An in vitro model of hematopoietic stem cell homing demonstrates rapid homing and maintenance of engraftable stem cells

Abstract

Hematopoietic stem cell (HSC) homing is believed to rely heavily on adhesion interactions between stem cells and stroma. An in vitro assay was developed for adhesion of engraftable HSCs in bone marrow suspensions to pre-established Dextertype long-term bone marrow culture stromal layers. The cell numbers in the adherent layer and supernatant were examined, along with the engraftment capability of adherent layer cells to indicate the number of HSCs that homed to in vitro stroma. The cell number in the supernatant declined over the 24-hour period. The number of test cells adhering to the stromal layer increased during the first hour and then fell at 6 and 24 hours. The number of test HSCs adhering to the stromal layer was substantial at 20 minutes, increased during the first hour, and then remained constant at 1, 6, and 24 hours of adhesion. These data indicate that adhesion of engraftable HSCs occurs quickly and increases during the first hour of contact with pre-established stroma, that adhesion plateaus within 1 hour of contact, and that HSCs maintain their engraftment capability for at least 24 hours of stromal adhesion. Long-term engraftment from test cells at more than 1 hour of adhesion represents 70.7% of the predicted engraftment from equivalent numbers of unmanipulated marrow cells, indicating that 2 of 3 test engraftable HSCs adhered. These findings demonstrate the usefulness of this model system for studying stemstromal adhesion, allowing further dissection of the mechanism of HSC homing and exploration of possible manipulations of the process. IntroductionHematopoietic stem cell (HSC) homing is the process by which HSCs, infused intravenously in the transplantation setting, specifically extravasate in the bone marrow to engraft and proliferate there. Homing has been studied extensively both in vivo and in vitro and is believed to rely on adhesion molecule interactions between stromata, which consist of stromal cells and extracellular matrix, and stem cells. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] In vivo homing studies typically rely on infusion of a labeled or detectable HSC population and subsequent detection at various times after the transplantation. 16,19 In this approach, the recipients must be examined early enough (approximately 24 hours or earlier) that only infused cells, and not their progeny, are detected. However, testing this early can make it difficult to ascertain that the cells are truly homed and not migratory. Also, to dissect the mechanisms of homing, it would be desirable to conduct various manipulations, such as adding an adhesion receptor blocking antibody, and this can be cumbersome or even impossible in vivo. On the other hand, in vitro systems are relatively easy to manipulate, but the assay result can be difficult to interpret. Most in vitro homing studies examine adhesion of a hematopoietic cell population or cell line to a test substrate, such as purified extracellular matrix molecule coatings or stromal cell lines, under various conditions. 20-29 ...

High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal

“…Multiple studies have associated a variety of cell projections with primitive hematopoietic cells (18,21,(41)(42)(43). In particular, Frimberger et al (16) observed several types of projections on the leading edge and periphery of cells in HSC-enriched populations using high-speed optical-sectioning microscopy and inverted fluorescent video microscopy.…”

“…The time of cytokine addition was set as 0 hours of culture time for all experiments. At the end of the 4 days of culture, the clones in the arrays were harvested individually, placed into separate 0.65-ml microcentrifuge tubes, and shipped via overnight courier at 4°C to Vancouver, where the cells in each tube were resuspended and injected into individual sublethally irradiated C57BL͞6J-W 41 ͞ W 41 recipients.…”

Murine allogeneic in vivo stem cell homing,

“…Nilsson et al (2001) have shown that injected purified stem cells rapidly leave the central vein area after infusion and reach the endosteal niches within a few hours. Frimberger et al (2001) have also shown in an in vitro Dexter model that bone marrow stem cells are capable of extremely rapid adherence to the stroma within 20 min after deposition on the flasks with secondary engraftability used as a read-out. These observations indicate that measurement of bone homing at 3 h after transplant is an adequate time to infer that the cell in the marrow space are indeed marrow stem/progenitor cells.…”

The characteristics of hematopoietic stem cells from autoimmune-prone mice and the role of neural cell adhesion molecules in abnormal proliferation of these cells in MRL/lpr mice

“…Since the ability of HSC to adhere to stromal cells correlates strongly to homing ability in vivo, [17][18][19][20] we investigated the ability of abnormal HSC to adhere to stromal cells. We found that abnormal HSC from MRL/lpr mice were significantly more adhesive than normal HSC (Figure 3).…”

“…[17][18][19][20] We cultured 1×10 5 HSC on a monolayer of FMS/PA6-P cells for 2 hours, and then assessed the adherent cells in a methylcellulose assay for CFU-C, which includes BFU-E, CFU-GM, CFU-GEMM, CFU-G and CFU-M. The total CFU-C counts developed from the HSC from abnormal MRL/lpr mice which adhered to the FMS/PA6-P cells were significantly higher than those from the HSC of normal B6 and C3H mice (Figure 3), indicating an enhanced adhesion (or homing) ability of HSC in MRL/lpr mice.…”