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Stem Cell Research Source

Hematopoietic Stem Cells

Historically, many of the earliest methods and attempts to isolate progenitor subsets from the blood and bone marrow involved separating cells based upon size and cell cycle characteristics. However, these techniques were not sufficient for successful analysis.

HSCs are found in the bone marrow of adult individuals, in the femur, hip, and other bones. Cells can be obtained directly by removal from the bone or from the blood following pretreatment with cytokines, such as G-CSF (granulocyte colony-stimulating factors). Other sources include umbilical cord blood and placenta. For experimental purposes, fetal liver or fetal spleen of animals also are useful sources of HSCs.

Experiment results

Identification and enrichment of adult mouse hematopoeitic stem cells with the BD FACSAria™ II cell sorter »

Early transplantation studies involving bone marrow derived hematopoietic stem cells (HSCs) revealed the need to distinguish blood cells derived from either the host or the graft to adequately assess cellular function from the transplanted cells. To qualitatively and quantitatively measure the impact and success of the transplanted cells, multiple cellular markers were analyzed, including intracellular proteins and cell surface receptors.

The development of the fluorescent cell sorter and the availability of monoclonal antibodies to cell surface markers

specific to hematopoietic stem cells enabled the analysis and cell isolation from heterogeneous cell populations.

Despite these breakthroughs, the field of hematopoietic stem cell research was slowed by the lack of a single biomarker to distinguish different stem cell subpopulations and specifically mark the pluripotent cell population.

Today, numerous cell surface specific antibodies, dyes, and fluorophores are available for use with multiparameter flow cytometric approaches to define different stem cell lineages.

Experiment Results

Identification and enrichment of adult mouse hematopoietic stem cells with the BD FACSAria™ II cell sorter. Side population (SP) analysis of CD34-, c-kit+ (CD117), Sca-1+, Lin- (CD34KSL) cells in mouse bone marrow (BM). Flow cytometric analysis of CD34KSL cells stained with Hoechst 33342 is shown.

A Unfractionated mouse BM cells with the sorting gate set for Lin-negative cells.

B Lin-negative cells are displayed for Sca-1 and c-kit expression with the sorting gate set for Sca-1+, c-kit+ cells.

C CD34 expression of KSL with the sorting gate set for the CD34- cell population.

D The CD34- cells displayed with Hoechst Red and Blue dyes. This CD34KSL/SP subset of cells makes up approximately 0.0025% of the nucleated cells found in the BM.

E To assess functionality of sorted cells, mouse colony-forming cell (CFC) assays were performed using Methocult™ GF M3534 medium, which has been formulated to support optimal growth of granulocyte (G) and macrophage (M) progenitor colonies. Representative colony forming units (CFUs) are displayed. The following antibodies were used for sorting: CD34 FITC (Cat. No. 560238), CD117 c-kit PE (Cat. No. 553355), LY-6 A/E Sca-1-PE-Cy™7 (Cat. No. 558162), and Lin- cocktail APC (Cat. No. 558074). Cells were sorted on a BD FACSAria II cytometer.

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