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In Vitro Stem Cell Analog Development: Image Cytometry Analysis of Hematopoietic Stem and Progenitor Cells In the Bone Marrow

Brendan A. Harley1, Elena Levantini2, John E. Mahoney3, Cesar Nombela Arrieta4, Daniel G. Tenen2, Alexei Protopopov3, and Leslie E. Silberstein5. (1) Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Roger Adams Lab, MC-712, Box C-3, 600 S. Mathews Ave., Urbana, IL 61801, (2) Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Harvard Institutes of Medicine, Room 907, 4 Blackfan Circle, Boston, MA 02115, (3) Dana-Farber Cancer Institute, Boston, MA 02115, (4) Laboratory Medicine, Children's Hospital Boston, One Blackfan Circle, Karp 10004D, Boston, MA 02115, (5) Children's Hospital, Karp Family Research Building, 10th Floor, Room 10217, 1 Blackfan Circle, Boston, MA 02115

Hematopoietic stem and progenitor cells (HSPCs) are an ideal platform for studying microenvironmental cues on stem cell behavior and for developing stem cell niche analogs. HSPC self-renewal and lineage differentiation depend on cues from distinct microenvironments, termed niches, in the bone marrow (BM) that are defined by cellular components, soluble regulators, and by the extracellular matrix (ECM) [1]. Here we employ Laser Scanning Cytometry (LSC) to define the anatomical localization of HSPCs within the femoral BM cavity as a first step towards quantifying HSPC-niche cell interactions within the BM microenvironment and then mimicking key interactions ex vivo.

HSPC niches exist within distinct anatomical regions: the femoral metaphyses and diaphysis. However, morphologically and functionally, the potential niches within these regions are significantly different. The diaphyseal BM cavity contains an endosteal region (ER), the region of BM in close association with the endosteal surface of the cortical bone, and the central medullary region (CMR) which contains the BM central sinus; the metaphyses are made up of cancellous bone. All three regions contain distinct populations of hematopoietic cells, BM stromal and reticular cells, adipose tissue, and disparate microvascular organizations. However, conventional fluorescence imaging techniques do not permit visualization of a large enough segment of the BM to definitively identify HSPCs or their niches, because of the rarity of HSPCs in the BM: LT-HSCs account for less than 0.005% of all BM cells [2].

LSC and confocal microscopy was used to quantify the distribution of HSPCs and differentiated hematopoietic cell populations in distinct anatomical locations (diaphyseal ER versus diaphyseal CMR versus the metaphyses) of the extravascular BM compartment. HSPCs were be identified using the bmi-1 gene targeted reporter (GFP) mouse model, where GFP levels are strongest for HSPCs, along with fluorescence immunostaining for antibodies (cKit, Sca-1) characteristic of primitive hematopoietic cell populations [3]. LSC allows complete longitudinal sections of the femoral BM cavity to be scanned and the entire cellular content (order 2.5x105 cells/image) mapped with single cell level precision, making objective quantification and statistical comparison of the localization of even extremely rare BM populations possible.

Combinations of antibodies (cKit, B220, IgM) were used to identify the morphological distribution of cell populations during HSPC to B cell development. More multipotent hematopoietic (cKit+) cells are predominantly located within the diaphyseal ER while more mature hematopoietic (IgM+) cells are predominantly within the diaphyseal CMR; B220+ cells, incorporating both precursor and mature hematopoietic cells are evenly distributed across the femoral BM cavity [4]. Further segregation has been observed for GFP+cKit+ cells (putative HSPCs); these cells are observed to be localized primarily within 10 cell diameters of the diaphyseal ER and preliminary results suggest a significant localization towards the metaphyseal regions of the BM microenvironment. These results suggest that cellular gradients exist within the BM, that these gradients may influence HSPC self-renewal and hematopoietic lineage differentiation processes, and that LSC techniques can be applied to quantify the morphological characteristics of HSC niches. Improved definition of HSPC niche(s) in the BM may have important implications for ex vivo expansion of clinically relevant HSPC populations, and will contribute to understanding the etiology of pathological conditions such as leukemogenesis, myelodysplasia and immunodeficiency.

These results will help to further characterize the cellular microenvironment surrounding the HSCs and their differentiated progeny. Further understanding the cell environment of HSCs in vivo provides important information regarding developing an appropriate in vitro analog of the BM microenvironment based on currently available collagen-glycosaminoglycan scaffold technology [5] for inducing maintenance (self-renewal) or lineage differentiation of primitive HSCs.

References:

1. Nagasawa T. Nat Rev Immunol 2006;6(2):107-16.

2. Wilson A, Trumpp A. Nature Revs Immunol 2006;6(2):93-106.

3. Hosen N, Yamane T, Muijtjens M, Pham K, Clarke MF, Weissman IL. Stem Cells 2007;25(7):1635-44.

4. Harley BA, Mahoney JE, Levantini E, Park S-Y, Le Y, Manis JP, Massberg S, von Andrian U, Tenen DG, Protopopov A, Silberstein LE. IABMR Annual Scientific Meeting; Boston, MA; 2007.

5. Harley BAC, Gibson LJ. Chemical Engineering Journal 2007;137(1):102-121.

Keywords: hematopoietic stem cell, niche, self-renewal, lineage development, microenvironment