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First published online November 16, 2006
Stem Cells Vol. 25 No. 3 March 2007, pp. 798 -806
doi:10.1634/stemcells.2006-0513; www.StemCells.com
© 2007 AlphaMed Press

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THE STEM CELL NICHE

Human Mesenchymal Stromal Cells Regulate Initial Self-Renewing Divisions of Hematopoietic Progenitor Cells by a β1-Integrin-Dependent Mechanism

Sandra Gottschling, Rainer Saffrich, Anja Seckinger, Ulf Krause, Kerstin Horsch, Katrin Miesala, Anthony D. Ho

Department of Medicine V, Ruprecht-Karls University, Heidelberg, Germany

Key Words. Hematopoietic progenitor cells • Marrow stromal cells • Asymmetric cell division • Self-renewal • β1-Integrins

Correspondence: Anthony D. Ho, M.D., Department of Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Telephone: 49-6221-568001; Fax: 49-6221-5633639; e-mail: anthony_dick.ho{at}urz.uni-heidelberg.de

Received August 16, 2006; accepted for publication November 7, 2006.
First published online in STEM CELLS EXPRESS   November 16, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
In previous reports, we have demonstrated that only direct cell-cell contact with stromal cells, such as the murine stromal cell line AFT024, was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because β1-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment, we have studied the role of β1-integrins in the regulation of self-renewing cell divisions. For this purpose, we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34+133+ cells in the absence or presence of an anti-β1-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. β1-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that β1-integrin-mediated contact was available within the first 72 hours, human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of β1-integrins by ligands alone (e.g., fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC, thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a β1-integrin-dependent mechanism.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Progenitor cells are characterized by their dual abilities to self-renew and to differentiate into various cell types. This requires asymmetric cell divisions generating two functionally different daughter cells. In previous experiments, we demonstrated that the cell division history and functional fate of hematopoietic progenitor cells (HPCs) are closely related: asymmetric cell division and slow division kinetics are associated with a primitive function and an immature genetic profile [1, 2].

Several groups have reported that cytokines such as stem cell factor (SCF), thrombopoietin, Flt-3L, interleukin (IL)-3, or IL-6 might support self-renewing cell divisions [3–5]. We and others have demonstrated that self-renewing cell divisions cannot be maintained or increased by soluble molecules, but only by direct cell-cell contact between HPC and stromal cells, such as the murine stromal cell line AFT024 [6–8]. The nature of these interactions and the mechanisms involved in these cell-cell contacts are, however, not yet defined.

Because β1-integrins, especially VLA4 ({alpha}4β1) and VLA5 ({alpha}5β1), have been reported to play a vital role in the early interaction of HPC with the bone marrow (BM) niche, we have addressed the question of whether these receptors might be involved in the regulation of initial cell divisions [9–11]. Other authors have demonstrated that β1-integrins are involved in the regulation of progenitor cell proliferation, survival, clonogenic growth, and maintenance during ex vivo culture and transduction [12–16]. Blocking of the {alpha}4- and {alpha}5-subunit of lymphomyeloid cell lines and human c-kit+ BM cells in long-term BM cultures leads to an inhibition of lymphopoiesis, retardation of myelopoiesis, and reduction of colony-forming progenitors [17, 18]. However, in vivo ablation of β1-integrins during fetal hematopoiesis has only minor effects on the generation, maintenance, and hematopoietic differentiation potential of HPC, although migration and colonization of the fetal liver, spleen, and bone marrow are severely impaired [19, 20]. In adult hematopoiesis, β1-integrin deficiency completely prevents engraftment of irradiated recipient mice, but deletion after engraftment results in a normal long-term hematolymphoid differentiation potential and a normal retention of progenitor cells in the bone marrow [21, 22]. This observation indicates that β1-integrins are either dispensable for hematopoiesis or play a role exclusively in the early phase of neonatal and adult hematopoiesis until alternative mechanisms of stem cell regulation are established.

Thus, although the significance of β1-integrins for the homing of HPC is indisputable, their role in regulating early hematopoiesis remains unclear. Moreover, data generated from the murine model should be validated in a human model system. To address these issues, we have used an anti-β1-integrin function blocking antibody and a novel in vitro system that allows the immediate identification and monitoring of individual human HPC by direct observation of the initial cell division behavior, followed by functional assessment through long-term culture-initiating cell (LTC-IC) assay. Human CD34+133+ cells, highly enriched in LTC-IC and NOD/SCID repopulating cells, were cocultured with primary human mesenchymal stromal cells (MS cells) as an in vitro surrogate niche. The latter have been shown to support HPC and reconstitute the complete human bone marrow environment in irradiated host mice [23–27]. In this study, we have demonstrated that β1-integrins play an important role in the regulation of initial self-renewing cell divisions and for the determination of the long-term fate of HPC by human MS cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Source and Preparation of HPC
HPC were obtained from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood of healthy voluntary donors after informed consent, following the guidelines of the 1996 Declaration of Helsinki and the local Ethics Committee of the University Hospital Heidelberg. Mononuclear cells (MNC) were isolated by Ficoll-Hypaque (Biochrom AG, Berlin, http://www.biochrom.de) centrifugation. CD34+ cells were enriched using magnet-associated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), subsequently labeled with a CD34-fluorescein isothiocyanate (FITC) (DakoCytomation, Hamburg, Germany, http://www.dakocytomation.com) and CD133-phycoerythrin antibody (Miltenyi Biotec) and sorted on a FACSVantage SE flow cytometry system (purity, 97.4% ± 2.5%). Dead cells were excluded by propidium iodide staining. For immunophenotyping (CD34+)133+ cells were labeled with a CD29-allophycocyanin (APC) antibody (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com).

Source and Preparation of Stromal Feeder Layers
The murine fetal liver stromal cell line AFT024 (a kind gift from I.R. Lemischka, Princeton University, Princeton, NJ) was maintained in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker, Apen, Germany, http://www.cambrex.com), 20% fetal calf serum (FCS) (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), supplemented with 50 µm 2-mercaptoethanol (Bio-Rad, Hercules, CA, http://www.bio-rad.com), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin (Pen/Strep) (Invitrogen, Karlsruhe, Germany, http://www.invitrogen.com). Cells were grown in 0.1% gelatin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany, http://www.sigmaaldrich.com)-coated culture dishes at 33°C.

MS cells were obtained from bone marrow aspirates of healthy voluntary donors after informed consent, following the 1996 guidelines of the declaration of Helsinki and the local Ethics Committee of the University Hospital Heidelberg. MS cells were prepared as published elsewhere [28]. In brief, MNC were plated at 1,000,000 cells per cm2 on 10 ng/ml fibronectin (Sigma-Aldrich Chemie)-coated tissue culture flasks. Nonadherent cells were discarded after 24 hours. Adherent cells were expanded in 58% DMEM, low glucose (BioWhittaker), 2% FCS (HyClone, Bonn, Germany, http://www.hyclone.com), 40% MCDB201, 1x insulin transferrin selenium, 1x linoleic acid bovine serum albumin, 10 nM dexamethasone, 0.1 mM L-ascorbic-acid-2-phosphate (all from Sigma-Aldrich Chemie), supplemented with 2 mM L-glutamine, 100 U/ml Pen/Strep (Invitrogen), platelet-derived growth factor-bb, and epidermal growth factor (10 ng/ml each; R&D Systems, Wiesbaden, Germany, http://www.rndsystems.com). Upon reaching 80% confluence, cells were trypsinized with 0.25% trypsin/1 mM EDTA (Invitrogen) and replated at 2,000 to 10,000 cells per cm2. Cells were expanded for 2–6 passages. To confirm their MS cell character, cells were successfully differentiated into bone, cartilage, and adipose tissue following standard protocols [29]. Immunophenotyping revealed a MS cells-typical marker pattern: CD13+/CD29+/CD34–/CD38–/CD44+/CD45–/CD73+/CD90+/CD105+/CD106+/CD166+/HLA-DR–. For all experiments, confluent MS cells of passage 5 were used.

Fixed MS cells were prepared by replacing the culture medium with a 2% glutaric dialdehyde solution (Sigma-Aldrich Chemie) in Hanks' balanced salt solution (Invitrogen). MS cells were fixed for 5 minutes, subsequently washed with medium, and incubated overnight at 37°C, 5% CO2. After another washing, MS cells were used for experiments.

Coating of Culture Plates
For some experiments cell culture dishes were coated with bovine serum albumin (BSA), fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1), or FN and VCAM-1. Dishes were incubated at room temperature overnight with 50 µg/ml fibronectin adhesion-promoting peptide (Sigma-Aldrich Chemie) in phosphate-buffered saline (PBS) (Invitrogen) or PBS containing 1% BSA. Alternatively, dishes were incubated with 1 mg/ml recombinant human VCAM-1 (R&D Systems) for 1 hour. Nonspecific binding sites were blocked by incubation with PBS/1% BSA for 30 minutes. For double coating, VCAM-1-precoated plates were incubated with 50 µg/ml fibronectin adhesion-promoting peptide before blocking. Proper coating results were verified by immunofluorescence microscopy with a CD106-FITC (Becton Dickinson) or fibronectin-rhodamine antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com).

Time-Lapse Microscopy and Assessment of Cell Division Symmetry
The method has been described in detail previously [7]. For monitoring cell division history, CD34+133+ cells were stained with the membrane dye PKH26 (Sigma-Aldrich Chemie). Individual CD34+133+PKH26bright cells were sorted into 96-well plates coated with BSA, FN, VCAM-1, FN, and VCAM-1, or MS cells using the automatic cell-depositing unit (ACDU) on a FACSVantage flow cytometry system. For noncontact culture, CD34+133+PKH26bright cells were sorted into 0.4-µm polycarbonate transwell inserts in 24-well plates (Costar GmbH, Bodenheim, Germany, http://www.corning.com). To ensure that only one cell per well was deposited, the ACDU was set up in a low throughput modus (200–500 events per second). After cell deposition, the dishes were checked by immunofluorescence microscopy: 84.0% ± 8.7% (n = 10) of the wells contained an intact single cell. Cells were cultured for 7 days in stroma-conditioned long-term bone marrow culture (SC-LTBMC) medium, consisting of Iscove's modified Dulbecco's medium (Invitrogen), 12.5% FCS, 12.5% horse serum (both from Stem Cell Technologies), supplemented with 2 mM L-glutamine, 100 U/ml Pen/Strep (Invitrogen), and hydrocortisone (Sigma-Aldrich Chemie) with half-medium exchanges three times per week. The cell division history of each individual cell was monitored every 12–24 hours by an Olympus IX70 inverted fluorescence microscope (Tokyo, http://www.olympus-global.com) (magnification, x10) equipped with an incubator (37°C, 5% CO2, 99% humidity) and a digital image acquisition and processing system (SiS, Klausdorf, Germany, http://www.sis-germany.com). Cell division symmetry was assessed according to the division kinetics of the first-generation daughter cells and the PKH26 dye distribution. A cell division was defined as asymmetric if one of the first-generation daughter cells remained quiescent and retained the initial fluorescence intensity, whereas the other first-generation daughter cell divided further. The asymmetric division index (ADI) was defined as number of asymmetrically dividing cells per total number of dividing cells.

LTC-IC Assay
To correlate the cell division history with the functional destiny of a cell, the entire progeny of each well was transferred into a LTC-IC assay. Cells were cultured in 96-well plates for 5 weeks on irradiated (20 Gy) MS cell feeder layers in LTBMC medium with half-medium exchanges three times per week. After 5 weeks, the medium was removed and replaced by clonogenic methylcellulose medium (Methocult GF H4434; Stem Cell Technologies). The plates were scored for the presence of secondary colony-forming cells between days 12 and 16.

Blocking Experiments
CD34+133+PKHbright cells were preincubated with the 4B4 monoclonal β1-integrin function-blocking antibody (mbAb) at a dilution of 1:100 or a IgG1-isotype control (both from Coulter Immunology, Hialeah, FL, http://www.beckmancoulter.com) for 15 minutes at 4°C in buffer containing PBS (Invitrogen), 2 mM EDTA (Sigma-Aldrich Chemie) and 1% FCS (Stem Cell Technologies) [30]. Excess antibody was removed by washing. Individual cells were deposited into 96-well plates and cultured in SC-LTBMC medium on the various adhesive layers. The division history and subsequent functional fate was assessed as described. The experiments were also performed in the continuous presence of the mbAb by adding the latter to the medium upon every medium exchange.

β1-Integrin Antibody Turnover and Adhesion Kinetics
CD34+133+ cells were preincubated with the mbAb and cultured in SC-LTBMC medium on fibronectin. The portion and fluorescence intensity of mbAb+ cells was determined at 0, 2, 48, 72, and 150 hours by fluorescence-activated cell sorting (FACS) analysis. Coupled antibody was visualized by FITC-conjugated goat-anti-mouse secondary antibody (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). To exclude nonspecific binding of the secondary antibody, the cells were blocked with 10% goat serum/PBS (Sigma-Aldrich Chemie) before staining. In parallel, the CD29 expression was determined at 0, 2, 48, 72, and 150 hours with an APC-conjugated antibody. The turnover of the mbAb was estimated by correlating the proportion of CD29+ cells with the proportion of mbAb+ cells.

The effect of the mbAb on cell adhesion was determined by seeding 100,000 CD34+133+PKH26bright cells per well that were pretreated with mbAb or IgG1-isotype onto MS cells or β1-integrin ligand-precoated 24-well plates. The cells were briefly spun down. After 2, 48, 72, and 150 hours, the adherent and nonadherent fractions were separated by four standardized washes on a horizontal shaker (30 seconds, 100 rpm). The proportion of adherent cells was determined by fluorescence microscopy in relation to an untreated control. Alternatively, the LTC-IC frequency of the adherent and nonadherent fractions of mbAb+ cells and an unmanipulated control probe was determined by a LTC-IC assay in limiting dilution.

Statistics
All experiments were performed at least three times, each measurement was performed in triplicate. Results are given as the mean ± SEM. Significance level (p = .05) was determined by paired Student's t tests.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Impact of Primary Human MS Cells on the Division History and Maintenance of Human HPC
For a precise characterization of the mechanisms regulating self-renewing divisions of human HPC, a surrogate niche consisting of human origin is preferred over that of murine origin. We have therefore verified whether primary human MS cells in lieu of the murine stromal cell line AFT024 have the same impact on the division behavior and subsequent functional fate of HPC. For this purpose, individually seeded CD34+133+ cells derived from G-CSF-mobilized peripheral blood were cocultured with either primary human MS cells or AFT024 and monitored by time-lapse fluorescence microscopy and subsequent LTC-IC assays (Fig. 1A, 1B).


Figure 1
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  		Figure 1. Experimental setup and division history of CD34+133+ cells under various culture conditions. (A): Experimental setup. CD34+133+ cells were individually cultured in 96-well plates on various adhesive molecules or feeder layers. The division history of each cell was monitored over 7d by fluorescence time-lapse microscopy. Subsequently, the progeny of each well was transferred into a LTC-IC assay and assessed for the presence of secondary colonies. (B): Assessment of the division symmetry. CD34+133+ cells were stained with the fluorescent dye PKH26, which distributes evenly among the daughter cells. A division was defined as asymmetric if one of the first generation daughter cells remained quiescent whereas the other one continued dividing. Scale bar = 5 µm. Magnification, x100. (C): Impact of various feeder layers and stromal elements on the division history of CD34+133+ cells. The division history of individually plated CD34+133+ cells is depicted. The cells were cultured with either AFT024, vital mesenchymal stromal (MS) cells, or fixed MS cells with or without SC-LTBMC medium, in transwells above vital MS cells, in SC-LTBMC, or in LTBMC. Plot a, columns represent the percentages of dividing cells per total number of dividing cells (first y-axis). The line represents the percentages of cells that have undergone asymmetric division per total number of dividing cells (second y-axis). Plot b, SDF (defined as cells with ≤2 divisions in 7d) and FDF (defined as cells with >2 divisions in 7d). AFT024 compared with MS cells: *, p = .017; {dagger}, p = .031; {ddagger}, p = .015. MS cells compared with other culture conditions: **, p < .05; {dagger}{dagger}, p < .05; {ddagger}{ddagger}, p < .05. Abbreviations: d, days; FDF, fast-dividing fraction; LTBMC, long-term bone marrow culture medium; LTC-IC, long-term culture-initiating cell; MSC, mesenchymal stromal cells; SC, stroma-conditioned; SDF, slow-dividing fraction; w, weeks.

 
AFT024 recruited 97.6% ± 2.5% of the HPC into cell cycle, of which 44.8% ± 2.3% underwent asymmetric cell division. In MS cell cocultures, significantly fewer cells were dividing (83.4% ± 5.7%; p = .017) and undergoing asymmetric division (33.5% ± 5.6%; p = .031) (Fig. 1C, plot a). As long-term culture-initiating cell assays showed that the vast majority of LTC-IC were derived from cells that had divided asymmetrically (71.2% ± 20.9%), this resulted in a significantly lower yield of LTC-IC (3.0% ± 1.0% vs. 5.7% ± 1.2%; p = .039) (Table 1).


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  		Table 1. Impact of various feeder layers and stromal elements on the total number of LTC-IC and their distribution in various proliferative subpopulations

 
Although significantly more cells were fast-dividing upon exposure to AFT024 (88.0% ± 3.0% vs. 62.3% ± 10.4%; p = .015), this did not lead to a loss of LTC-IC. Under both culture conditions, 30.2% ± 6.8% and 29.7% ± 14.0%, respectively, of the LTC-IC were recruited from fast-dividing cells, demonstrating that both stromal feeder layers were able to counteract contact-mediated inhibition of proliferation and, above all, to recruit HPC into cell cycle without loss of primitive function (Fig. 1C, plot b; Table 1).

Although the murine fetal liver stromal cell line AFT024 has a slightly higher capacity to support self-renewing cells divisions of HPC, these data have demonstrated for the first time that primary human MS cells are able to consistently alter the cell division symmetry of HPC in favor of self-renewing cell divisions and to recruit HPC into the cell cycle without loss of primitive function. Human MS cells might therefore represent an appropriate surrogate niche to study the regulation of human HPC.

Impact of Stromal Cytokines, Cell Surface-Associated Molecules, and Bidirectional Interaction on the Division History and Maintenance of Human HPC
Using human MS cells, we then examined the impact of the various stromal elements (cytokines, cell surface-associated molecules, and bidirectional interaction) controlling the division history and maintenance of HPC. Maximal cell division (83.4% ± 5.7%) was induced upon coculture with vital MS cells (Fig. 1C, plot b). Exposure to cell surface-associated molecules (i.e., fixed MS cells) induced only 30.8% ± 4.2% of HPC into cell cycle. The addition of stroma-conditioned medium increased the mitotic rate to 47.8% ± 3.2%. Similar proportions of dividing cells were induced by cytokines alone in SC-LTBMC medium cultures (56.1% ± 1.5%) and transwell cultures (45.0% ± 7.1%). Lacking exposure to stromal elements as in LTBMC medium cultures resulted in a low rate of spontaneous cell divisions (13.6% ± 4.4%). Thus, although cell surface-associated molecules of MS cells are able to increase the mitotic rate of HPC to some extent, cytokines are the major promoters of cell division and differentiation.

In addition to alteration of the mitotic rate, MS cells favored asymmetric cell division (Fig. 1C, plot a). In LTBMC medium, all cells underwent symmetric cell division. MS cell-conditioned medium induced 10.9% ± 1.8% and MS cells in transwell setting induced 12.3% ± 0.5% of HPC into asymmetric division. Exposure to direct contact with fixed MS cells or with fixed MS cells plus conditioned medium increased the ADI to 22.5% ± 3.5% and 18.4% ± 2.3%, respectively. Maximal asymmetric division (33.4% ± 5.6%) and maintenance of functionally primitive cells (Table 1) was induced by exposure to vital MS cells, demonstrating the significance of bidirectional interaction through cell surface-associated molecules between human HPC and human MS cells for the regulation of cell division and self-renewal.

Vital MS cells also significantly accelerated the cell division kinetics (Fig. 1C, plot b). Although the majority of HPC (71.7% ± 4.7%) in coculture with MS cells underwent their first division later (48 hours after deposition) than HPC in stroma-free cultures (24 hours after deposition), the latter mainly divided only one or two times during the initial 7 days. Vital MS cells induced 62.3% ± 10.4% of the HPC to undergo ≥2 divisions per 7 days. Although the majority of LTC-IC was recruited from quiescent or slow-dividing cells, a significant proportion of the LTC-IC was derived from fast-dividing cells in cultures supported by the presence of vital MS cells (Table 1).

Turnover and Functional Kinetics of the Monoclonal β1-Integrin Function-Blocking Antibody
To verify the role of β1-integrins for the regulation of self-renewing divisions of HPC, we applied an mbAb. We first determined its turnover and functional kinetics by FACS analysis and adhesion assays. CD34+133+ cells were incubated with the mbAb, and the proportion of mbAb+ cells, visualized by a FITC-conjugated secondary antibody, was determined every 24 hours by FACS analysis. As a control, the proportion of β1-integrin+ (CD29+) cells was monitored to discriminate between mbAb uncoupling and β1-integrin downregulation.

During the first 72 hours, 87.0% ± 4.1% of the CD34+133+ cells expressed CD29 (Fig. 2A, plot a). The presence of the mbAb on the surface of CD34+133+ cells showed a similar time course. Of the CD34+133+ cells, 85.1% ± 0.4% were mbAb+ (Fig. 2A, plot b). Moreover, the cells showed an equal fluorescence profile (proportion of cells per fluorescence level) during this time period, thus indicating a stable ratio of antibody molecules per cell. After 72 hours, the proportion of CD29+ cells declined to 65.7% ± 8.1% (Fig. 2A, plot a), indicating either a downregulation of CD29 or the generation of CD29– cells through cell division. The proportion of mbAb+ cells declined to a greater extent to 50.1% ± 6.3% mbAb+ cells at 120 hours (Fig. 2A, plot b), indicating an uncoupling of the mbAb. Although the fluorescence profile shifted toward lower fluorescence intensities on the whole, the distribution of the cells within the fluorescence profile remained the same, demonstrating an equal uncoupling of the antibody. In summary, these data demonstrated an efficient and stable coupling of the mbAb during the first 72 hours.


Figure 2
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  		Figure 2. Turnover and impact of the mbAb on the adhesion of CD34+133+ cells. (A): Turnover of the mbAb. The mbAb was visualized by a fluorescein isothiocyanate-conjugated secondary antibody at 0, 48, and 72 hours by fluorescence-activated cell sorting analysis. In parallel, the CD29 expression was determined. Plot a, CD29+ cells at 0, 48, and 72 hours. Plot b, mbAb+ cells at 0, 48, and 72 hours. (B): Impact of the mbAb on the adhesion of CD34+133+ cells. Native (open bars) and mbAb-treated (hatched bars) CD34+133+PKH26+ cells were cultured on mesenchymal stromal cells and β1-integrin ligands. The proportion of adherent cells was determined at 2 and 72 hours by fluorescence microscopy. Abbreviations: FN, fibronectin; IT, isotype control; LTBMC, long-term bone marrow culture medium; mbAb, monoclonal β1-integrin function-blocking antibody; MSC, mesenchymal stromal cells; NC, negative control; SC, stroma-conditioned; VCAM-1, vascular cell adhesion molecule-1.

 
Corresponding to the data of Liesveld et al. [30], treatment of the CD34+133+ cells with this mbAb reduced their adhesion to MS cells (29.1% ± 3.9%), fibronectin (22.4% ± 0.8%), VCAM-1 (29.8% ± 4.2%), and both of the β1-integrin ligands (31.6% ± 5.0%) by 52.3% ± 6.8%, to the level of nonspecific adhesion to BSA (11.7% ± 4.1%) (Fig. 2B).

Functional analysis of the adherent and nonadherent fractions of mbAb-treated probes showed that the entire LTC-IC activity was contained in the nonadherent fractions, indicating a complete blockade of the adhesion of functionally primitive cells by the mbAb. In untreated probes, the majority of LTC-IC (90.6% ± 5.5% on average) was found in the adherent fractions. These data demonstrate an efficient and stable binding of the mbAb and a specific and sufficient inhibition of β1-integrin-mediated adhesion of CD34+133+ cells and LTC-IC to MS cells and β1-integrin ligands over a period of 72 hours.

Significance of β1-Integrins for the Regulation of Self-Renewal and Maintenance of Stemness of HPC
To analyze the role of β1-integrins in regulating self-renewing divisions of HPC and maintenance of stemness induced by MS cells, we have monitored the division history and subsequent functional fate of individually plated CD34+133+ cells that were preincubated with the mbAb. Whereas mbAb treatment did not cause any significant alteration of the mitotic rate of HPC in MS cell cultures (83.4 ± 5.7% vs. 88.1 ± 4.6%) and stroma-conditioned medium cultures (56.1 ± 1.5% vs. 58.6 ± 10.6%), the proportion of asymmetrically dividing HPC in MS cell cultures was significantly reduced by the mbAb (20.2% ± 1.5% vs. 33.4% ± 5.6%; p = .017) (Fig. 3, plot a). Moreover, antibody treatment caused a significant increase of fast-dividing cells (62.3% ± 10.4% vs. 81.2% ± 3.1%; p = .036) and acceleration of the cell cycle entry (36 vs. 48 hours). The division history and asymmetric divisions of HPC cultured in stroma-conditioned medium were not affected by the antibody treatment, thus excluding an effect of the mbAb on cell cycle entry and division (Fig. 3, plot b).


Figure 3
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  		Figure 3. Impact of β1-integrins on the division history of CD34+133+ cells. The division history of native (open bars) and mbAb-treated (dotted bars) CD34+133+ cells was monitored. (A): Total and asymmetric division. (B): SDF and FDF. *, p = .017; {dagger}, p = .039 compared with mbAb-treated HPC. Abbreviations: FDF, fast-dividing fraction; LTBMC, long-term bone marrow culture medium; mbAb, monoclonal β1-integrin function-blocking antibody; MSC, mesenchymal stromal cells; SC, stroma-conditioned; SDF, slow-dividing fraction.

 
Corresponding to the decrease in asymmetric divisions among HPC cocultured with vital MS cells after treatment with the mbAb, there was a significant decrease of LTC-IC from 3.0% ± 1% to 1.2% ± 0.3% (p = .040). Moreover, upon antibody treatment, less LTC-IC could be derived from fast-dividing cells (15.9% ± 4.5% vs. 31.7% ± 7.6%) (Table 2), indicating a significant role of β1-integrins for the recruitment of HPC into cell cycle without loss of primitive function. In addition, β1-integrins promoted the survival of primitive cells. The number of nonviable HPC was significantly higher in mbAb-treated MS cell cultures than in untreated controls (24.1% ± 2.6% vs. 13.5% ± 3.5%; p = .014). The viability of HPC in stroma-conditioned medium cultures was not affected by mbAb treatment, thus excluding a direct toxic effect on the HPC.


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  		Table 2. Impact of β1-integrins on the distribution of LTC-IC in various proliferative subpopulations

 
Because preincubation of CD34+133+ cells with the mbAb against β1-integrin was sufficient to alter the long-term fate of HPC, and binding of the mbAb was limited to the first 72 hours, our hypothesis is that β1-integrin-dependent adhesion triggers a cascade of reactions in the HPC that leads to maintenance of self-renewing capacity. Although all of the HPC were finally transferred onto an irradiated MS cell feeder layer for the LTC-IC assay, the cell fate determined by the initial contact could no longer be altered. This demonstrates the crucial role of initial β1-integrin-mediated contact for the long-term fate of HPC.

Moreover, the lack of any difference between HPC that were preincubated with the mbAb versus those kept in continuous presence of it makes a significant effect of stromal β1-integrins on the regulation of HPC in this experimental setup unlikely. In summary, these experiments have demonstrated for the first time that β1-integrins play an important role in the regulation of the long-term fate of HPC by favoring initial self-renewing cell divisions and survival of primitive HPC.

The Effect of β1-Integrins on HPC Is Indirect
To determine whether activation of β1-integrins alone is sufficient to alter the cell division behavior and maintenance of HPC, the division history and functional fate of individual CD34+133+ cells were monitored in the presence of β1-integrin ligands. Because fibronectin and VCAM-1, both expressed by various stromal cells, represent the major ligands for the selective, β1-integrin-mediated adhesion of primitive hematopoietic cells to the bone marrow stroma, they were used for the following experiments [10, 11, 31]. Compared with vital human MS cells, neither fibronectin, nor VCAM-1, nor both ligands together were able to increase the mitotic rate, proportion of self-renewing cell divisions, and maintenance of HPC (Fig. 4, plots a and b; Table 3).


Figure 4
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  		Figure 4. Impact of β1-integrin ligands on the division history of CD34+133+cells. The division history of CD34+133+ cells cultured on β1-integrin ligands was monitored. (A): Total and asymmetric division. (B): SDF and FDF. Mesenchymal stromal cells compared with other culture conditions: *, p < .05; {dagger}, p < .05; {ddagger}, p < .05. Abbreviations: FDF, fast-dividing fraction; FN, fibronectin; LTBMC, long-term bone marrow culture medium; MSC, mesenchymal stromal cells; SC, stroma-conditioned; SDF, slow-dividing fraction; VCAM-1, vascular cell adhesion molecule-1.

 


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  		Table 3. Impact of β1-integrin ligands on the distribution of LTC-IC in various proliferative subpopulations

 
Compared with cultures using MS cell-conditioned medium alone, the presence of β1-integrin ligands significantly reduced the mitotic rate (35.8% ± 4.0% to 43.6% ± 4.4% vs. 56.1% ± 1.5%; p < .05) and retarded the cell division kinetics. The majority of HPC (69.8% ± 10.4%) cultured on β1-integrin ligands underwent their first division later than HPC cocultured with MS cells (72 vs. 48 hours). Moreover, upon coculture with β1-integrin ligands, none of the cells underwent more than two divisions during the initial 7 days.

Despite the alteration of the mitotic rate and the division kinetics, the β1-integrin ligands had no impact on the division symmetry. The proportion of cells undergoing asymmetric division remained unchanged in a range between 2.4% ± 1.5% and 7.1% ± 2.9%, resulting in a low number of LTC-IC.

Thus, although β1-integrins played a significant role in the regulation of self-renewing cell divisions and maintenance of HPC by MS cells, they did not act directly on this process.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
In this study, we have provided for the first time evidence that β1-integrins play a significant role in the regulation of initial self-renewing cells divisions of human HPC by human mesenchymal stromal cells.

Although the crucial role of β1-integrins in homing and adhesion of human HPC to the bone marrow niche has been extensively studied in immunocompromised animal transplantation models, studies on the significance of β1-integrins between human HPC and the niche of the same species are scanty [10, 14, 16, 18, 31, 32]. We propose that observations made in animal models need to be validated in the human system. Therefore, we have used primary human MS cells as in vitro surrogate niche. Several authors have demonstrated the significant role of MS cells in maintaining stemness of human HPC in vitro and in vivo. Other studies have shown that MS cells were able to reconstitute the complete human bone marrow environment in irradiated mice [24–27].

Using the murine stromal cell line AFT024 as in vitro surrogate niche, previous experiments by our group have demonstrated that maintenance of stem cell function is associated with asymmetric cell division that could only be induced by the cellular microenvironment [1, 2, 7, 8]. In this study, we have verified that primary human MS cells show similar properties [1, 8]. By monitoring the division history and subsequent functional fate of HPC at a single cell level, we could confirm that human MS cells—although slightly inferior to AFT024—maintain primitive HPC. Mounting evidence derived from the murine model has shown that osteoblasts at the endosteal areas of the bone might represent the HPC niche [33, 34]. However, in early phases of fetal development, as well as in diseases such as myelofibrosis, human HPC survive and proliferate in extramedullary sites. Since MS cells are ubiquitous in the human BM, spleen, and liver and generate osteoblasts, fibroblasts, and osteoclasts, they might represent an alternative or even the essential niche in the human system to precisely define the role of β1-integrins [28, 35–37].

To verify whether soluble or cellular determinants play a role for this process, we have exposed the HPC to cytokines, cell surface-associated molecules (i.e., fixed MS cells), or complete cellular determinants (vital MS cells). Cytokines were able to induce cell divisions and, to a very limited extent, if at all, self-renewing cell divisions. Only direct contact with vital MS cells was able to increase the self-renewing divisions significantly. Whereas some authors suggest that soluble regulatory molecules released by stromal cells might play a role [24, 28, 38–43], we have unequivocally demonstrated the significance of cell-surface-associated molecules and vital MS cells. This is in alignment with previous results of our group and other authors [1, 2, 6–8, 37, 45–48]. Srour et al. have recently confirmed that the initial HPC divisions were independent of the presence of cytokines such as SCF, Flt-3L, IL-3, IL-6, and other soluble molecules [49]. Calvi et al. [33] and Zhang et al. [34] have corroborated the significance of direct contact between HPC and osteoblasts as stem cell niche for the regulation of the hematopoietic self-renewal capacity in the murine bone marrow.

By applying an β1-integrin function blocking antibody and a novel in vitro assay allowing the immediate identification and follow-up of HPC, we have demonstrated that β1-integrins play a significant role not only in the interaction between HPC and MS cells but also in the regulation of the long-term fate of HPC by favoring initial self-renewing divisions and the survival of primitive HPC. Treatment of CD34+133+ HPC with the mbAb against β1-integrins inhibited the adhesion of HPC to MS cells and induced a significant reduction in the proportion of self-renewing divisions and yield of LTC-IC. Moreover, β1-integrin-mediated contact in the first hours was crucial for the maintenance of stemness. Other authors have confirmed the critical significance of the initial cell divisions [48, 49]. β1-Integrin ligands alone (e.g., fibronectin, VCAM-1), however, showed no effect on the cell division symmetry and self-renewal of HPC, indicating an indirect effect of these receptors.

Although antibody blocking of VLA4 and VLA5 of human c-kit+ BM cells has been shown to suppress human hematopoiesis in long-term bone marrow cultures, studies in murine models have led to conflicting results [18]. In vivo experiments with β1-integrin-deficient fetal HPC showed only a minor significance of these receptors for the generation, maintenance, and differentiation of HPC, although their migration and homing to primary and secondary sites of hematopoiesis was severely impaired [19, 20]. Arroyo et al. reported that during embryonic development in the murine model, multipotent progenitors could migrate to the fetal liver, spleen, thymus, and bone marrow in the absence of {alpha}4-integrins and concomitantly β1- and β7-integrins but their differentiation into erythroid, myeloid, and lymphoid lineages was inefficient and severely compromised at perinatal and postnatal stages [51, 52]. Whereas the early phase of hematopoiesis in fetal development was circumvented by transplantation of {alpha}4–/– embryonic HPC into adult bone marrow, long-term hematopoiesis was not affected, suggesting different requirements for {alpha}4-integrins during different phases of ontogeny [53].

For adult hematopoiesis, Brakebusch et al. have shown that β1-integrin deficiency completely prevents engraftment of irradiated recipient mice, but deletion after engraftment results in a normal long-term hematolymphoid differentiation potential and a normal retention of progenitor cells in the bone marrow, with the exception of a transient defect in thymus colonization and T-cell-dependent IgM-antibody response [21]. Similar results have recently been reported by Bungartz et al. using mice with a deletion of the β1- and β7-integrin genes restricted to the hematopoietic system. Although the absence of {alpha}4β1- and {alpha}4β7-integrins causes alterations in numbers and in distribution of progenitor cells, they are not essential for the differentiation of lymphocytes or myelocytes or the proceeding of hematopoiesis [22].

Nevertheless, in all these studies, the β1-integrin deficiency was induced after engraftment had occurred. Thus, the impact of β1-integrins in early hematopoiesis cannot be deduced by these experiments. We have used an in vitro assay that permitted the monitoring of primitive cells during the initial phase of cell divisions and have demonstrated a pivotal role for β1-integrins in the promotion of self-renewing cell divisions during that period. This is in alignment with data from Priestley et al. [54] and Scott et al. [55], who have also recently shown that {alpha}4β1- and {alpha}4β7-integrins were essential for the self-renewal of stem cells during regenerative stress but not during homeostasis in the adult bone marrow.

In summary, our data have provided evidence that β1-integrins play an essential role in the regulation of the initial self-renewing cell divisions of HPC by the stromal environment and the maintenance of stemness within the first 72 hours of homing.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
This work was supported by Deutsche Forschungsgemeinschaft HO 914/2-1, HO 914/2-3, HO 914/3-1, Joachim Siebeneicher-Stiftung, Bundesministerium für Bildung und Forschung FKZ01GN0107, and the National Genome Research Network NGFN-2 (N3NV-S19T05, EP-S19T01). We thank Dr. Renate Alexi and Heinrich Lannert for providing leukapheresis samples and Klara R. Jurutka for proofreading.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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