Rational design of in vitro hematopoietic stem cell niches: Identifying cellular gradients within the bone marrow microenvironment

Brendan A. Harley^*, John E. Mahoney^#, Shin-Young Park^*, Yi Le^*, Alexei Protopopov^#, Leslie E. Silberstein^*,+

* Joint Program in Transfusion Medicine, Children's Hospital, Boston, MA  02115

# Dana-Farber Cancer Institute, Boston, MA  02115

+ The CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA  02115

Hematopoiesis is a complex physiological process in which a small number of pluripotent hematopoietic stem cells (HSCs) residing in the bone marrow (BM) proliferate to maintain their number, but also differentiate to generate a full complement of blood and immune cells. This process occurs in concert with a host of specialized (osteoblast, osteoclast, endothelial cells, BM stromal cells) and primitive (mesenchymal stem cells – MSCs) cell populations, as well as in an extracellular matrix (ECM) environment with distinct chemical, microstructural, and mechanical processes.

Taken out of physiological (morphological) context, many of the processes associated with B cell lineage development (surface antigen presentation, mechanisms regulating steps along the development pathway) have been studied [1]. The development process of B cells from HSCs is perhaps one of the best-documented areas of stem cell biology, and this level of knowledge informs critically on developing tools and materials appropriate for modeling and recapitulating the HSC lineage development and self-renewal microenvironments. Understanding the process within the in vivo microenvironment can provide a vast array of important new data relevant to transfusion medicine, immunology, and the development of biomaterials appropriate for stem cell engineering. Specific cells have already been identified in situ that may affect or be part of specific HSC maintenance and differentiation. HSCs are suggested to be associated with osteoblast and vascular endothelial cell subsets [2,3], multipotent progenitors and pre-pro-B cells are seen in direct contact with CXCL12 (but not IL-7) expressing stromal cells, and pro-B cells appear to associate with IL-7 (but not CXCL12) expressing stromal cells [4]. However, the majority of these data were acquired using conventional imaging techniques without the sufficient rigor or a large enough field of view in order to properly identify the (stromal) cell types within the HSC niche in the BM or to define the morphological arrangement of HSCs, differentiating B cells, and the surrounding niche cells that are responsible for HSC quiescence, self-renewal, and B cell lineage differentiation.

Laser Scanning Cytometry (LSC) uses laser-based opto-electronics and automated analysis capabilities to simultaneously measure biochemical constituents and evaluate cell or tissue morphology in conventionally prepared histology specimens. The technology allows automated analyses of large tissue sections at high magnifications, preserving the tissue morphological organization while performing flow cytometry-like analyses of cellular and tissue markers using fluorescent and chromatic dyes. Post-scan analytical tools allow analyses of single cells or cell populations having defined biochemical or morphological properties or at specific anatomical regions within the tissue. LSC techniques were used to characterize the morphological distribution of HSC-derived cell types in the BM. Longitudinal sections from the murine femur were used to simultaneously image the endosteal surface, central sinus region, and metaphyseal regions at either ends of the femur. Conventional immunohistochemistry techniques combined with fluorescent secondary antibodies allowed concurrent visualization of multiple surface antigens within the BM microenvironment and identification of distinct cellular subpopulations within a morphological context. The cKit (CD117) surface antigen was used to identify a population of cells enriched for HSCs and less mature (HSCs through pre-B cells) cells along the B cell lineage development pathway. IgM was used to identify a population of cells enriched for more mature cells (Immature-B and Mature-B cells) along the B cell lineage development pathway. Good agreement was observed for the overall fraction of positive cells within the BM measured using LSC and conventional flow cytometry, performed on single cell suspensions prepared from BM from the opposite femur. A strong morphological distribution of more differentiated (IgM⁺) vs. less differentiated (cKit⁺) cells was observed. cKit⁺ cells were observed highly concentrated towards the endosteal surface and within the metaphyseal regions while IgM⁺ cells were observed highly concentrated towards the central sinus region of the bone.

Figure 1. Top Left: Longitudinal image of mouse femur stained with DAPI (nuclei, blue) and an antibody for cKit (red); bone edge delineated by white dashed line. Top Right: Morphological distribution of cKit⁺ cells (green) in bone marrow (blue) after LSC analysis. The majority of cKit⁺ cells are found along the bone endosteal surface or at the metaphyses. Bottom Left: Longitudinal image of mouse femur stained with DAPI (nuclei, blue) and an antibody for IgM (red) and IgD (green). Bottom Middle, Right: Morphological distribution of cells of increasing maturity along the B cell lineage development pathway are found towards the center of the bone marrow cavity: all cells (blue), IgM⁺IgD^- (green), IgM⁺IgD⁺ (red). These results suggest a gradient of maturity within the bone marrow from the most primitive at the bone edge to the most differentiated in the central region of the marrow.

These results suggest that cellular gradients exist within the BM and that these gradients may at least partially influences HSC self-renewal and lineage differentiation processes. Ongoing research is incorporating antibodies for further surface antigens (CD150, flk2, GR-1, B220) and specific sequences of these antibodies to identify the morphological distribution of additional cell populations that are part of the B cell development pathway (from HSCs to fully differentiated B cells). 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:

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2. Zhang J, Niu C, Ye L, Huang H, He X, Tong W-G, Ross J, Haug J, Johnson T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Li L. Identification of the haematopoietic stem cell niche and control of the niche size. 2003;425(6960):836-841.

3. Kiel MJ, Iwashita T, Yilmaz OH, Morrison SJ. Spatial differences in hematopoiesis but not in stem cells indicate a lack of regional patterning in definitive hematopoietic stem cells. Developmental Biology 2005;283(1):29-39.

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

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