A New Technique for the Isolation and Surface Immobilization of Mesenchymal Stem - 《stemcells》干细胞学杂志干细胞之家



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作者：Ke-Tai Guoa, Richard Schferb, Angela Paula, Annika Gerberb, Gerhard Ziemera, Hans P. Wendela作者单位：aDepartment of Thoracic, Cardiac, and Vascular Surgery, University Hospital of Tuebingen;bInstitute of Clinical and Experimental Transfusion Medicine, University of Tuebingen, Tuebingen, Germany
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      【摘要】  d' u& ^* C* l  X1 l% k
   Adult mesenchymal stem cells (aMSCs) are a stem cell population present in bone marrow, which can be isolated and expanded in culture and characterized. Due to the lack of specific surface markers, it is difficult to separate the MSCs from bone marrow directly. Here, we present a novel method using high-specific nucleic acids called aptamers. Porcine MSCs were used as a target to generate aptamers by combinatorial chemistry out of a huge random library with in vitro technology called systematic evolution of ligands by exponential enrichment (SELEX). After cloning and sequencing, the binding affinity was detected using a cell-sorting assay with streptavidin-coated magnetic microbeads. We also used 12-well plates immobilized with aptamers to fish out MSCs from the cell solution and a fluorescein isothiocyanate-labeled aptamer to sort MSCs from bone marrow using high-speed fluorescence-activated cell sorting. The cells showed high potency to differentiate into osteogenic, as well as into adipogenic, lineages with typical morphological characteristics. Surface marker staining showed that the attached cells were CD29 , CD44 , CD45¨C, CD90 , SLA class I , SLA DQ¨C, and SLA DR¨C. Compared with existing methods, this study established a novel, rapid, and efficient method for direct isolation of aMSCs from porcine bone marrow by using an aptamer as a probe to fish out the aMSCs. This new application of aptamers can facilitate aMSC isolation and enrichment greatly, thereby enhancing the rate of aMSC-derived cells after in vitro differentiation for various applications in the emerging field of tissue engineering and regenerative medicine.
      【关键词】 Mesenchymal stem cells Aptamer Fluorescence-activated cell sorting analysis# D# Z4 P: K" p* h! y, H$ }
   INTRODUCTION3 q5 ^$ W3 K( j

Adult mesenchymal stem cells (aMSCs) are commonly regarded as multipotent CD29 , CD44 , CD90 , CD11b¨C, CD34¨C, and CD45¨C progenitor cells with broad differentiation potential into mesodermal and ectodermal, as well as entodermal cells and tissues, respectively . However, the biological impacts of the antibody binding to aMSCs are unknown, and this technique cannot be used to fix the living cells onto scaffolds for the regeneration of structural and functional tissues.  e3 T. q0 Z6 s3 c* R" j, I4 u$ i5 e

In this study, we present a new class of capture molecules called aptamers, which can be used to isolate aMSCs from whole bone marrow directly and easily. Furthermore, the analysis of freshly aptamer-isolated aMSCs from bone marrow will reveal novel insights into the aMSC subpopulations and their antigenic profile in their natural environment.1 ~1 e, g* q: l2 W8 d

Aptamers are single-stranded DNA (ssDNA) or RNA molecules that can fold into a three-dimensional structure to bind to a variety of targets, including proteins, peptides, enzymes, antibodies, and various cell surface receptors . These favorable properties of aptamers might also be applicable in the emerging field of regenerative medicine for isolation and selective binding of aMSCs.

MATERIALS AND METHODS
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Isolation and Culture of aMSC
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Fresh bone marrow was extracted from porcine femur under sterile conditions. The animals (pigs . Briefly, MNCs were isolated from bone marrow aspirate by centrifugation over a Ficoll histopaque layer (30 minutes, 400g, density 1.077 g/ml). After centrifugation, the cells were cultivated under standard culture conditions with low-glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% fetal calf serum, penicillin (50 U/ml), and streptomycin (50 µg/ml). The medium was changed after the first 24 hours and then twice a week. When cells reached 80% confluence, they were detached using 0.25% Trypsin-EDTA and replated in preparation for SELEX and differentiation potential assessments.

For the specificity tests (fluorescence-activated cell sorting  with aptamer), rat and human aMSCs were isolated and characterized as above. The animals (Sprague-Dawley rats) were kept and treated according to the animal welfare instructions of the University of Tuebingen. The human bone marrow was taken in the course of orthopedic operations with approval of the local ethics committee of the University of Tuebingen according to the Declaration of Helsinki. The murine P19 cells were purchased from American Type Culture Collection (Manassas, VA, http://www.atcc.org).1 \- J5 K/ _7 P0 W; M3 N

aMSC Characteristics3 M# [: l0 ]) E- x3 N. o' s; Z

The potential of aMSCs to differentiate into adipogenic and osteogenic lineages was assayed as follows. For osteogenic differentiation, aMSCs were cultured in an osteogenic culture medium that included 0.2 mM L-ascorbic acid 2-phosphate magnesium salt N-hydrate, 0.01 µM dexamethasone (all reagents were from Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and 10 mM ß-glycerophosphate. After 21 days, the subcultured cell layers were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained according to the alkaline phosphatase (ALP) staining kit (kit number 85; Sigma-Aldrich). Five weeks after culturing, the deposition of mineralized bone matrix was identified using Von Kossa staining. Cell layers were fixed with 4% paraformaldehyde, incubated with 2% silver nitrate solution (wt/vol) for 10 minutes in the dark, washed thoroughly with deionized water, and exposed to UV light for 15 minutes .
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For adipogenic differentiation, aMSCs were stimulated with growth medium supplemented with 0.5 µM hydrocortisone, 0.5 mM 3-isobutyl-1-methylxanthine, and 60 µM indomethacin (Sigma-Aldrich) for 3 weeks. During these 3 weeks, the medium was changed twice a week. Cells were then rinsed twice with PBS, fixed with 10% formalin for 10 minutes, washed with distilled water, rinsed in 60% isopropanol, and covered with a 0.3% oil red O solution (Sigma-Aldrich) in 60% isopropanol for 10 minutes. Cultures were then briefly rinsed in 60% isopropanol and thoroughly in distilled water and then left to dry at room temperature . The surface marker identification of the cultured MSCs was performed by fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies staining to CD29, CD44, CD45, CD90, SLA-class I, SLA DQ, and SLA DR (Becton, Dickinson and Company, Heidelberg, Germany, http://www.bd.com). For the isotype controls, nonspecific mouse immunoglobulin G was substituted for the primary antibody.7 w6 X1 j0 U1 w
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Selection of Aptamer Binding to aMSCs
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DNA Library and Primers. The DNA oligonucleotide library contains a 40-base central random sequence flanked by primer sites on either side (5'-GAATTCAGTCGGACAGCG-N40-GATGGACGAATATCGTCTCCC-3'). The size of the library is approximately 1015. The FITC-labeled forward primer (5'-C12-FITC-GAATTCAGTCGGACAGCG-3') and biotin-labeled reverse primer (5'-Bio-GGGAGACGATATTCGTCCATC-3') were used in polymerase chain reaction (PCR) to get the double-labeled DNA and separate the ssDNA by streptavidin-coated magnetic beads (M-280 Dynabeads; Invitrogen). The library and all primers were synthesized by Operon Technologies (Cologne, Germany, http://www.operon.com).

SELEX Procedure. The selection of the DNA aptamers of porcine aMSCs was performed as follows. Four nanomoles of ssDNA pools were denatured by heating at 80¡ãC for 10 minutes in a selection buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM KCl, 100 mM NaCl, 1 mM MgCl2, and 0.1% NaN3 and then renatured at 0¡ãC for 10 minutes. To reduce background binding, a fivefold molar excess of yeast tRNA (Invitrogen) and bovine serum albumin (BSA; Sigma-Aldrich) were added. The MSCs (passage 2, 106 cells for the first round and 105 cells for subsequent rounds) were incubated with the ssDNA at 37¡ãC for 30 minutes in the selection buffer. Partitioning of bound and unbound ssDNA sequences was done by centrifugation. After centrifugation and washing three times with 1 ml selection buffer (with 0.2% BSA), cell-bound ssDNAs were amplified by PCR (Master Mix from Promega, Mannheim, Germany, http://www.promega.com). FITC- and biotin-labeled primers were used in PCR amplification (25 cycles of 1 minute at 94¡ãC, 1 minute at 48¡ãC, and 1 minute at 72¡ãC, followed by 10 minutes at 72¡ãC). For flow cytometric analysis, FITC-labeled ssDNA was prepared as described above. Aptamers obtained from the 10th round of selection were PCR-amplified using unmodified primers and cloned into Escherichia coli using the TA cloning kit (Invitrogen). Plasmids of individual clones were isolated by the plasmid extraction kit (Qiagen, Duesseldorf, Germany, http://www1.qiagen.com), and inserts were amplified by PCR and sequenced with the ABI PRISM 377 DNA Sequencer (Applied Biosystems, Darmstadt, Germany, http://www.appliedbiosystems.com). Individual FITC-aptamers were prepared to perform the binding affinity tests . As a rule, the SELEX procedure takes 1 to several months.9 K: d, c0 o. I- E  \. J

Aptamer Binding to MSCs8 H8 u! y7 [* q
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FACS Assay of Aptamer Binding Affinity to aMSCs. Two hundred picomoles of FITC-labeled aptamer was incubated with 105 aMSCs at 37¡ãC for 30 minutes, washed three times, and analyzed using flow cytometry (Becton, Dickinson and Company). The same amounts of murine P19 cells, rat aMSCs, and human aMSCs incubated with aptamer separately were used as a control. The aptamer that had the best binding affinity was chosen to continue the further identification process. The secondary structure of the aptamer was analyzed using DNASYS software (version 2.5; Hitachi Software Engineering Co., Ltd., Tokyo, http://www.hitachi-sk.co.jp).
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Aptamer Binding to aMSCs. Biotinylated aptamer G-8 was synthesized by Operon Technologies and incubated with 105 aMSCs for 30 minutes at 37¡ãC, washed three times, and incubated with anti-biotin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) for 15 minutes at 0¡ãC. The same number of aMSCs without aptamer were incubated with anti-biotin microbeads and acted as a negative control. The mixture was washed three times and filtered through a magnetic column. Then the column was removed from the magnet holder, and the beads were put into cell culture medium./ h2 |- n3 i* T

Aptamer Binding to aMSCs in Whole Bone Marrow: Flow Cytometry. Ten milliliters of fresh bone marrow was lysed with ammonium chloride and incubated with FITC-labeled aptamer (200 pmol) for 30 minutes at 37¡ãC. After being washed three times, the cells were analyzed using FACS. The same amount of peripheral blood was treated identically to act as a control.4 s- x) X; B+ [8 v' d% ]

Aptamer Binding to aMSCs in Whole Bone Marrow: Capture Experiment. Twenty milliliters of fresh bone marrow was lysed with ammonium chloride and resuspended using PBS (2% fetal bovine serum, 1 mM EDTA). FcR blocking antibody and 1 nmol aptamer were added to the bone marrow solution for 30 minutes at room temperature. EasySep biotin selection cocktail (CellSystems, St. Katharinen, Germany, http://www.cellsystems.de) was added to the solution and incubated for 15 minutes. EasySep magnetic nanoparticles were added, and the mixture was incubated for 10 minutes. Then the mixture was put into the magnet and set aside for 5 minutes. The supernatant was poured out, and the magnetically labeled cells were washed twice with buffer and further cultured.3 `9 F* }6 E2 I
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Aptamer-Mediated aMSC Adhesion on a Solid Surface. A 12-well cell culture plate (Greiner Bio-One International AG, Nuertingen, Germany, http://www.gbo.com/en) was coated with streptavidin, incubated overnight at 4¡ãC, and then washed with PBS-T (0.05% Tween-20) three times. The biotinylated aptamer and the biotinylated library (control) (1 nmol) were added to different wells and incubated at 30¡ãC for 4 hours. The plate was washed with PBS-T and incubated with aMSCs at 37¡ãC for 30 minutes with gentle shaking. The medium was then removed from the plate, and the nonadherent cells were discarded. Cell attachment was observed under an inverse microscope (Zeiss Axiovert 135; Zeiss, Oberkochen, Germany, http://www.zeiss.com).$ z4 O6 }# Q: I( n7 L$ x5 d
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FITC Aptamer-Mediated aMSC Sorting. Twenty milliliters of bone marrow was lysed to remove the red blood cells. Four nanomoles of FITC-labeled aptamer G-8 was then incubated with the bone marrow for 30 minutes under 37¡ãC, followed by three washing steps. Then the bone marrow cells were analyzed using flow cytometry. The FITC-positive cells were sorted and collected for further analyses.
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Characterization of the Sorted aMSCs- a' F( C0 z. v. U6 t

Phenotype Identification of the Sorted aMSCs. Twenty milliliters of whole bone marrow from an adult pig was lysed to remove the red blood cells. The FITC-labeled aptamer G-8 was added and incubated for 30 minutes at 37¡ãC. After being washedthree times, the cells were analyzed under sterile conditions using high-speed FACS (FACS-Sort; Becton, Dickinson and Company), and the FITC-positive cells were sorted and collected in PBS. Some of the sorted cells were analyzed the second time using the phycoerythrin (PE)-labeled CD4, CD8, CD29, CD44, CD45, and CD90; the rest of the sorted cells were cultured for 2 weeks and then analyzed using PE-labeled antibodies CD29, CD44, CD45, and CD90 (Becton, Dickinson and Company).

Differentiation of the Sorted aMSCs. The sorted aMSCs were cultured in osteogenic and adipogenic culture mediums. The ALP staining and oil red staining were performed as noted.
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Comparison of Efficiency of aMSC Isolation Between Conventional Plastic Adherence and Aptamer-Based aMSC Isolation! H1 @& ~7 Y( w7 e

To evaluate the efficiency of aMSC isolation, the adipogenic and osteogenic differentiation potential and the quantities of isolated cells were compared. MNCs were isolated from fresh porcine whole bone marrow via density gradient and plated at a density of 500 cells per well. After 24 hours, medium was changed to remove nonadherent cells. Then, adipogenic, osteogenic, or normal medium was added. Aptamer-sorted aMSCs were plated at the same density (500 cells/well). After 24 hours, medium was changed, and adipogenic, osteogenic, or normal medium was added. After 5 weeks, when the aptamer-sorted cells reached confluency, the adipogenic and osteogenic staining procedures were started.
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Plasma Stability

Fresh human plasma was prepared by centrifugation of 3,000g whole blood for 20 minutes. Eight nanomoles of the aptamer G-8 and a random ssDNA (GGGAGCTCAGCCTAAACGCT-CAAGGATCGTTCGCAACGGTTCGACGCAGTTCGTTCG-ACATGAGGCCCGGATC) were incubated separately at 37¡ãC in a final volume of 0.5 ml of freshly prepared heparinized human plasma. Samples of 50 µl were removed after 0.5, 1, 2, 4, 6, and 24 hours. Reactions were terminated by adding 5 µl of loading buffer and subsequently storing the mixture on ice. Full-length and digested oligonucleotides were separated on a 2% agarose gel and photodocumented .

RESULTS) ^  q0 R: w, _7 B

aMSC Isolation and Characteristics( J; L5 i% b, h+ d2 s$ Y

Porcine aMSCs were successfully isolated from bone marrow via gradient centrifugation, expanded in monolayer culture, and evaluated for osteogenic differentiation potential. Spindle bipolar to polygonal fibroblastic cells was observed 4 days after the first seeding. The cells reached confluence after 12 days. On initial inspection (first passage), the cells showed a uniform monolayer. The aMSCs cultured in osteogenic medium showed ALP-positive and Von Kossa-positive (calcium mineral precipitation) after 8 days and 28 days, respectively. The aMSCs cultured in adipogenic differentiation medium showed positive for oil red staining, whereas all of the controls were negative (Fig. 1 A¨C1F). Surface marker staining showed that the attached cells were CD29 , CD44 , CD45¨C, CD90 , SLA class I , SLA DQ¨C, and SLA DR¨C (Fig. 1G).
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Figure 1. Characterization and identification of adult MSCs from bone marrow via gradient centrifugation and plastic adherence. The cells showed high potency to differentiate into osteogenic, as well as into adipogenic, lineages with typical morphological characteristics. (A, B): Osteogenic differentiation and Von Kossa staining (x100); (A) is control. (C, D): Cells stained with alkaline phosphatase hematoxylin (x200); (C) is control. Cells in (E, F) were differentiated adipogenic and stained with oil red hematoxylin (x400); (E) is control. (G): Epitope identification of the cultured aMSCs. The adult porcine aMSCs were CD29 , CD44 , CD90 , SLA class I , SLA class II DQ¨C, and SLA class II DR¨C. The red curve is the isotype control.
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Selection of Aptamers with High Affinity to aMSCs
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aMSCs derived from porcine bone marrow were used as the target for in vitro selection of aptamers from a random pool of DNA molecules. The starting library consisted of 79-mer ssDNA molecules containing randomized 40-nucleotide inserts. This library was applied to a number of cultured cells in the same passage, which minimized nonspecific interaction. To monitor the enrichment of specific cell-binding aptamers during selection, SELEX pools of the second and subsequent rounds were analyzed using flow cytometry after incubation with aMSCs. In each round of the selection, the concentration of competitor DNA was increased to further selection toward a high-affinity and high-specificity aptamer pool. Analysis of the histograms of the fluorescence-labeled pools in successive cycles of selection showed a shift beginning with the second round toward higher fluorescent intensity. After 10 rounds of selection, the fluorescence of the aMSC-selected round-11 pool showed no further increase. This pool was then cloned and sequenced.
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Sequences from 36 clones were obtained, and their inserts were analyzed and sorted into putative families using the alignment of consensus motifs. Motifs were identified by inspection with the aid of computer-assisted search engines (data not shown). On the basis of the demonstrable binding to aMSCs, one aptamer, G-8, was chosen for further characterization.

Binding of Aptamers to aMSCs" Z# `7 B6 ~2 h. d, D4 e

Flow Cytometric Tests. Fluorescence of G-8 binding to aMSCs is shown in Figure 2A¨C2C, which illustrates that the aptamer binds only to aMSCs and not to murine P19 cells or human MSCs.0 X/ C4 }# w  W/ `
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Figure 2. Affinity of aptamer G-8 to adult mesenchymal stem cells (aMSCs) from different species tested using flow cytometry, which shows that aptamers bind to aMSCs, not to murine P19 cells or human MSCs. (A): The green curve represents the porcine aMSCs incubated with fluorescein isothiocyanate (FITC) G-8; the red curve represents the murine P19 cells incubated with FITC G-8. (B): The green curve represents the porcine aMSCs incubated with FITC G-8; the red curve represents the rat aMSCs incubated with FITC G-8. (C): The green curve represents the porcine aMSCs incubated with FITC G-8; the red curve represents the human aMSCs incubated with FITC G-8. (D, E): As negative control, the aptamer G-8 shows no affinity to peripheral blood. (D): The binding of the aptamer to whole bone marrow. (E): The binding of the aptamer to peripheral blood. The red curve represents the aptamer incubated with cells; the green curve represents the cell control.5 {! j# E5 N: \6 \( a% R+ I
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Fishing Experiment. aMSCs that bound with the biotinalyted aptamer could be sorted and congregated using anti-biotin microbeads. When filtered through a magnetic column, aMSCs could be fixed by the biotinalyted aptamer. After 2 weeks in culture, as shown in Figure 3A and 3B, the anti-biotin microbeads alone were not able to fish out aMSCs, which means there were no cells growing in the culture flask (Fig. 3A, negative control). The anti-biotin microbeads with biotinalyted aptamer fixed on their surfaces can bind with aMSCs, and growing cells could therefore be detected (Fig. 3B). This result shows that the aptamer is able to fish out aMSCs from the cell solution.

Figure 3. Aptamer-based cell sorting. The cells that bind to the biotinylated aptamer can be pulled down together with anti-biotin microbeads (B) and grow well in the culture flask, whereas the noncoated microbeads did not bind to the cells. The cells were washed through the magnetic filter, and no cells were held on the magnetic columns, resulting in fewer cells in the culture flask (A) (x100). (C, D): Captured adult mesenchymal stem cells (aMSCs) from bone marrow. (C) is the control, only beads incubated with whole bone marrow, in which there were only very few cells growing on the culture flask. The right picture shows the whole bone marrow incubated with the aptamer (fixed on the magnetic microbeads); there are more cells congregated and growing (x100). (E, F): Surface binding of aMSCs on aptamer-coated plates. (B): After 30 minutes of incubation of aMSCs, the aptamer-coated culture plate captured many aMSCs (216.7 ¡À 13.3 cells per eyeshot) (F). The culture plate coated with the library captured only very few aMSCs (34.3 ¡À 5.1 cells per eyeshot) (E) (x100).' `# `" V3 P9 t- o( p- F
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Binding of Aptamers to aMSCs in Whole Bone Marrow2 C  N) B" D. h

Flow Cytometric Assay. The aptamer G-8 showed almost no binding to peripheral blood cells compared with the whole bone marrow (Fig. 1G).

Capture Experiment. With the EasySep biotin selection kit, aMSCs from whole bone marrow could be fished out using streptavidin magnetic beads that coat with biotinylated aptamer and grow well in culture flasks (Fig. 3C, 3D).) g$ D# P1 C/ c, }. u. O8 ]

Aptamer-Mediated aMSC Adhesion on Solid Surfaces
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The biotinylated aptamer was immobilized onto a streptavidin-coated plate. The aMSC solution was added to the plate and incubated for 30 minutes with gentle shaking. Compared with the plate without coated aptamer, significantly (p 8 T" N9 }! i; j, |' l" V
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Characterization of the Sorted aMSCs
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Phenotypic Identification of the Sorted aMSCs. MNCs from bone marrow were collected with the FITC-labeled aptamer G-8 using high-speed FACS (1% yield) and analyzed using PE-labeled antibodies. The results showed two subpopulations of sorted cells. The first subpopulation (R1), containing small granular cells, was CD4¨C (82.2%), CD8¨C (80.5%), CD29¨C (70.7%), CD44  (90.9%), CD45  (86.4%), and CD90¨C (77.6%). The second subpopulation (R2), containing small and densely granular cells, was CD4¨C (98.9%), CD8¨C (98.9%), CD29¨C (83.7%), CD44  (87.7%), CD45  (99.2%), and CD90  (91.8%). The sorted cells were cultured for 14 days (passage 0) and also stained using PE-labeled antibodies. The results were CD29  (98.0%), CD44  (99.6%), CD90  (99.5%), and CD45¨C (87.6%), which are consistent with previously described markers of aMSCs in culture (Fig. 4). In contrast to the freshly sorted cells, no distinct subpopulation could be detected, and the cultured cells upregulated CD29 and lost the CD45 antigen.
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Figure 4. Phenotypic identification of aptamer-sorted aMSCs from porcine bone marrow either directly after the sorting process or after 2 weeks in culture. (A): The subpopulation R1 of the sorted aMSCs was stained with PE-labeled antibodies immediately after sorting. The results showed that they were CD4¨C, CD8¨C, CD29¨C, CD44 , and CD90¨C. The subpopulation R2 of the sorted aMSCs was stained with PE-labeled antibodies immediately (B) after sorting. The results showed CD4¨C, CD8¨C, CD29¨C, CD44 , and CD90 . The red curves show the isotype controls. After 2 weeks in culture, the sorted aMSCs were stained with PE-labeled antibodies. The cells were CD29 , CD44 , CD45¨C, and CD90 . The red curves show the isotype controls. Abbreviations: aMSC, adult mesenchymal stem cell; PE, phycoerythrin.
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Efficiency of aMSC Isolation+ _2 j: m- c3 x' W
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No cell growth could be detected in the wells in which MNCs from whole bone marrow were seeded (initially plated: 500 cells/well; conventional 24-hour plastic adherence procedure for isolation of aMSCs; Fig. 5A, 5G), whereas aptamer-sorted cells grew well and showed adipogenic (Fig. 5B) and osteogenic (Fig. 5F) differentiation (initially plated: 500 cells/well; medium change after 24 hours).
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Figure 5. Efficiency of adult mesenchymal stem cell (aMSC) isolation and differentiation capacity. Adipogenic (A¨CD) and osteogenic (E¨CH) differentiation of the aptamer-sorted porcine aMSCs (passage 0) versus plastic adherence procedure for isolation of aMSCs (passage 0). Mononuclear cells were isolated from fresh whole bone marrow according to density gradient and plated at a density of 500 cells per well (A, C, E, G). After 24 hours, the medium was changed to remove nonadherent cells. Then adipogenic, osteogenic, or normal medium was added. Aptamer-sorted aMSCs were plated at a density of 500 cells per well (B, D, F, H). After 24 hours, the medium was changed, and adipogenic, osteogenic, or normal medium was added. The staining procedure was started after 5 weeks, once the aptamer-sorted cells reached confluence: (A¨CD): Adipogenic differentiation. (A): Whole bone marrow: 24-hour adherence, adipogenic medium. (B): Aptamer-sorted aMSCs: 24-hour adherence, adipogenic medium. (C): Whole bone marrow: 24-hour adherence, control (normal medium). (D): Aptamer-sorted aMSCs: 24-hour adherence, control (normal medium). Staining with oil red O, hematoxylin counterstaining. (E¨CH): Osteogenic differentiation. (E): Whole bone marrow: 24-hour adherence, osteogenic medium. (F): Aptamer-sorted aMSCs: 24-hour adherence, osteogenic medium. (G): Whole bone marrow: 24-hour adherence, control (normal medium). (H): Aptamer-sorted aMSCs: 24-hour adherence, control (normal medium). Staining for alkaline phosphatase, hematoxylin counterstaining. No cell growth could be detected in wells (A, C, E, G) (plastic adherence procedure for isolation of aMSCs), whereas aptamer-sorted cells (A, B, D, F, H) grew well and showed adipogenic (B) and osteogenic (F) differentiation.5 T7 P5 a6 z. {

Plasma Stability( ?9 z% E7 k6 C7 U! C' J

For clinical or therapeutical applications, aptamers should be resistant against rapid degradation by exo- and endonucleases. Human plasma predominantly contains high 3'-exonuclease activity . In human blood plasma, the random sequence cannot resist the degradation of plasma (Fig. 6A). The unmodified aptamer G-8 can resist the degradation of nucleases for 24 hours, which was detected using agarose gel analysis (Fig. 6B) and does not need extra modification to improve its stability./ W4 L5 t" r! m! c1 f/ d0 \# }

Figure 6. To proof the plasma stability against fast enzymatic degradation, the aptamer G-8 was incubated in human blood plasma for up to 24 hours at 37¡ãC and visualized using agarose gel electrophoresis. A random single-stranded DNA (ssDNA) sequence was chosen as a control. (A): Random ssDNA sequences cannot resist the plasma digestion for 30 minutes (positive control). (B): Aptamers can resist degradation for at least 24 hours. Abbreviation: h, hour(s).1 N) _- k6 P8 N6 [, ]

DISCUSSION
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This study established a new, rapid, and efficient method for direct isolation of aMSCs from porcine bone marrow. Using an aptamer as a probe to fish out the aMSCs, this method is quick and efficient compared with existing methods. Due to the lack of a special surface marker, the traditional method of isolating aMSCs from bone marrow mainly depends on the plastic adherence characteristics of MSCs, on negative selection, or on removing other cells that expose surface markers . However, the first (plastic adherence) method is time-consuming. Identification of the functional differentiation of aMSCs takes between 2 and 4 weeks. The observation that not a single cell could be cultured with the conventional plastic adherence method after seeding 500 MNCs from fresh bone marrow confirms the known low frequency of real aMSCs in bone marrow./ L  i, J/ l3 p

In contrast, seeding 500 aptamer-isolated cells leads to confluent cultures of aMSCs with adipogenic and osteogenic differentiation potential. Our experiments show that the MSCs indicate an antigen shift from isolation to culture. Like previous reports of CD45 loss in culture . Furthermore, using CD29 or CD90 magnetic beads to select CD29- or CD90-positive MSCs, or using CD45-negative selection to isolate and purify MSCs, leads to the loss of significant amounts of MSCs. Therefore, the negative isolation method is expensive and inefficient.
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Our method shows the advantage of isolating very young MSCs, which have strong potential to differentiate into osteoblasts and adipose. Additionally, aptamer synthesis is easy and aptamers can resist enzymatic digestion for several hours. aMSCs are considered a readily accepted source of stem cells due to their efficacy in multiple types of cellular therapeutic strategies, including bone tissue regeneration . Here, we have pioneered the use of aptamers in the isolation of aMSCs from bone marrow using an aptamer with high specificity for porcine aMSCs. Moreover, using the scaffold-immobilized aptamer as a probe to fish out circulating cells is a new application for material and tissue-engineering research. The cell adhesion experiments show that immobilized aptamers can enhance aMSC attachment in the very short term. The aMSC differentiation assay indicates the long-term compatibility between an aptamer and aMSCs without affecting stem cell-specific plasticity. Compared with the established method of aMSC isolation based on plastic adherence, the aptamer-based isolation of aMSCs shows a much higher efficiency combined with high specificity without affecting stem cell plasticity. We have done experiments on aptamer-protein precipitation using lysed MSCs and looked at the target of the aptamer using proteomics tools. However, we could not find any known protein sequence, perhaps due to the limited porcine protein library. We anticipate that target protein characterization will work much better using aptamers against human MSCs. Because our long-term goal is to create a scaffold coated with aptamers, we chose an aptamer-coated surface to mimic the target condition.

CONCLUSION4 F8 P1 L; t1 i+ E! }- u
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This new aptamer against aMSCs can improve the current scaffold design by modifying the surface of the scaffold, increasing environmentally mediated stem cell plasticity and making the cells respond well to the microenvironmental conditions. Furthermore, this new technique could be useful for the identification and isolation of aMSCs and their various desired subpopulations.8 T" z5 \0 w: x& C  T* J0 k' y

DISCLOSURES

The authors indicate no potential conflicts of interest./ D; z) y; t- x* e+ d; {% l4 J) e
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ACKNOWLEDGMENTS* n+ w6 X! `6 [) o
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We thank Dr. D. Bail, Dr. A. M. Scheule (Department of Thoracic and Cardiovascular Surgery, University of Tuebingen, Germany), and Dr. C. Stahl (Department of Cardiology, University of Tuebingen, Germany) and Dr. T. Kluba (Department of Orthopedics) for their supply of porcine or human bone marrow and C. Grimmel and Prof. Dr. T. Biedermann (Department of Dermatology, University of Tuebingen, Germany) for their kind help with the FACS technology.4 _1 k* c2 q6 J! z( H
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