Characterization of engraftable hematopoietic stem cells in murine long-term bone marrow cultures

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 ...

Section: Discussionsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 50%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Frimberger

Stering

Quesenberry

2001

“…LTBMC was performed essentially as described by Frimberger et al 49 Briefly, BMMNCs at 3 ϫ 10 6 /mL in 10 mL IMDM supplemented with 20% horse serum, 10 Ϫ5 M hydrocortisone, 10 Ϫ5 M 2-mercaptoethanol, 100 units/mL penicillin, and 100 g/mL streptomycin were subjected to hyperoxic or hyperoxic-hypoxia-hyperoxic treatments as described in "Isolation of BM Lin Ϫ cells and short-term culture." The cells were overlaid on the pre-established FBMD-1 feeder stoma layers, and fed weekly by removal of one-half of the supernatant medium and replacement with fresh medium.…”

Section: Long-term Bm Culture (Ltbmc)mentioning

confidence: 99%

Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells

Zhang

Sejas

et al. 2005

Hematopoietic cells are often exposed to transient hypoxia and reoxygenation as they develop and migrate. Given that bone marrow (BM) failure occurred in patients with Fanconi anemia (FA), we reason that hypoxia-then-reoxygenation represents a physiologically relevant stress for FA hematopoietic progenitor/stem cells. Here we show that expansion of Fancc-/- BM cells enriched for progenitor and stem cells was significantly decreased after 2 continuous cycles of hyperoxic-hypoxic-hyperoxic treatments compared with wild-type (WT) BM cells. This inhibition was attributable to a marked decrease of lineage-depleted (Lin-) ScaI- c-kit+ cells and more primitive Lin- ScaI+ c-kit+ cells in Fancc-/- BM cells following reoxygenation. Evaluation of the cell-cycle profile of long-term BM culture (LTBMC) revealed that a vast majority (70.6%) of reoxygenated Fancc-/- LTBMC cells was residing in the G0 and G1 phases compared with 55.8% in WT LTBMC cells. Fancc-/- LTBMC cells stained intensely for SA-beta-galactosidase activity, a biomarker for senescence; this was associated with increased expression of senescence-associated proteins p53 and p21(WAF1/CIP1). Taken together, these results suggest that reoxygenation induces premature senescence in Fancc-/- BM hematopoietic cells by signaling through p53, up-regulating p21, and causing senescent cell-cycle arrest. Thus, reoxygenation-induced premature senescence may be a novel mechanism underlying hematopoietic cell depletion and BM failure in FA.

“…In the past 2 decades, researchers have attempted to mimic the native BM environment in 2-dimensional culture systems by adding the proper cytokines and growth factors to cell cultures, coculturing HSCs with stromal cells, or both. [24][25][26] However, this method does not recapitulate the development and self-renewal of hematopoietic or leukemic stem cells. Recently, several 3-dimensional bone-tissue-like models have been developed with scaffolds that better mimic the physiologic in vivo situation [27][28][29] ; however, assembly of such bone-tissue analogs is challenging owing to the complexity of the BM microenvironment.…”

mentioning

confidence: 99%

Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment

Chen

Jácamo

Shi

et al. 2012

109

114

The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1␣ was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ؎ 6%; P ‫؍‬ .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment. IntroductionThe relevance of the bone marrow (BM) microenvironment in regulating hematopoietic stem cell (HSC) behavior has only recently been established. [1][2][3][4][5] The maintenance of HSC quiescence and normal hematopoiesis requires complex bidirectional interactions between the BM niche and HSCs. [6][7][8] Moreover, much evidence supports the idea that the BM microenvironment also plays a pivotal role in the initiation and propagation of leukemia. [9][10][11] Leukemic cells have been shown to hijack the homeostatic mechanisms of normal HSCs and take refuge within the BM niche. This mechanism is pivotal during chemotherapy and contributes to disease relapse. 12,13 Although human HSCs can be genetically modified and transplanted into immunodeficient mice, the BM microenvironment is not transplantable. Numerous studies in patients and mice undergoing bone marrow transplantation have failed to demonstrate consistent engraftment of donor BM stroma cells. [14][15][16] A better understanding of the BM niche will not only improve our understanding of HSC self-renewal and hematopoiesis in general but also accelerate the development of new therapeutic modalities and targeted agents for the therapy of hematopoietic malignancies. Although the concept of a BM "niche" was formulated in 1978, it remains largely unidentified owing to technical limitations. 13,[17][18][19] Currently, the xenotransplant NOD/SCID and NOD/SCID/IL-2r␥ null mouse repopulation assays are the most widely used and relevant readout systems for studying human normal and malignant hematopoiesis. H...