Mouse hematopoietic stem cell identification and analysis

Abstract

Hematopoietic stem cells (HSCs) remain by far the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains difficult because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in bone marrow is about 0.01% of total nucleated cells and $5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. This prohibits the study of processes in HSCs, which require large amounts of starting material. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. This report will review different hematopoietic stem and progenitor purification strategies and compare flow cytometry profiles for HSC sorting and analysis on different instruments. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology. ' International Society for Advancement of CytometryKey terms hematopoietic stem cell; flow cytometry; peripheral blood; cell cycle HEMATOPOIETIC stem cells have tremendous therapeutic potential and have been harnessed in the clinic for more than 40 years in the context of bone marrow transplantation. Multipotent long-term HSCs (LT-HSCs) reside in the bone marrow and through a process of asymmetric cell division, can self-renew to sustain the stem cell pool or differentiate into short-term HSCs (ST-HSCs) or lineage-restricted progenitors that undergo extensive proliferation and differentiation to produce terminally differentiated, functional hematopoietic cells. ST-HSCs or multipotent progenitors (MPPs) are only able to sustain hematopoiesis in the short term, whereas the LTHSCs must persist for the lifespan of the organism to perpetually replenish the hematopoietic system. HSCs can be isolated from bone marrow or peripheral blood using enrichment (magnetic cell separation-MACS) and/or single-cell sorting (fluorescence-activated cell sorting-FACS) based on cell surface markers and/or vital dye staining. The HSC has served as the paradigm for adult stem cell populations by virtue of a well-defined differentiation cascade with distinct intermediaries connecting the differentiation of LT-HSCs into mature, functional hematopoietic cells. Each of the cell stages of HSC differentiation can be purified from the bone marrow or peripheral blood using characteristic cell surface markers, which has greatly facilitated the study of hemato...

Possible mechanisms of retinal function recovery with the use of cell therapy with bone marrow-derived stem cells

“…Other sources for clinical and scientific use include umbilical cord blood and placenta (10)(11) . In reference to phenotype, hematopoeitic stem cells are identified by their small size, lack of lineage (lin) markers, low staining (side population) with vital dyes such as rhodamine 123 (rhodamine DULL , also called rho lo ) or Hoechst 33342, and presence of various antigenic markers on their surface, many of which belong to the cluster of differentiation series: CD34, CD38, CD90, CD133, CD105, CD45 and also c-kit, the receptor for stem cell factor (12)(13)(14)(15)(16)(17) .…”

“…Cells from BM could be easily and safely aspirated. After administering local anesthesia, about 10 ml of the bone marrow is aspirated from the iliac crest using a sterile bone marrow aspiration needle, and mononuclear bone marrow stem cells is separated using the Ficoll density separation method (14)(15)(16)(17) (Figure 1). Stem cell-based therapy has been tested in animal models for several diseases, including neurodegenerative disorders, such as Parkinson disease, spinal cord injury, and multiple sclerosis.…”

Phenotypes of stem cells from diverse origin

“…Therefore, we asked all authors to revisit the table and upgrade their part either by own data or based on additional literature, as necessary. This resulted in the upgraded Table 1 showing phenotypes of very small embryonic-like stem cells (vSELs) (5,6), neural stem cells (NSCs) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16), hematopoietic stem cells (HSCs) from two organs (4,17), MSCs (4,18), EpSC (4), limbal epithelial stem cells (LSCs) (19), endothelial progenitor cells (EPCs) from different organs (3,4,(20)(21)(22)(23)(24)(25), supra-adventitial adipose stromal cells (SA-ASCs) (3,(22)(23)(24)(25), adipose pericytes (pericyte) (3,(22)(23)(24)(25), and finally cancer stem cells (CSCs) (26).…”

Where new approaches can stem from: Focus on stem cell identification

“…We have gained vast knowledge in the field of hematopoietic stem cells and this area is probably the most established field of stem cell research. The state of the art in identifying hematopoietic stem cells is summarized in this issue by Challen et al (1).…”