Hematopoietic Bone Marrow Cell SortingWithout Compromise: 7 Lasers, Eight Colors, Six Way Sorting A Novel Approach

Authors: Vasilis Toxavidis, John Tigges & Meritxell Alberich Jorda

Affiliation: Beth Israel Deaconess Medical Center Harvard Stem Cell Institute

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Beckman Coulter Scientific Symposium

Hematopoietic Bone Marrow Cell SortingWithout Compromise: 7 Lasers, Eight Colors, Six Way Sorting A Novel Approach

PRINCIPAL OF THE TECHNIQUE

Background:

Hematopoiesis is a highly dynamic and tightly regulated process responsible for producing blood cells. The hematopoietic system, due to its well characterized cell hierarchy and well defined cell types, has been extensively used to study cell renewal and differentiation.

All blood cells are produced from a small population of cells known as hematopoietic stem cells (HSC), which reside in the bone marrow. HSCs, divided into long-term (LT-HSC) and short-term (ST-HSC), are capable to self-renew, as well as, to commit to the distinct progenitor lineages: common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), MEP (megakaryocyte-erythroid progenitor) and CLP (com-mon lymphoid progenitor). These populations will further differentiate and give rise to mature and functional hematopoietic cells.

Research Applications:

Flow cytometry analysis and sorting are valuable techniques for studying the distinct hematopoietic bone marrow populations. Identification, isolation and purification of HSCs and progenitors provide the possibility to study the biology and regulation of each population independently, during normal hematopoiesis and in disease.

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PROTOCOL

Standard Procedure:

Murine bone marrow cells were isolated from femurs and tibias of C57/BL6J mice. Bones were crushed in 5 mL PBS/2%FBS, and cell suspensions spun down at 1600 rpm for 8 min. Subsequently, red blood cells were lysed with 1 mL ACK buffer for 2 min at RT and 20 mL of PBS were then added to each tube before filtering suspensions using a 70μM cell strainer. Cells were spun down as before and pellets were resuspended in 200 μL PBS/2%FBS. Staining for long-term and short-term hematopoietic stem cells were done using the following anti-mouse antibodies: lineage allophycocyanin-conjugated antibodies specific for B220, CD3, Gr1, mac1 and Ter119, allophycocyanin-Cy7-conjugated CD117/c-kit, pacific blue-conjugated Ly-6A/E/Sca-1, fluo-rescein isothiocyanate-conjugated CD34, phycoerythrin-Cy7-conjugated CD150, CD48-conjugated PE-Texas Red or CD48-conjugated DyLight594, FCgR-conjugated phycoerythrin, and DAPI for viability.

Cells were stained for 30 min on ice, followed by a wash with 3 mL PBS before centrifugation. Cell pellets were resuspended in 500 μL PBS and acquired using a Beckman Coulter MoFlo Astrios* high-speed cell sorter. Single stained controls were prepared using 10,000 total bone marrow cells using the same antibod-ies (1:100 v/v), except for allophycocyanin and phycoerythrin-Cy7, where specific allophycocyanin-conju-gated B220 and phycoerythrin-Cy7-conjugated B220 antibodies were used. Dead cells were identified and excluded by DAPI staining.

Beckman Coulter MoFlo Astrios optimization:

The instrument was compensated using the single-color stained controls, listed above.

In order to gather enough data points for either the LT-HSC/ST-HSC populations or GMP/CMP/MEP popula-tions, 1-2x10e6 total WBM cells should be acquired.

*For Research Use Only. Not for use in diagnostic procedures.

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HSC & Progenitor Analysis of WBM:

• WBM gated to exclude debris and cell fragments

• Mouse Whole Bone Marrow gated to exclude all Lineage+ (LIN+) markers

• Lineage negative (LIN-) population expected to be < 5% of WBM

• LIN- population (Gate B) is 2.45%

• To clearly see the LIN- population, increase the plot resolution and increase plot size (this can be a useful technique for the identification of HSC and Progenitor cell populations as well).

ST & LT HSC:

• HSC low % of WBM

• LT expected to be 10x to 15x lower in quantity than ST

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Differentiated Progenitor Cells:

• Results are typical of published data for % distribution of Progenitor cells [2,3]

• The MEP/CMP/GMP gates can be more clearly defined by increasing the Progenitor Cell gate to include the double negatives. This increases the CD150 population and is a useful guide for gating.

The Beckman Coulter MoFlo Astrios was our preferred sorter of choice during hematopoietic stem and pro-genitor cell analysis. We easily identified previously described bone marrow populations, including LT-HSC, ST-HSC, MEP, CMP and GMP. In conclusion, we present the Beckman Coulter MoFlo Astrios as providing the necessary technological advancements to analyze hematopoietic stem cell and progenitor cell popula-tions, with ease.

Compensation strategies: For individuals attempting HSC and Progenitor cell population sorting, Fluores-cence Minus One (FMO) controls are suggested to verify compensation and gating strategy. FMO’s are con-trol cocktails in which only one fluorescent marker is excluded at a time. Essentially, all antibody conjugates are present in the sample, except one for which, the FMO provides a gating control.

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REFERENCES

1. Menzel S, Thein SL. Curr Opin Hematol. 2009 16(3):179-186.

2. Kaufmann KB, Gründer A, Hadlich T, Wehrle J, et al. (2012). A novel murine model of myeloproliferative disorders generated by over expression of the transcription factor NF-E2. J. Ex Med. 10.1084/jem.20110540.

3. M. Alberich Jorda, S. Jiang, R. Zagozdzon, K. Parmar, et al. (2009). The peripheral cannabinoid receptor regulates human and mouse hematapoiesis, bone marrow recovery, and hematopoietic stem and progenitor cell mobilization. Haematologica, 94[suppl.2]:208 abs. 0511.

NOTES

The results demonstrated represent those generated on the Beckman Coulter MoFlo Astrios. As differences exist in the performance between instruments, the author cannot guarantee a similar result with the use of other flow cytometry instruments. Refer to instrument configuration and set up below.

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MoFlo Astrios Instrument Configuration

Compensation Matrix

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Post Sort