Kuanyin K. Lin

Mouse Hematopoietic Stem Cell Identification And Analysis

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.

Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice

Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cell types. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, Tgf-β signaling, Pten/Akt signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology.

Purification of Hematopoietic Stem Cells Using the Side Population

Hematopoietic stem cells (HSCs) primarily reside in bone marrow, are defined by their ability to maintain blood homeostasis, and replenish themselves through self-renewal. Although HSC purification schemes vary from laboratory to laboratory, the resulting cell populations are similar, if not the same. This chapter will discuss different enrichment methods for HSCs and provide a detailed protocol for staining HSC with Hoechst 33342 for the side population (SP).

Imprinted genes that regulate early mammalian growth are coexpressed in somatic stem cells

Lifelong, many somatic tissues are replenished by specialized adult stem cells. These stem cells are generally rare, infrequently dividing, occupy a unique niche, and can rapidly respond to injury to maintain a steady tissue size. Despite these commonalities, few shared regulatory mechanisms have been identified. Here, we scrutinized data comparing genes expressed in murine long-term hematopoietic stem cells with their differentiated counterparts and observed that a disproportionate number were members of the developmentally-important, monoallelically expressed imprinted genes. Studying a subset, which are members of a purported imprinted gene network (IGN), we found their expression in HSCs rapidly altered upon hematopoietic perturbations. These imprinted genes were also predominantly expressed in stem/progenitor cells of the adult epidermis and skeletal muscle in mice, relative to their differentiated counterparts. The parallel down-regulation of these genes postnatally in response to proliferation and differentiation suggests that the IGN could play a mechanistic role in both cell growth and tissue homeostasis.

CD48 on hematopoietic progenitors regulates stem cells and suppresses tumor formation

The proliferation and differentiation of adult stem cells is balanced to ensure adequate generation of differentiated cells, stem cell homeostasis, and guard against malignant transformation. CD48 is broadly expressed on hematopoietic cells but excluded from quiescent long-term murine HSCs. Through its interactions with CD244 on progenitor cells, it influences HSC function by altering the BM cytokine milieu, particularly IFNγ. In CD48-null mice, the resultant misregulation of cytokine signaling produces a more quiescent HSC, a disproportionate number of short-term progenitors, and hyperactivation of Pak1, leading to hematologic malignancies similar to those found in patients with X-linked lymphoproliferative disease. CD48 plays a vital role as an environmental sensor for regulating HSC and progenitor cell numbers and inhibiting tumor development.

CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway.

A protein that is thought to orchestrate the distribution of other signaling molecules on the cell membrane, CD81, is critical to maintaining the functional integrity of hematopoietic stem cells during their regeneration.

Isolation and characterization of mouse side population cells.

The side population (SP) is a subpopulation of mouse bone marrow cells highly enriched for hematopoietic stem cell activity. The SP is identified using flow cytometry as a minor population that efficiently effluxes the DNA-binding dye Hoechst 33342 relative to the rest of the bone marrow. Phenotypic and functionally analysis has established SP cells as highly phenotypically homogeneous and functional active. In this chapter we describe a detailed protocol for the purification of murine bone marrow SP cells based on Hoechst dye efflux in combination with the presence of HSC surface markers.

Hematopoietic Stem Cell Properties, Markers, and Therapeutics

This chapter provides an overview on hematopoietic stem cells (HSCs), which primarily reside in bone marrow, maintain blood formation, and replenish themselves throughout the adults lifespan. Adult HSCs are relatively dormant under homeostasis but can extensively proliferate when they encounter regenerative stresses. Adult HSCs comprise only ∼0.02% of whole bone marrow cells but possess abilities to self-renew and differentiate (hematopoiesis) to replenish the whole hematopoietic system. A single HSC is sufficient to establish long-term multilineage engraftment, which would only be possible through a self-renewal process. During adult hematopoiesis, BM-HSCs generate both lymphoid and myeloid cells. Lymphoid cells are comprised of primarily T-cells, B cells, and natural killer (NK) cells. Myeloid cells include granulocytes, macrophages, megakaryocytes, and erythrocytes. Hematopoiesis is a gradual differentiation process that involves multiple decision points beginning with HSCs and ending with terminally differentiated lineages. In adults, HSCs reside in the bone marrow cavity, closely associated with surrounding stromal cells. The most primitive HSCs localize to the interior surface of bone (periosteum/endosteum border) on the basis of colony-forming assays and Brd-U label retention, bringing them within close contact with osteoblasts. Molecules including N-cadherin, Notch-1, Tie2, and CXCR-4 are implicated in the HSC-to-niche interface. The identification and purification of long-term (LT)-HSCs relies on combinations of cell surface markers, with the presence or absence of specific antigens allowing discrimination of these cells from other bone marrow cell types including the immediately downstream short-term (ST)-HSCs and progenitor cell compartments.