-relevant growth factors to obtain a larger number of cells or to elucidate mechanisms and signaling pathways driving self-renewal or differentiation. Early acting cytokines – mouse stem cell factor (SCF), Flt3-Ligand, and interleukin-6 (IL-6) – expand primitive HSCs, although current HSC culture systems allow only limited expansion and maintenance of primitive HSCs that can engraft and completely restore the entire blood system.

MACS Handbook:

Cell culture

2.6.2 Viral transduction of HSCs

At a glance: Kits and reagents for viral transduction of HSCs

Use	Comment	Product
Viral transduction	Surface of cell culture dishes does not have to be pre-coated with Vectofusin-1.	Vectofusin-1

Viral transduction is a fast and efficient method to study gene function or modulate gene expression. It is also a potential approach for gene modification in the context of gene therapies. Transduction of HSCs is especially interesting because of the cells' potential to eliminate hematopoietic defects. The modification of HSCs with retroviral vectors often requires the presence of a transduction-enhancing reagent. Polycationic reagents induce aggregation of vector particles and facilitate binding of the vectors to target cells via electrostatic interaction. Bridging molecules, such as recombinant fibronectin, interact with both vector particles and cell membrane. Transduction performance can be enhanced by centrifugation.

Miltenyi Biotec offers the novel transduction enhancer Vectofusin-1®, a fully synthetic non-toxic cationic amphipathic peptide that supports high transduction levels with small amounts of retroviral vector. When added to culture medium, Vectofusin-1 promotes the entry of several retroviral pseudo-types into target cells.

Hematopoietic stem cells