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THE STEM CELL NICHE

Molecular and Secretory Profiles of Human Mesenchymal Stromal Cells and Their Abilities to Maintain Primitive Hematopoietic Progenitors

^aDepartment of Medicine V, University of Heidelberg, Heidelberg, Germany;
^bDepartment of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany;
^cCytonet GmbH, Heidelberg, Germany;
^dDepartment of Pediatrics, Johann-Wolfgang Goethe-University, Frankfurt, Frankfurt am Main, Germany

Key Words. Mesenchymal stromal cells • Hematopoietic progenitor cells • Feeder layer • Adhesion • Secretory profile • Molecular profile

Correspondence: Wolfgang Wagner, M.D., Ph.D., Department of Medicine V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Telephone: +49 6221 56 8001; Fax: +49 6221 56 5813; e-mail: wolfgang_wagner{at}med.uni-heidelberg.de

Received on April 16, 2007; accepted for publication on July 1, 2007.

First published online in STEM CELLS EXPRESS  July 5, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self-renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively, and this may influence their biologic properties. In this study, we have compared human MSC isolated from bone marrow (BM) using two culture conditions, from cord blood (CB), and from adipose tissue (AT). The ability to maintain long-term culture-initiating cell frequency and a primitive CD34⁺CD38^– immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays, enzyme-linked immunosorbent assay, and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations, but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin-11, N-cadherin, vascular cell adhesion molecule 1, neural cell adhesion molecule 1, and integrins were highly expressed in MSC preparations derived from BM and CB. Thus, MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell-cell junctions but not with secretory profiles.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Regulation of self-renewal versus differentiation of hematopoietic progenitor cells (HPC) is controlled by specific interactions with the microenvironment in the bone marrow. In the murine model, specialized spindle-shaped N-cadherin-expressing osteoblasts located in the endosteum have been suggested to play an important role [1]. Other cell types, such as osteoclasts, stromal cells, and endothelial cells, as well as extracellular matrix components, might also play an essential role [2, 3]. Most of these studies on interactions between hematopoietic stem cells and the niche have been performed in the murine model. For human hematopoiesis and especially for the specific interactions between the human hemopoietic stem cells (HSC) and the cellular determinants of the niche, it is preferable to use cellular determinants derived from human origin.

Numerous reports have demonstrated the vital role of stroma feeder layers for maintenance of multipotency of HPC ex vivo [4–7]. Various stroma cell preparations, including mesenchymal stem cells (alternatively named mesenchymal stromal cells [MSC]) [8], have been shown to maintain HPC in an undifferentiated state with varying degrees of efficiency [6, 9–14]. Moreover, there are ongoing clinical studies using MSC for the ex vivo expansion of HPC prior to transplantation.

MSC populations have been isolated from various human tissues using different culture methods [15–21]. MSC preparations are commonly defined by plastic adherent growth, by a panel of surface markers, and by their in vitro differentiation capacity. Isolation methods have a tremendous impact on the composition of these cell preparations [22–24]. In this study, we have compared the hematopoiesis supportive potential of MSC preparations derived from human bone marrow (BM), umbilical cord blood (CB), and human adipose tissue (AT). Combined with gene expression analysis and chemokine secretion profiles, we have provided evidence that specific molecular profiles are associated with hematopoiesis supportive function.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Cultivation of Feeder Layer Cells
Mesenchymal stromal cells from human BM, human umbilical CB, and human AT were isolated as described before [22]. All samples were taken after informed consent using guidelines approved by the Ethics Committee on the Use of Human Subjects at the University of Heidelberg. BM-MSC were obtained from the same donors using two different growth conditions: BM-MSC^M1 were isolated in culture medium M1 previously described by M. Reyes and colleagues [25]. BM-MSC^M2 were cultivated in the commercially available Poietics Human Mesenchymal Stem Cell Medium (M2; PT-3001; Cambrex, Walkersville, MD, http://www.cambrex.com) following the manufacturer's instructions. Culture of CB-MSC was initiated as previously described by Kogler et al. [21] and, after the initial passages, expansion of the cells was performed in MesenCult basal medium (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) as previously described by L. Hou and colleagues [26]. AT-MSC were obtained from elective liposuction procedures and isolated in the culture medium M1. HS68 cells (human newborn foreskin fibroblasts) (CRL-1635; American Type Culture Collection, Manassas, VA, http://www.atcc.org) were cultured in Dulbecco's modified Eagle's medium (DMEM)-HG (Cambrex) with 2 mM L-glutamine, 100 U/ml penicillin/streptomycin (Gibco, Grand Island, NY, http://www.invitrogen.com), and 10% vol/vol fetal calf serum (FCS) (Stem Cell Technologies). The murine fetal liver cell line AFT024 (a kind gift from I. R. Lemischka, Princeton University, Princeton, NJ, U.S.) was maintained in DMEM (Gibco) and supplemented with 20% FCS, 50 µM 2-Mercaptoethanol (Bio-Rad, Hercules, CA, http://www.bio-rad.com), 1% vol/vol penicillin/streptomycin, and 1% vol/vol L-glutamine as described before [27].

Isolation of Hematopoietic Progenitor Cells
HPC were collected from fresh umbilical cord blood after informed consent using guidelines approved by the Ethics Committee on the Use of Human Subjects at the University of Heidelberg. Mononuclear cells were isolated after centrifugation on Ficoll-Hypaque (Biochrom AG, Berlin, http://www.biochrom.de). CD34⁺ cells were enriched with a monoclonal anti-CD34 antibody labeled using magnetic beads on an affinity column (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). After additional staining with anti-CD34-allophycocyanin (APC) (Becton Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), further purification was achieved using the FACS-Vantage-SE flow cytometry system. Staining with propidium iodide (PI) was performed to allow exclusion of nonviable cells.

Adhesion Assay
To analyze cell-cell adhesion, we used our previously described assay based on gravity force [28]. In brief, adhesive press-to-seal silicone isolators with eight wells, 9-mm diameter, 1.0 mm deep (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), were fixed on glass slides, and confluent feeder layer were grown in these wells. CD34⁺ HPC were stained with the fluorescent membrane dye PKH26, and approximately 10,000 HPC were seeded in each well. After 1 hour, HPC were analyzed on the feeder layer cells. The adhesion array was then inverted (180° flip), and after 1 hour adherent cells remained attached to feeder layer cells, whereas nonadherent cells dropped down and could be observed on the focus level of the cover slide. Single images were acquired of the same region on the adhesion array before the inversion (all cells) as well as after the inversion in the focus level of feeder layer cells (adherent cells) and on the lower glass slide level (nonadherent cells) [28]. Mean and standard deviation of the proportion of adherent cells have been calculated for each well, and these values have been considered to determine the overall mean and SD of all experiments with the same conditions. We have adopted the two-sided, unpaired Student's t test to estimate the probability of differences in adherence under different experimental conditions. A p value of p < .05 was considered to be statistically significant.

Maintenance of CD34⁺CD38^– Immunophenotype
Confluent layers of BM-MSC^M1, BM-MSC^M2, CB-MSC, AT-MSC, HS68 cells, or AFT024 cells were irradiated (20 Gy) in 24-well plates. CD34⁺ cells were then cocultured with these feeder layers in RPMI medium with 10% FCS. All cells were harvested after 4 and 8 days by vigorous pipetting, washed in phosphate-buffered saline (PBS), and stained with CD34-APC and CD38-fluorescein isothiocyanate (FITC). Expression of CD34 and CD38 antigens was then analyzed by flow cytometry. Reliable discrimination between MSC and HPC was possible according to their different forward-scatter and side-scatter signals. In addition, feeder cells demonstrated a high autofluorescence in the PI channel [27]. The total number of PI negative events was determined as a parameter for cell proliferation after 4, 8, and 12 days of cocultivation either directly on the irradiated feeder layer or in transwells (0.4 µm pore size; Corning, Corning, NY, http://www.corning.com) above the feeder layer (noncontact conditions).

Long-Term Culture-Initiating Cell Assay
Long-term culture-initiating cell (LTC-IC) frequency [29] was compared within purified CD34⁺ cells upon cocultivation with different feeder layer cells. Confluent layer of BM-MSC^M1, BM-MSC^M2, CB-MSC, AT-MSC, HS68 cells, or AFT024 cells were irradiated (20 Gy) in 96-well plates. CD34⁺ cells were plated in limiting dilutions (11 replicates per concentration: 30, 10, 3, 1 cell per well) on these feeder layer and cultured in long-term culture medium (Iscove's modified Dulbecco's medium [Gibco] with 12.5% FCS, 12.5% horse serum [Terry Fox Laboratories, Vancouver, Canada, http://www.bccrc.ca/tfl], 2 mmol/l L-glutamine [Gibco], penicillin [1,000 U/ml], streptomycin [100 U/ml; Gibco], and 10^–6 mmol/l hydrocortisone). After 5 weeks, cells were overlaid with clonogenic methylcellulose medium (1.12% methylcellulose [Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com] supplemented with 3 IU/ml erythropoietin [Amgen, Thousand Oaks, CA, http://www.amgen.com] and 7.5% supernatant of the bladder carcinoma cell line 5637) as described before [30]. Cultures were scored for secondary colony-forming cells after an additional 2 weeks of growth. Three independent experiments were performed in duplicate. To estimate the probability of differences in LTC-IC frequency, we used the paired Student's t test.

Analysis of Microarray Data
Global gene expression profiles were determined using the Human Genome Microarray with 51,145 different cDNA clones of the UnigeneSet-RZPD3 [31]. Microarray experiments of BM-MSC^M1, BM-MSC^M2, CB-MSC, AT-MSC, and HS68 cells have been described before (four donor samples for each MSC isolation protocol and a technical replica for each hybridization) [22]. The final microarray data, including the description of all spotted expressed sequence tags (ESTs) (according to Minimal Information About Microarray Experiments requirements), were submitted to the public microarray database ArrayExpress (http://www.ebi.ac.uk/arrayexpress; accession number: E-EMBL-4). These data were now reanalyzed with regard to differences in the hematopoiesis supporting activity of MSC preparations as follows. Genes were selected that were significantly higher expressed in BM-MSC^M1 versus AT-MSC, BM-MSC^M2 versus AT-MSC, and CB-MSC versus AT-MSC (t test: p < .01 for each comparison). The list of the 478 different ESTs that fulfilled these criteria is presented in supplemental online Table 2. These genes were further classified by Gene Ontology analysis using GoMiner software (http://discover.nci.nih.gov/gominer), and representation in functional categories was analyzed by Fischer's exact p value test (p < .01).

Immunoblot
Cell lysates were obtained by 15 minutes of incubation with lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton, 1% protease inhibitor cocktail [Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com]). Protein concentration of all samples was determined by Bradford assay, and equal amounts were resolved on each lane of 4%–12% Tris-glycine gradient gels (anamed, Darmstadt, Germany, http://www.anamed.org). Proteins were transferred on a polyvinylidene difluoride membrane (Millipore, Billerica, MA, http://www.millipore.com), labeled with monoclonal anti-cadherin-11 antibody (clone 5H2H5; Zymed Laboratories, San Francisco, http://www.invitrogen.com/antibodies) or anti-N- cadherin (clone 32; Becton Dickinson), and detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com).

Assessment of Chemokines in Conditioned Culture Media
Chemokine secretion of MSC preparations was analyzed in conditioned culture media. BM-MSC^M1, BM-MSC^M2, CB-MSC, AT-MSC, or HS68 cells were seeded in 75-cm² flasks and grown to 70%–80% confluency. Thereafter, culture media were exchanged for RPMI (1% vol/vol glutamine; 1% vol/vol penicillin/streptomycin). Using the same defined culture medium was necessary to compare chemokine production of the different cell preparations. After 72 hours, the conditioned medium was carefully harvested, centrifuged for 10 minutes at 5,000g to remove debris, and stored at –80°C for further analysis.

Qualitative assessment of 120 cytokines was performed using RayBio Human Cytokine Antibody Array C (membranes VI and VII; Hölzel Diagnostica, Köln, Germany, http://www.hoelzel-biotech.com) according to the manufacturer's instructions. Horseradish peroxidase luminescence was detected by Lumi-Imager F1 (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) and mean signal intensity determined for each spot. For comparison of different experiments, data were background subtracted (blanks) and normalized by positive controls that are included on the antibody arrays. The chemokine profile was further analyzed using the microarray tool TIGR MeV Version 2.2 Software (Institute of Genomic Research, Rockville, MD, http://www.tigr.org) for an unsupervised clustering of data sets (Spearman rank correlation).

The presence of granulocyte macrophage–colony-stimulating factor (GM-CSF), interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12p70, interferon (IFN)-, macrophage inflammatory protein-1 (MIP-1), and tumor necrosis factor (TNF)- was quantitatively determined using a multiplex cytokine bead array (Flex-Set Cytometric Bead Array [CBA]) according to the manufacturer's protocol (Becton Dickinson). Flow cytometric analysis was performed using the Becton Dickinson FACSArray Bioanalyzer and CBA analysis FCAP software (Becton Dickinson). A total of 2,700 events were acquired following the protocol supplied. The minimum detection levels for each cytokine were: GM-CSF, 4.9 pg/ml; IL-2, 8.4 pg/ml; IL-4, 0.3 pg/ml; IL-6, 0.2 pg/ml; IL-10, 2.3 pg/ml; IL-12p70, 2.2 pg/ml; IFN-, 0.3 pg/ml; MIP1, 4.6 pg/ml; and TNF-, 0.7 pg/ml.

Concentrations of stromal cell-derived factor (SDF)1, insulin-like growth factor binding protein (IGFBP)2, and osteoprotegerin (OPG) were quantified by RayBio enzyme linked immunosorbent assays (ELISAs; Hölzel Diagnostica) in conditioned culture medium. Sensitivities of the individual assays were as follows: IGFBP2, <5 pg/ml; OPG, <1 pg/ml; SDF1, <3 pg/ml. All assays were performed according to manufacturer's instructions.

Cultivation of HPC with IGFBPs
Five hundred CD34⁺ cells were plated in 96-well plates and cultured either in serum-free Stemline II expansion medium (Sigma-Aldrich) or in RPMI medium with 10% FCS. The Stemline II cultures contained thrombospondin (80 ng/ml), stem cell factor (50 ng/ml), and FL-3 ligand (50 ng/ml). IGFBP1 (RayBiotech, Norcross, GA, http://www.raybiotech.com), IGFBP2 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), or IGFBP3 (RayBiotech) were supplemented as indicated (10 ng/ml or 100 ng/ml). After 7 and 14 days, cells were harvested, washed in PBS, stained with CD34-APC and CD38-FITC, and analyzed by flow cytometry. Cell numbers were assessed by counting in a counting chamber.

	RESULTS

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Adhesion of HPC to MSC Preparations
We have isolated human MSC from BM, AT, and CB. The marrow-derived MSC were cultured under two culture media (M1 and M2). Immunophenotypic analysis, in vitro differentiation assays, and microarray analysis of these cell preparations have been reported in previous studies [22, 23]. Here, we have quantified cell-cell interaction between these MSC preparations and HPC using our novel adhesion assay based on gravitational force [28, 32]. This standardized assay involves no shear stress and provides a reliable quantification of the adherent and nonadherent fraction of CD34⁺ cells; 86% ± 6% of CD34⁺ cells adhered to BM-MSC^M1, 63% ± 8% to BM-MSC^M2, and 75% ± 8% to CB-MSC. Adhesion was also high on HS68 fibroblasts (81% ± 11%) and AFT024 cells (79% ± 4%). In contrast, only 46% ± 10% of the CD34⁺ cells adhered to MSC that were derived from adipose tissue, and this was significantly less in comparison with all other cell preparations (BM-MSC^M1: p = .002; BM-MSC^M2: p = .044; CB-MSC: p = .005; HS68: p = .001; and AFT024: p = .005; Fig. 1).

View larger version (14K): [in this window] [in a new window]   Figure 1. Adhesion of CD34+ cells to different mesenchymal stromal cell (MSC) preparations. The percentage of adherent CD34+ cells was determined on different feeder layer cells using our standardized adhesion assay based on gravitational force [28]. Adhesion to MSC from adipose tissue was significantly lower in comparison with MSC from bone marrow, cord blood, HS68 fibroblasts, and AFT024 cells (n = 6; * p < .05; ** p < .01; *** p < .005). Abbreviations: AT-MSC, adipose tissue-mesenchymal stromal cells; BM-MSC-M1, bone marrow-mesenchymal stromal cells in medium 1; BM-MSC-M2, bone marrow-mesenchymal stromal cells in medium 2; CB-MSC, cord blood-mesenchymal stromal cells.

View larger version (41K): [in this window] [in a new window]   Figure 2. Mesenchymal stromal cells (MSC) from bone marrow and cord blood maintain a more primitive immunophenotype. CD34+ cells were cultured on different feeder layers in RPMI with 10% fetal calf serum. After 0, 4, and 8 days, cells were harvested and stained with CD34-APC, CD38-FITC (a representative experiment after 8 days is presented in [A]). For statistical analysis, median expression of CD34 and CD38 was determined in relation to cocultivation with HS68 cells within each experiment (B, C). CD34 expression was significantly lower and CD38 expression was significantly higher upon cocultivation with MSC from adipose, whereas MSC from bone marrow and cord blood retained a more primitive immunophenotype (n = 3; paired Student's t test; * p < .05; ** p < .01; *** p < .001). Abbreviations: APC, allophycocyanin; AT-MSC, adipose tissue-mesenchymal stromal cells; BM-MSC-M1, bone marrow-mesenchymal stromal cells in medium 1; BM-MSC-M2, bone marrow-mesenchymal stromal cells in medium 2; CB-MSC, cord blood-mesenchymal stromal cells; d, day; FITC, fluorescein isothiocyanate.

View larger version (11K): [in this window] [in a new window]   Figure 3. Mesenchymal stromal cells (MSC) from bone marrow and cord blood maintain more LTC-IC. Maintenance of long-term culture-initiating cells was compared with MSC from bone marrow (two culture conditions: BM-MSCM1 and BM-MSCM2), cord blood (CB-MSC), adipose tissue (AT-MSC), and fibroblasts (HS68). LTC-IC frequency was significantly lower upon cocultivation with MSC from adipose tissue. Standard deviation is presented as error estimate (n = 6; paired Student's t test; * p < .05; ** p < .01; *** p < .001). Abbreviations: AT-MSC, adipose tissue-mesenchymal stromal cells; BM-MSC-M1, bone marrow-mesenchymal stromal cells in medium 1; BM-MSC-M2, bone marrow-mesenchymal stromal cells in medium 2; CB-MSC, cord blood-mesenchymal stromal cells; LTC-IC, long-term culture-initiating cell.

View larger version (20K): [in this window] [in a new window]   Figure 4. Proliferation of CD34+ cells under contact versus noncontact conditions. Proliferation of CD34+ cells upon either cultivation in contact with stromal cells or under noncontact growth conditions in transwells was determined according to the total number of counted events by flow cytometry. There was a continuous increase in counted events in a time course of 4, 8, and 12 days, and this was significantly higher under contact conditions with all types of stromal cells (n = 4; *** p < .001; ** p < .01, * p < .05). Furthermore, proliferation was significantly higher on each of the mesenchymal stromal cell preparations in comparison with HS68 fibroblasts and AFT024 cells. Abbreviations: AT-MSC, adipose tissue-mesenchymal stromal cells; BM-MSC-M1, bone marrow-mesenchymal stromal cells in medium 1; BM-MSC-M2, bone marrow-mesenchymal stromal cells in medium 2; CB-MSC, cord blood-mesenchymal stromal cells; d, day.

View larger version (86K): [in this window] [in a new window]   Figure 5. Analysis of conditioned culture medium. Culture medium was conditioned by the different mesenchymal stromal cell (MSC) preparations for 3 days and analyzed by RayBiotech Chemokine arrays representing 120 different chemokines. The results were normalized by the positive controls and further analyzed by unsupervised clustering. The measurements of two biological replicas (1 and 2) clustered together, indicating that there are reproducible differences in the cytokine expression of these MSC preparations. However, there was not a clear concordance of chemokine profiles with the potential to maintain stemness. Abbreviations: AT-MSC, adipose tissue-mesenchymal stromal cells; BM-MSC-M1, bone marrow-mesenchymal stromal cells in medium 1; BM-MSC-M2, bone marrow-mesenchymal stromal cells in medium 2; CB-MSC, cord blood-mesenchymal stromal cells.

View larger version (47K): [in this window] [in a new window]   Figure 6. Gene expression of supportive feeder layer. To correlate differences observed in supportive function of MSC preparations, we have identified those genes that were significantly higher expressed in BM-MSCM1 versus AT-MSC, BM-MSCM2 versus AT-MSC, and CB-MSC versus AT-MSC. Four hundred and seventy-eight expressed sequence tags passed these filter criteria (A). These included 161 nonredundant genes that were classified by Gene Ontology terms. The most significantly over-represented Gene Ontology groups are presented ([B]; p < .01). A selection of relevant genes is depicted in (C). As a reference, all MSC samples were cohybridized with RNA of HS68 fibroblasts (green: higher expression in HS68 fibroblasts; red: higher expression in the corresponding MSC sample). Immunoblot analysis of cadherin-11 and N-cadherin also revealed differential expression of these adhesion proteins on protein level. A representative experiment of three is shown (D). Abbreviations: Acc.No, accession number; AT-MSC, adipose tissue-mesenchymal stromal cells; BM-MSCM1, bone marrow-mesenchymal stromal cells in medium 1; BM-MSCM2, bone marrow-mesenchymal stromal cells in medium 2; CB-MSC, cord blood-mesenchymal stromal cells; GO, Gene Ontology; MSC, mesenchymal stromal cells.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

A multitude of preparative protocols for the acquisition, separation, in vitro cultivation, and expansion of MSC has been described, and it is still controversial whether these cell preparations are related to each other on a molecular basis [16, 21, 34–40]. Isolation methods of MSC might therefore affect their biologic properties and especially their ability to maintain stemness of HPC. In this study, we have shown that the potential of MSC from BM and CB is significantly higher as compared with MSC from AT. This potential has been reflected in the adhesiveness of HPC, in the impact on alterations in immunophenotype of HPC, and above all in the maintenance of LTC-IC. A recent study by Corre and coworkers has also reported that adipose cells support differentiation but not self-renewal of HPC [41].

The essential role of direct cell-cell contact for the regulation of self-renewal and differentiation of adult stem cells has been shown in various cell systems. Specific junctional complexes might play a similar role in the hematopoietic system [42]. We have previously demonstrated that blocking antibodies for ITGA5 and ITGB1 reduced adhesion of HPC to MSC feeder layers and that ITGB1 is involved in the maintenance of self-renewal upon interaction [28, 33]. In this study, we have shown that CD34⁺ cells adhered significantly more to BM-MSC^M1, BM-MSC^M2, CB-MSC, and AFT024 cells than to AT-MSC. This adhesiveness for HPC correlated with the ability of these feeder layers to maintain stemness. However, CD34⁺ cells adhered also to the HS68 cell line, indicating that cell-cell adhesion alone is not sufficient as a measure for maintenance of stemness. Nor does the cytokine secretion pattern correlate with maintenance of stemness as shown by the secretory profiles simultaneously studied.

For the first time we have described a combination of gene expression profiles of MSC with different abilities to maintain stemness of HPC. Similar studies have previously been described using murine cell lines [43–45]. In contrast to these studies, we have used primary feeder layer cells that have not been immortalized through transfection and that are suitable for clinical use. Genes upregulated in MSC preparations with the ability to maintain stemness included cadherin-11; N-cadherin; integrins -1 (ITGA1), -5 (ITGA5, CD49e), and β-1 (ITGB1, CD29); VCAM1; NCAM1; and THBS1. Most of these proteins have already been demonstrated to play an essential role in maintenance of self-renewal capacity and stemness of HPC [33]. Strikingly, the higher expression of genes involved in Gene Ontology categories "cell adhesion," "cell-cell adhesion," and "integrin complex" in supportive MSC preparations was in line with the higher adhesion of CD34⁺ cells to these feeder layers compared with MSC from AT. Cadherin-11, NCAM, and thrombospondin 2 were also among the genes more highly expressed in AFT024 cells than in nonsupportive murine cell lines [43, 46]. This observation indicates that the previously mentioned proteins are involved in direct cell-cell contact and in maintenance of stemness. Zhang et al. have provided evidence that the specific interaction of HPC and osteoblasts in the murine model is mediated by N-cadherin [1]. On the other hand, we have previously examined adherent and nonadherent fractions of CD34⁺ cells on BM-MSC^M1 [28]. Gene expression analysis revealed that cadherin-11, VCAM1, thrombospondin 2, ITGBL1, and CTGF were among the genes with highest overexpression in the adherent fraction of CD34⁺ cells. It is intriguing that these adhesion molecules are highly expressed in adhesive feeder layers and in the adherent fraction of HPC. These results imply that molecular mechanisms essential in maintenance of stemness are mediated by an orchestra of cell-cell junction proteins (cadherin-11, N-cadherin, NCAM1, VCAM1) and cell-matrix junction proteins (ITGA5, ITGB1).

Besides cell-cell adhesion, additional mechanisms such as soluble molecules have been reported to play a role in the interaction between HPC and their niche. Various genes encoding for secreted proteins were higher expressed in BM-MSC^M1, BM-MSC^M2, and CB-MSC than in AT-MSC, and these included IGFBP-3 and -4, pleiotrophin (PTN), and osteopontin (SPP1). All of these secreted proteins have previously been described as highly expressed in embryonic murine supportive stromal clones by Oostendorp and his colleagues [44]. Furthermore, PTN was also higher expressed in AFT024 cells compared with nonsupportive murine cell lines [43]. However, soluble regulatory molecules alone were not enough to maintain stemness of HPC, and the ability to maintain self-renewal capacity did not correlate with the chemokine secretory profiles. In all cell lines, we observed a high expression of G-CSF, GRO, IL6, IL8, MCP1, TIMP1, and TIMP2, and this was in line with previous publications [41, 47]. The only significant difference was found in IGFBP-1 and -2, which were higher expressed in supportive BM-MSC^M1, BM-MSC^M2, and CB-MSC compared with nonsupportive AT-MSC and HS68 cells. It has been suggested that IGFBPs are involved in the regulation of pro-B cell development as well as regulation and proliferation of T cells [48, 49]. Furthermore, IGFBP-3 has been suggested to promote proliferation of primitive HPC in vitro, whereas other authors did not observe an effect of IGFBP-3 on GFU-GM or BFU-E [50, 51]. We have demonstrated that IGFBP-1, IGFBP-2, and IGFBP-3 had no effect on proliferation or maintenance of a primitive immunophenotype in culture medium without cellular support. The lack of consistency of different hematopoietic supportive function of MSC with their chemokine secretory profile underlines the significance of direct cell-cell contact among HPC with very specific cellular determinants in maintaining stemness and that human MSC are not just more efficient fibroblasts.

Our comparative study highlights various adhesion proteins that are higher expressed in feeder layers that maintain primitive HPC. In the future, the functional role of these proteins needs to be systematically analyzed. Various approaches including small interfering RNA, blocking function antibodies, and overexpression need to be performed for each of these adhesion proteins alone as well as in combinations to asses their relative significance in regulative cell-cell interaction.

	SUMMARY

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Our results have provided evidence that direct cell-cell contact with specific mesenchymal stromal cells derived from the bone marrow or cord blood is essential for maintenance of stemness and that secretory profiles did not elucidate their ability to maintain self-renewal. The essential role of junctional proteins in this process is implicated by differences in cell-cell adhesion as well as by differential expression of adhesion proteins such as cadherin-11, N-cadherin, NCAM1, VCAM1, and various integrins.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank Kerstin Horsch and Katrin Miesala for excellent technical assistance in cell culture and cell sorting and Sophia Derdak (dkfz, Heidelberg, Germany) for the valuable contribution in the analysis of RayBio Human Cytokine Antibody Arrays. We are grateful to Dr. I. R. Lemischka (Princeton University) for providing AFT024 cells. This work was supported by the German Ministry of Education and Research (BMBF) within the National Genome Research Network NGFN-2 (EP-S19T01) and within the supporting program "cell-based regenerative medicine" (START-MSC), the German Research Foundation DFG (HO 914/2–3), and the Joachim Siebeneicher-Stiftung, Germany. W.W. and C.R. contributed equally to this article.

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