Bone marrow-derived mesenchymal stromal cells differ in their attachment to fibronectin-derived peptides from term placenta-derived mesenchymal stromal cells
© Maerz et al. 2016
Received: 5 August 2015
Accepted: 18 November 2015
Published: 11 February 2016
Human mesenchymal stromal cells (MSCs) can be isolated from different sources including bone marrow and term placenta. These two populations display distinct patterns of proliferation and differentiation in vitro. Since proliferation and differentiation of cells are modulated by cell–matrix interactions, we investigated the attachment of MSCs to a set of peptide-coated surfaces and explored their interactions with peptides in suspension.
Human MSCs were isolated from bone marrow and term placenta and expanded. Binding of MSCs to peptides was investigated by a cell-attachment spot assay, by blocking experiments and flow cytometry. The integrin expression pattern was explored by a transcript array and corroborated by quantitative reverse transcription polymerase chain reaction and flow cytometry.
Expanded placenta-derived MSCs (pMSCs) attached well to surfaces coated with fibronectin-derived peptides P7, P15, and P17, whereas bone marrow-derived MSCs (bmMSCs) attached to P7, but barely to P15 and P17. The binding of bmMSCs and pMSCs to the peptides was mediated by β1 integrins. In suspension, expanded bmMSCs barely bind to P7, P13, P15, and less to P14 and P17. Ex vivo, bmMSCs failed to bind P7, but displayed a weak interaction with P13, P14, and P15. In suspension, expanded pMSCs displayed binding to many peptides, including P4, P7, P13, P14, P15, and P17. The differences observed in binding of bmMSCs and pMSCs to the peptides were associated with significant differences in expression of integrin α2-, α4-, and α6-chains.
Human bmMSCs and pMSCs show distinct patterns of attachment to defined peptides and maintain differences in expression of integrins in vitro. Interactions of ex vivo bmMSCs with a given peptide yield different staining patterns compared to expanded bmMSCs in suspension. Attachment of expanded MSCs to peptides on surfaces is different from interactions of expanded MSCs with peptides in suspension. Studies designed to investigate the interactions of human MSCs with peptide-augmented scaffolds or peptides in suspension must therefore regard these differences in cell–peptide interactions.
KeywordsMesenchymal stromal cells Cell attachment Integrins Bone marrow stem cells Placenta stem cells
Multipotent mesenchymal stromal cells (MSCs) have been detected in and isolated from various tissues and niches. Today the main source for isolation of MSCs is the bone marrow in the iliac crest or in the femoral shaft [1, 2]. Closely related mesenchymal cells have been described in the white pulp of teeth [3–5] and as pericytes along the vasculature of adipose tissue [6–9], the endometrial and fetal parts of placenta [10–13], in muscle tissue [14, 15], and in inner organs [16, 17]. Furthermore, related cells have been isolated from umbilical cord blood  and peripheral blood [19, 20], urine , amniotic fluid and Wharton’s jelly of the umbilical cord [22, 23], and even from avascular tissue [24, 25]. The niches for these MSCs or MSC-like cells differ significantly in their mechanical and chemical composition. Bone marrow, for instance, is rather stiff (E ≈ 100 kPa), cartilage is considerably softer (E ≈ 30 kPa), muscle is quite elastic (E ≈ 12 kPa), and adipose tissue very flexible (E <10 kPa) . Moreover, in bone marrow, type I, III, V and VI collagen, laminin isoforms containing the α4-, and α5-chains, fibronectin, and glycosaminoglycans dominate the stem cell niche [27–31], whereas pericytes of placenta are found in contact with laminin α2- and α5-chains and type IV collagen of the basal lamina and adjacent to fibronectin .
The MSCs from bone marrow (bmMSCs) express a significantly different transcriptome compared to MSCs from pancreas or placenta [16, 33]. Human bmMSCs differ in their growth kinetics and expression of integrin α4 from placenta-derived MSCs (pMSCs) . Moreover, MSCs from adipose tissue express CD34 [35, 36], an antigen not found on bmMSCs [37–39]. Our recent studies are in line with these reports as we find significant differences between bmMSCs and pMSCs in their osteogenic differentiation capacities , expression of Runx2, WISP2, osteoglycin and osteomodulin , and expression of the stem cell markers alkaline phosphatase and CD146 .
Previously we investigated the binding and attachment of bmMSCs to proteins and peptides in comparison to fibroblasts . There, fibroblasts differed from bmMSCs in both binding, as determined by the multiple substrate array technique , and short-term attachment . Based on the fact that bmMSCs and pMSCs differed in their proliferation and differentiation capacities [13, 33, 34], and proliferation and differentiation of MSCs are modulated by the extracellular matrix and integrin signaling [43–50], we investigated the interaction of bmMSCs versus pMSCs with a set of peptides and the expression of integrins in more detail.
Our results suggest that i) bmMSCs and pMSCs differ significantly in their expression of integrins, and therefore in attachment to distinct peptides. In addition, ii) interactions of MSCs with peptides on a solid phase via attachment follow different kinetics or thermodynamics compared to interactions of MSCs with the same peptides in suspension, and iii) the expression of matrix-binding receptors on bmMSCs ex vivo seems be modulated by the in vitro culture condition. This may have interesting consequences when, for instance, attachment assays are performed in vitro to investigate the mobilization and migration of MSCs in the circulation and homing to specific niches.
Preparation of MSCs from femoral bone marrow and term placenta tissue
Aspirates from human femoral bone marrow (n = 15 patients, nine females, six males, mean age 67 years, average volume 12–15 mL) were obtained from the Clinic for Trauma and Restorative Surgery, BG Trauma Center Tübingen, University of Tübingen, after written and informed consent. The fraction of mononuclear cells was enriched by density gradient centrifugation and the cells were expanded as described recently . Human term placenta was obtained from the Department of Gynecology and Obstetrics, University of Tübingen Hospital, from mothers undergoing planned Caesarean delivery after written and informed consent (n > 15 donors, mean age 34 years). The MSCs were isolated, purified and cultured in a good manufacturing practice (GMP)-compliant expansion medium as described recently . Both types of MSCs were characterized according to the criteria defined by the International Society for Cellular Therapy by flow cytometry to confirm the expression of CD73, CD90, CD105, and CD146 as well as documenting lack or very low expression of CD11b or CD14, CD34, and CD45 (not shown) [33, 37, 40, 52]. The differentiation capacities of the MSCs investigated were confirmed in vitro by induction of osteogenic, adipogenic, and chondrogenic (bmMSCs), or adipogenic and chondrogenic differentiation (pMSCs), respectively  (not shown). Dermal fibroblasts were isolated from surgical waste from the skin of patients and expanded as described (n = 4, ). In some experiments MSCs were washed (2 × phosphate-buffered saline (PBS)) detached by mild proteolysis (5 min, 37 °C, Accutase®, PAA Laboratories), washed again, counted, and aliquots of 5 × 105 viable MSCs were resuspended in 1 mL cold freezing medium (Dulbecco's modified Eagle's medium, 20 % fetal bovine serum (FBS), 10 % dimethyl sulfoxide), cooled further and stored in the gas phase of liquid N2 tanks. For additional studies, frozen aliquots of MSCs were rapidly thawed, washed twice with 25 mL medium, seeded in culture vessels, and cultured over night in GMP-compliant expansion medium. Then the cells were washed twice with PBS, detached by proteolysis (5 min, 37 °C, Accutase®), counted to confirm yield and viability, and utilized for the corresponding experiment. The study was approved by the Ethics Committee of the Medical Faculty of University of Tübingen (# 453/2011B02).
Attachment of MSCs to proteins and peptides
Attachment of MSCs or fibroblasts to proteins and peptides immobilized on plastic surfaces was explored as described previously [30, 53, 54]. As substratum for cell attachment, bovine serum albumin (BSA) was activated by maleimide-ester, coupled with peptides, and separated from residual peptides by dialysis and gel filtration . The peptide-modified BSA served as substrate for the cell attachment assays. Then cells were harvested as described above, viability was confirmed (>90 %, trypan blue dye exclusion) and 3 × 106 cells were resuspended in 1.3 mL medium supplemented with 0.1 % PSA buffer (final concentration 0.9 % BSA/PSA) and ion-mix (final concentration 1 mM CaCl2,1 mM MgCl2, 0.025 mM MnCl2); 200 μL of this cell suspension was added to the substratum spots. After incubation at ambient temperature for 15 min, attachment of the cells was analyzed by microscopy. Cells not adhering to the spots were removed by washing (4 × PBS), and the cells attached recorded by dark-field and phase-contrast optics (Leica DM IRB, Leica Wetzlar Germany). Attachment of cells to laminin-111 (LM), fibronectin (FN) or BSA (all from Sigma-Aldrich) served as controls, respectively.
In a second line of experiments, attachment of MSCs to peptides or proteins was blocked by pre-incubation of 5 × 105 MSCs per spot in 100 μL media with a function blocking monoclonal antibody (mAb) to human β1 integrin (anti-CD29, clone: 4B4, dilution 1:20, 4 °C for 30 min; Beckman Coulter Inc, USA). MSCs incubated with mAb to CD90 (clone: Thy1-A1, dilution 1:5, 4 °C for 30 min; R&D Systems) and mock-treated MSCs served as controls. Then the cells were added to the substratum spots, incubated, washed and recorded as described above.
To label cells by fluorescent dyes, 1 × 106 cells in the second passage of in vitro culture were detached by mild proteolysis (5 min, 37 °C, Accutase®), washed twice with PBS and incubated with PKH26 (red label) or PKH67 (green label) as described  using standard reagent kits (Sigma-Aldrich, Taufkirchen, Germany). To confirm high viability of MSCs in the attachment assays, cells were loaded with Calcein-AM and Ethidium-homodimer (Live/Dead cell Staining Kit II, PromoCell, Heidelberg, Germany). Then attachment of cells was explored with the labeled MSCs as described above. Live cells emitting green fluorescence at 515 nm and dead cells emitting red fluorescence at 620 nm were recorded by a fluorescence microscope.
Flow cytometry of MSCs after expansion in vitro and ex vivo
List of reagents employed: monclonal antibodies for exploring mesenchymal stromal cells by flow cytometry
CD29 / ITGB1
CD49b / ITGA2
CD49d / ITGA4
CD49f / ITGA6
List of reagents employed: peptides
Amino acid sequence
Estimated molecular weight
GEFYF DLRLK GDK
Human collagen IV α1 chain
LAIKN DNLVY VY
Human laminin α4 chain G domain
WQPPR ARITG Y
AASIK AVAVS ADR
Human laminin α1
DVISL YNFKH IY
Human laminin α4 chain G domain
EILDV (part of P17)
Human fibronectin type III repeat
DELPQ LVTLP HPNLH GPEIL DVPST
Human fibronectin type III repeat
In other experiments cells were prepared ex vivo from fresh samples of bone marrow by Ficoll® gradient centrifugation , washed, counted and directly stained with fluorescently labeled mAb to the MSC-specific antigen CD271 as described recently  (Table 1). Then, the cells were washed twice with FACS buffer and resuspended in FACS buffer with the diluted labeled peptides (Table 2), and incubated on ice. The bmMSCs were washed twice again and subjected to flow cytometry. Double labeled cells were analyzed in the dot blot mode with four quadrants. Unstained cells and cells incubated with anti-CD271 only served as controls and to set the gates.
Differences in transcripts encoding integrins
To explore the differences in the expression of integrins between bmMSC and pMSC two data sets generated by gene array using the Affymetrix GeneChip technology were employed in this study as described recently . Transcripts encoding integrins that were expressed significantly different in bmMSC versus pMSC were investigated further by quantitative reverse transcription polymerase chain reaction (qRT-PCR).
qPCR of transcripts after reverse transcription
To enumerate the steady state mRNA expression in bmMSCs versus pMSCs, cells were harvested by Accutase® (PAA) and washed by cold PBS; 1 × 106 MSCs were collected in 1.5 mL microtubes and mRNA was extracted (RNeasy, Qiagen, Hilden Germany). Reverse transcription was performed from 1 μg of total RNA (oligo-(dT)npriming, Advantage RT for PCR Kit, Clontech, Mountain View, USA) to generate the cDNA substrate for PCR. Gene-specific cDNA was enumerated by qRT-PCR (LightCycler, Roche, Mannheim, Germany)  using transcript-specific primers (MWG Eurofins, Ebersberg, Germany). Quantification of transcripts encoding GAPDH and PPIA served as references in each of the amplifications to normalize the amounts of the target gene by the FitPoint (ΔΔCt–) method . The mean values of replicate experiments and standard deviations were calculated by Excel® spread sheet software, and statistical significances between groups of data were computed with a two-sided paired Student’s t-test. Probability values (p) equal to or less than 0.05 were considered to be statistically significant.
Attachment of human MSCs to proteins and peptides
Comparison of cells attaching to peptides and proteins
In addition, we investigated the attachment of MSCs to peptides P7, P14, P15, and P17 that facilitated sufficient binding of cells prior to deep freezing (naïve) and after cryopreservation (cryo) and revitalization (Fig. 2b). In all cases, attachment of cryopreserved bmMSCs and pMSCs was lower compared to the same cells prior to deep freezing, but it was not different in bmMSC compared to pMSC (p = 0.64). The mean normalized attachment of naïve MSC dropped 2.2-fold from 43 ± 18 % to 19.2 ± 14.2 % (n = 10, p ≤ 0.0048; Fig. 2b), suggesting that cryopreservation strongly influences the interaction between cells and the extracellular matrix.
Interaction of human MSCs with peptides in suspension
Interactions of peptides with bmMSCs ex vivo
Expression of integrins in bmMSCs and pMSCs in vitro
Expression of integrins in human MSCs in vitro
Gene array (CFC)
−2.5 (p < 0.05)
(p = 0.063, s.n.s.)
−12.4 (p < 0.0001)
(p < 0.0006)
−19.3 (p < 0.0001)
(p < 0.005)
+6.8 (p < 0.0001)
(p < 0.0001)
+4.6 (p < 0.0006)
(p < 0.41, s.n.s.)
Our study provided evidence that MSCs can bind to small peptides in a specific way through integrins, that patterns of MSC–peptide interactions depend on the type of MSCs investigated, and that such interactions are modified by cell culture conditions. In addition, the attachment of cells to peptide-augmented surfaces differs from the patterns of staining of MSCs in suspension. Integrins are important receptors for anchoring cells in a tissue. They play an important role in pattern formation, tissue organization, differentiation, mobilization and homing of cells and many other physiological and pathological processes [48, 49, 63–66]. The composition of the extracellular matrix in a given tissue and even more in a (stem) cell niche is important to maintain the integrity of the tissue and at the same time its function [27, 67]. By choice of peptides such a niche or environment can possibly be mimicked on or in scaffolds to facilitate the homing or seeding of distinct cells in an in vivo situation.
We also provide evidence that fibroblasts, bmMSCs and pMSCs will attach to biomaterials modified with specific peptides in an ordered way. Therefore two- or even three-dimensional patterns of different cells and even closely related cells can be generated in vitro by modification of the scaffolds' surfaces with peptides. Blocking β1-integrins by a mAb abolished the attachment of the MSCs to both proteins and peptides. This confirmed that the peptide–cell interactions investigated in this study are highly selective and depend on β1-integrins.
However, the interaction of peptides with integrins yielded a higher dissociation constant KD compared to the KD measured for a naïve protein . The RGD peptide–a popular motif used in many attachment assays–bound with a more than 50-fold lower affinity to its receptor (KD = 1.7) compared, for instance, to fibrin, the prototype protein containing the RGD motif . Moreover, attachment of cells to peptides by integrins depends not only on the avidity of the individual ligand (i.e., peptide/protein)–receptor (i.e., integrin) interaction, but also on the blend of different integrins and expression level of integrins on a given cell surface. Therefore, in the context of regenerative medicine or tissue engineering, a selectivity of peptide-modified scaffolds means a lower affinity of such a scaffold to the cell when compared to protein-coated surfaces. This can possibly be compensated for by distributing or patterning the peptides on scaffold surfaces according to the density of integrins on the cells . Then cooperative effects will “glue” the cells to the scaffold.
As shown in one of our recent studies, expression of integrins on human MSCs is regulated by transforming growth factor beta (TGF-β) . Others reported on the role of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and epidermal growth factor in regulation of integrins on MSCs or other cells [70–72]. These cytokines are found in the sera used to complement the growth medium for human MSCs , and their titers in media may differ from the concentrations recorded in situ. Therefore, expression of integrins on cells may differ in vivo compared to in vitro. We used GMP-compliant media enriched with human plasma and platelet extract for expansion of MSCs to keep our preclinical experiments close to a future clinical application . Human platelet extract is a rich source for PDGF, TGF-β, bFGF and other factors . This explains in part the differences observed in peptide binding between MSCs ex vivo and MSCs after expansion. This finding also suggests that binding assays performed with MSCs expanded in media containing xenobiotic serum cannot completely reflect the interaction of MSCs in situ. This problem applies to MSC-specific antigens used for ex vivo staining as well since expression of CD271 on human bmMSCs was elevated on cells ex vivo, but disappeared in primary cultured cells [13, 38]. Comparably, expression of CD34 on adipose tissue-derived human MSCs varies and depends on the cell culture conditions . In addition, the concentration of growth factors detected in serum depends on the age of the donor . Therefore, not only MSC proliferation, but also integrin expression, will depend on the quality of platelet extract used to enrich the MSC media. Expression of integrins on MSCs may change during in vitro culture and expression of integrin α6 chain was lost on human umbilical cord-derived MSCs after 5 passages expansion . We used bmMSCs and pMSCs in their second passage of culture. Therefore loss of integrins on bmMSCs due to extended expansion in this time frame is not a likely event. A recent study on expression of integrins on human bmMSCs reported minimal or no detection of integrin α6, but intermediate to high expression of other integrins . In contrast, we find some expression of integrin α6 chain on bmMSCs in our experiments. This seemingly conflict of results may be caused by the differences in preparation and maintenance of the bmMSCs since we used—unless specified differently—non-cryopreserved MSCs expanded in GMP-compliant medium for two passages. Danmark and colleagues used a commercial source for bmMSCs and therefore cryopreserved cells, and a commercial MSC medium of undisclosed composition . Our data suggest that cryopreservation significantly reduced attachment of MSCs to peptides. These important differences in the experimental design may account for the different outcomes.
Moreover, short-term attachment of MSCs to surfaces is modulated in vitro by proteins found in the cell culture medium used. This is relevant when MSCs are in contact with surfaces that contain physiological ligands for high-affinity attachment of cells, such as RGD peptides. Biodegradable polymers such as lactate esters are widely used compounds for tissue engineering that do not contain natural cell binding motifs. Attachment of cells to such polymers depends therefore on the adsorption versus desorption of peptides or proteins to the polymer to which the cells then subsequently bind in the second place . However, long-term binding of MSCs to any substratum in vitro and in vivo is not only modified by the composition of the pericellular milieu, but in addition modulated by proteins and extracellular matrix components produced by the MSCs themselves. For instance, integrin α5β3- and αvβ3-mediated binding of MSCs to FN triggers migration of the cells through PDGF-BB and PDGF-R signaling . Binding of MSCs to FN or fibrin modulated their osteogenic differentiation in two- and three-dimensional cultures . Laminin-322 modulated osteogenesis of MSCs , whereas type II collagen hydrogels together with TGFβ1 promoted chondrogenesis . Our preliminary data suggest that pMSCs express, at least on a transcript level, less LAMA5, COL4A5 and COL13A1, but more COL5A3, COL14A1 and COL11A1 than bmMSCs. These differences in expression of extracellular matrix proteins by bmMSCs versus pMSCs add to the integrin-mediated differences discussed above by occupying the binding sites of their respective integrins. Moreover, MSCs also produce cytokines and growth factors including TGFβ1 thus modifying expression and conformation of integrins in an autocrine way [36, 53]. Therefore dissimilarities reported for attachment, migration, proliferation or differentiation of MSCs in different studies can be explained by the inconsistencies in the various protocols employed [45, 46, 48].
Covering β1 integrins on MSCs by a mAb blocked their migration to an infarcted heart , and others provided evidence for integrin α4 receptors in the context of MSC homing . Expression of integrins differs between stromal cells from bone marrow and adipose tissue . Our data complement these studies as we find differences in cell–peptide interactions observed between bmMSCs and pMSCs in vitro and between bmMSCs ex vivo and in vitro, and add to the evidence for the role of integrins in tissue and/or niche-specific homing of these cells.
In summary, human MSCs derived from bone marrow or placenta maintain distinct differences in expression of integrins after expansion in fully GMP-compliant medium for at least two passages of in vitro culture. bmMSCs display differences in peptide binding ex vivo compared to bmMSCs in vitro. We conclude that studies investigating the interaction of MSCs with peptides are biased by the growth conditions utilized, and even GMP-compliant media enriched with human plasma and platelet extract do not reflect the milieu a MSC is exposed to in bone marrow or in other tissues in vivo. Studies involving peptide-augmented scaffolds designed for in vivo applications must take these differences in MSC–peptide interactions into account.
Basic fibroblast growth factor
Bone marrow-derived mesenchymal stromal cell
Bovine serum albumin
- E :
Fluorescence-activated cell sorting
Fetal bovine serum
Good manufacturing practice
Mean intensity of fluorescence
Mesenchymal stromal cell
Platelet-derived growth factor
(term) Placenta-derived mesenchymal stromal cell
Phosphate buffer solubilized albumin
Quantitative reverse transcription polymerase chain reaction
arginine–glycine–aspartic acid (peptide motif)
Transforming growth factor beta
The authors express their great gratitude to Alicia Owen for her invaluable help in computing the gene array data and critically reading the manuscript, and to Chaim Goziga for assistance in preparing the artwork. We thank the surgeons and midwives at the University Hospital for providing tissue samples. This study was supported by the Landesstiftung Baden-Württemberg, the Deutsche Forschungsgemeinschaft (KFO273), the German Society for Orthopaedic Research, a donation from the Prostata Netzwerk at UKT, and in part by institutional funding.
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