Comparative Analysis of Mesenchymal Stem Cells from Bone Marrow, Umbilical Cord - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:06 |只看该作者 |倒序浏览 |打印

作者：Susanne Kerna, Hermann Eichlera, Johannes Stoeveb, Harald Kltera, Karen Biebacka作者单位：a Institute of Transfusion Medicine and Immunology, German Red Cross Blood Service of Baden-WrttembergCHessen, Mannheim, Germany;b Department of Orthopaedic Surgery of University Hospital Mannheim, University of Heidelberg, Faculty of Clinical Medicine, Mannheim, Germany ) g! |7 w3 {; O) O# c# C

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      【摘要】
   Mesenchymal stem cells (MSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. Bone marrow (BM) was the first source reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and the decline in MSC number and differentiation potential with increasing age. More recently, umbilical cord blood (UCB), attainable by a less invasive method, was introduced as an alternative source for MSCs. Another promising source is adipose tissue (AT). We compared MSCs derived from these sources regarding morphology, the success rate of isolating MSCs, colony frequency, expansion potential, multiple differentiation capacity, and immune phenotype. No significant differences concerning the morphology and immune phenotype of the MSCs derived from these sources were obvious. Differences could be observed concerning the success rate of isolating MSCs, which was 100% for BM and AT, but only 63% for UCB. The colony frequency was lowest in UCB, whereas it was highest in AT. However, UCB-MSCs could be cultured longest and showed the highest proliferation capacity, whereas BM-MSCs possessed the shortest culture period and the lowest proliferation capacity. Most strikingly, UCB-MSCs showed no adipogenic differentiation capacity, in contrast to BM- and AT-MSCs. Both UCB and AT are attractive alternatives to BM in isolating MSC: AT as it contains MSCs at the highest frequency and UCB as it seems to be expandable to higher numbers.   [: R/ Y$ G( W9 V. {8 S6 o8 V3 S. j$ l
      【关键词】 Mesenchymal stem cells Bone marrow Umbilical cord blood Adipose tissue Comparative analysis Multilineage differentiation( N7 \# |9 p/ D" A% k
   INTRODUCTION$ `- d5 \, W8 W

Mesenchymal stem cells (MSCs) found in many adult tissues are an attractive stem cell source for the regeneration of damaged tissues in clinical applications because they are characterized as undifferentiated cells, able to self-renew with a high proliferative capacity, and possess a mesodermal differentiation potential .
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Although bone marrow (BM) has been the main source for the isolation of multipotent MSCs, the harvest of BM is a highly invasive procedure and the number, differentiation potential, and maximal life span of MSCs from BM decline with increasing age . Therefore, alternative sources from which to isolate MSCs are subject to intensive investigation.

One alternative source is umbilical cord blood (UCB), which can be obtained by a less invasive method, without harm for the mother or the infant .

Adipose tissue (AT) is another alternative source that can be obtained by a less invasive method and in larger quantities than BM. It has been demonstrated that AT contains stem cells similar to BM-MSCs, which are termed processed lipoaspirate (PLA) cells .

As BM-MSCs are best characterized, we asked whether MSCs derived from other sources share the characteristics of BM-MSCs. The aim of our study was to compare MSCs isolated from the three sources under identical in vitro conditions with respect to their morphology, frequency of colonies, expansion characteristics, multilineage differentiation capacity, immunophenotype, and success rate of isolating the cells.+ S  ~, w/ f! C8 F, l, h. V3 j

MATERIALS AND METHODS. u+ M" w# K# i( c6 B+ ^

Collection of BM

BM aspirates of 18 patients ranging in age from 68¨C84 years were obtained from the femoral shaft undergoing total hip replacement at the orthopedic department of the University Hospital Mannheim. Additional BM aspirates were obtained from three patients ranging in age from 44¨C71 years by puncturing the iliac crest. The BM aspirates were received in accordance with the ethical standards of the local ethical committee.
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Isolation and Culture of Mononuclear Cells from BM$ T/ F$ a& P( y, C' r
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The aspirates were diluted 1:5 with 2 mM ethylenediaminetetraacetic acid (EDTA)-phosphate-buffered saline (PBS) (Merck & Co., Whitehouse Station, NY, http://www.merck.com; Nexell, Baxter, Unterschleissheim, Germany, http://www.baxter.de). The MNC fraction was isolated by density gradient centrifugation at 435g for 30 minutes at room temperature using Ficoll-Hypaque-Plus solution (GE Healthcare BioSciences Corp., Piscataway, NJ, http://www.gehealthcare.com) and seeded at a density of 1 x 106 cells per cm2 into T75 or T175 cell culture flasks (Nunc, Rochester, NY, http://www.nuncbrand.com; Greiner Bio-One, Frickenhausen, Germany, http://www.gbo.com). The first change of medium was accomplished within 3 days after isolation. The resulting fibroblastoid adherent cells were termed BM-derived fibroblastoid adherent cells (BM-FACs) and were cultivated at 37¡ãC at a humidified atmosphere containing 5% CO2. The expansion medium consisted of either mesenchymal stem cell growth medium Bullet-Kit(MSCGM; Cambrex, Walkersville, MD, http://www.cambrex.com) or Dulbecco¡¯s modified Eagle¡¯s medium-low glucose (DMEM-lg) containing 10% mesenchymal stem cell growth supplements (MSCGS) (Cambrex). FACs were maintained in MSCGM or DMEM-lg 10% MSCGS until they reached 70% to 90% confluency. Cells were harvested at subconfluence using Trypsin (PromoCell, Heidelberg, Germany, http://www.PromoCell.com). Cells at the second passage and thereafter were replated at a mean density of 1.3 ¡À 0.7 x 103/cm2.3 f8 h- G0 W; E' j1 c
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Generation of single separated, fibroblastoid colonies termed fibroblastoid colony-forming units (CFU-F) was achieved by initially seeding the MNCs at a low density (1 103 to 1 x 104 cells per cm2). CFU-F were selected and isolated either using Trypsin (PromoCell) or by scraping them off from the surface of the culture plate with the tip of a pipette. Sub-cultivation was performed as described for the BM-FACs.
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Collection of UCB) _, y/ z1 P$ a' H/ i. m: [) X

UCB units (n = 59) were collected from the unborn placenta of full-term deliveries in a multiple bag system containing 17 ml of citrate phosphate dextrose buffer (Cord Blood Collection System; Eltest, Bonn, Germany)  and processed within 24 hours of collection. The collection was performed in accordance with the ethical standards of the local ethical committee.) g6 o( _2 G" k3 ?5 h

Isolation and Culture of MNC from UCB

The isolation of MSCs was performed as described for BM with a few exceptions. Prior to the isolation of MNC, the anticoagulated cord blood was diluted 1:1 with 2 mM EDTA-PBS. The MNC fraction was initially seeded at a density of 1 x 106( j' n, {/ p' E: a! }  R- o6 R
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MNC/cm2 into fetal calf serum (FCS)-precoated culture plates (FCS batches S0113/1038E and S0113/892E; Biochrom, Berlin, Germany, http://www.biochrom.de) (Falcon, Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) . Nonadherent cells were removed 12¨C18 hours after initial plating. The same culture conditions and media were applied as described for BM-FACs. Adherent fibroblastoid cells only appeared as CFU-F and were harvested at subconfluence using Trypsin (PromoCell). Cells at the second passage and thereafter were replated at a mean density of 3.5 ¡À 4.8 x 103/cm2.5 r3 C5 t" X5 J# }( M% J: e( F
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Collection of AT

AT was obtained from 18 donors ranging in age from 26¨C57 years undergoing liposuction procedures. Lipoaspirates were obtained in accordance with the ethical standards of the local ethical committee.
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Isolation and Culture of PLA cells from AT
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The raw lipoaspirate (50¨C100 ml) was processed as described previously . To isolate the stromal vascular fraction (SVF), lipoaspirates were washed intensely with PBS. Thereafter the lipoaspirates were digested with an equal volume of 0.075% collagenase type I (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 30¨C60 minutes at 37¡ãC with gentle agitation. The activity of the collagenase was neutralized with DMEM-lg containing 10% FCS. To obtain the high-density SVF pellet, the digested lipoaspirate was centrifuged at 1,200g for 10 minutes. The pellet was resuspended in MSCGM or DMEM-lg containing 10% MSCGS and filtered through a 100 µm nylon cell strainer (Falcon). The filtered cells were centrifuged at 1,200g for 10 minutes. The resuspended SVF cells were plated at a density of 1 x 106/cm2 into T75 or T175 culture flasks. Nonadherent cells were removed 12¨C18 hours after initial plating by intensely washing the plates. The resulting fibroblastoid adherent cells were termed AT-derived fibroblastoid adherent cells (AT-FACs). AT-FACs were cultivated under the same conditions as described for BM-FACs. AT-FACs were harvested at subconfluence using Trypsin (PromoCell). Cells at the second passage and thereafter were replated at a mean density of 1.8 ¡À 3.1 x 103/cm2.

The generation of single separated CFU-F was achieved by initially seeding the SVF cells at a low density (1 x 102 to 1 x 103 cells per cm2). CFU-F were selected and isolated as described for BM-CFU-F. Subcultivation of the cells was performed as described for the AT-FACs.
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Primary Dermal Fibroblasts as Controls& j3 Y5 A7 T" W, I

Primary normal dermal fibroblasts (PromoCell) served as negative controls in the differentiation studies. The cells were cultured in 10% supplemented fibroblast growth medium (PromoCell).
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Expansion Characteristics

Fibroblastoid cells from BM, UCB, and AT were harvested at subconfluence as described in the previous paragraphs. The cultures were abandoned as soon as they showed a senescent phenotype defined by two criteria: 1) when at least 90% of the cells adopted an altered morphology resembling senescent cells within 3¨C4 weeks after subcultivation (Fig. 1E¨C1G); and 2) when they ceased proliferation. The senescence ratio was determined up to passage 2 calculating the ratio of samples adopting a senescent phenotype to the total number of samples cultured. Cumulative population doublings were calculated using the formula x = , where N1 is the inoculum cell number and NH the cell harvest number. To yield the cumulated doubling level, the population doubling for each passage was calculated and then added to the population doubling levels of the previous passages. As the cell number of fibroblastoid cells of all three tissues could be determined for the first time at passage 1, the cumulative doubling number was first calculated for passage 2.8 c% S$ ^  L$ z& e

Figure 1. Morphology of adherent cells after initial plating and at the senescent phase. (A¨CC): Fibroblastoid adherent cells after initial plating derived from the fibroblastoid adherent cell (FAC) monolayer of BM at day 14 (A), from a UCB-fibroblastoid colony-forming units (CFU-F) at day 16 (B), and from the FAC monolayer of AT at day 8 (C). (E¨CG): Senescent cells from the FAC monolayer of BM at passage 2 (E), from an UCB-CFU-F at passage 4 (F), and from the FAC monolayer of AT at passage 6 (G) were identified by an altered morphology. All representative examples are shown at a magnification of x 100. (D): Mean values of the cumulative population doublings. Population doublings were determined at each subcultivation; mean values of BM-mesenchymal stem cells (MSCs) are shown in black; mean values of UCB-MSCs are shown in gray; mean values of AT-MSCs are shown by open bars. The starting sample numbers at passage 2 are given in the diagram. As samples underwent senescence, the number of samples went down at proceeding passages; the number of samples at distinct passages gave rise to the following mean values. BM: p3, n = 9; p4 and p5, n = 7; p6, n = 1. UCB: p4, n = 9; p5, n = 7; p6¨C8, n = 4; p9, n = 1. AT: p3, n = 6; p4, n = 5; p5 and p6, n = 2; p7, n = 1. *, p
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In Vitro Differentiation

Primary normal human dermal fibroblasts (PromoCell) served as negative controls in all three differentiation studies.$ K7 P7 c6 v1 Q/ C9 P0 D6 I

Osteogenic Differentiation/ I6 v# X8 b( D2 i2 m5 T6 w4 O8 v
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Differentiation of each sample was performed at defined passages as follows: BM-FACs, passages 0¨C5 (n = 7); BM-CFU-F, passages 2¨C6 (n = 14); UCB-CFU-F, passages 1¨C7 (n = 8); AT-FACs, passages 0¨C5 (n = 14); AT-CFU-F, passages 2¨C6 (n = 28). To promote osteogenic differentiation, the cells were seeded at a density of 3.1 x 103 cells per cm2 into eight-chamber-slides (Nunc) and cultured in MSCGM or DMEM-lg 10% MSCGS until they reached 70%¨C80% confluence. As soon as subconfluence was reached, osteogenic differentiation of the cells was induced by feeding them for 2.5 weeks, twice a week, with osteogenic induction medium consisting of 100 nM dexamethasone, 10 mM ß-glycerophosphate, 0.2 mM ascorbate, and 10% FCS in osteogenic basal medium (Cambrex). For the negative control, the cells were kept in MSCGM or DMEM-lg 10% MSCGS. Osteogenic differentiation was confirmed by the increase of alkaline phosphatase (AP) expression by histochemical staining following the manufacturer¡¯s instructions (leukocyte alkaline phosphatase kit 85L-3R; Sigma-Aldrich). Later differentiation stages were detected by the von Kossa stain, demonstrating the deposition of a hydroxyapatite matrix. The von Kossa stain was performed according to the protocol described previously  with a few modifications. The cells were fixed with 10% formalin (Sigma-Aldrich) for 15 minutes at room temperature and stained for 10¨C15 minutes with 5% silver nitrate (Sigma-Aldrich). The stain was developed incubating the cells in 1% pyrogallol (Merck) and then fixed with 5% sodium thiosulfate (Sigma-Aldrich) for 5 minutes.
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Adipogenic Differentiation
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Differentiation of each sample was performed at defined passages as follows: BM-FACs, passages 0¨C5 (n = 9); BM-CFU-F, passages 2¨C6 (n = 14); UCB-CFU-F, passages 1¨C7 (n = 11); AT-FACs, passages 0¨C5 (n = 16); AT-CFU-F, passages 2¨C6 (n = 28). To induce adipogenic differentiation, the cells were seeded at a density of 2.1 x 104 cells per cm2 into eight-chamber slides (Nunc) and cultured in MSCGM or DMEM-lg 10 MSCGS until reaching 100% confluence or postconfluence. Then, the cells were induced by three cycles of induction/maintenance  using adipogenic induction medium consisting of 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-xanthine, 10 µg/ml recombinant human insulin, 100 mM indomethacin, and 10% FCS (Cambrex) and using adipogenic maintenance medium consisting solely of 10 µg/ml recombinant human insulin and 10% FCS (Cambrex). After completing the three cycles of induction and maintenance, the induced cells were incubated for another 7 days in adipogenic maintenance medium. The noninduced control cells were fed only with adipogenic maintenance medium. Adipogenic differentiation was confirmed by the formation of neutral lipid-vacuoles stainable with Oil Red O (Sigma-Aldrich). For the Oil Red O stain, cells were fixed with 10% formalin (Sigma-Aldrich), washed, and stained with a working solution of 0.18% Oil Red O for 5 minutes. The nuclei were counterstained with Mayer¡¯s hematoxylin solution (Sigma-Aldrich).7 j4 R1 X; F* P0 r0 `
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Chondrogenic Differentiation
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Differentiation of each sample and CFU-F was performed at defined passages as follows: BM-FACs, passages 0¨C5 (n = 8); BM-CFU-F, passages 2¨C6 (n = 14); UCB-CFU-F, passages 1¨C7 (n = 15); AT-FACs, passages 0¨C5 (n = 18); AT-CFU-F, passages 2¨C6 (n = 28). For chondrogenic differentiation, cells were cultured in a micromass culture. Therefore 2.5 x 105 cells were centrifuged in a 15-ml polypropylene tube (Greiner) at 150g to form a pellet. Without disturbing the pellet, the cells were cultured for 4 weeks in 0.5 ml of complete chondrogenic differentiation medium (Cambrex) including 10 ng/ml TGF-ß-3 (Strathmann Biotec AG, Hamburg, Germany, http://www.strathman-biotec-ag.de). Cells were fed twice a week. After the culture period, cryosections were analyzed by Safranin O staining. For the staining, the sections were fixed with ice-cold acetone (Sigma-Aldrich) and stained with 0.1% aqueous Safranin O for 5 minutes and the nuclei were counterstained with Weigert¡¯s iron hematoxylin (Sigma-Aldrich).

Immunophenotypic Analyses
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For further characterization, cell surface antigen phenotyping was performed on BM-MSCs at passages 0¨C7, of UCB-MSCs at passages 3¨C7, and of AT-MSCs at passages 1¨C5. The following cell-surface epitopes were marked with anti-human antibodies: CD14-fluorescein isothiocyanate (FITC), CD34-phycoerythrin (PE), CD73-PE, CD90-Cy5 (Becton Dickinson), CD29-PE, CD44-FITC, CD45-Peridium-chlorophyll protein complex (PerCP), HLA-class I-FITC, HLA-class II-FITC (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), CD133-PE (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), CD105-FITC, and CD106-PE (Immunokontakt; AMS Biotechnology, Wiesbaden, Germany, http://www.immunok.com). Mouse isotype antibodies served as control (Becton Dickinson; Beckman Coulter). 10,000 labeled cells were acquired and analyzed using a FACScan flow cytometer running CellQuest software (Becton Dickinson).7 _& x: n% q, D  w. x8 C
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Statistical Analysis4 e+ Z4 B9 g; M8 N- s; X7 i1 G
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Data are presented as mean ¡À standard deviation. A two-sided, nonpaired t test was used to analyze the flow cytometry and the cumulative doubling data. A Mann-Whitney U test was used to compare the isolation efficacy of CFU-F derived from UCB, BM, and AT. A 2 test was applied to compare the differentiation capacities of cells isolated from the different tissues and ratios of samples with a senescent phenotype. Differences were considered significant at p

RESULTS8 C- B" z; H4 x' w9 c( C4 ~* A, m

Isolation of Adherent Cells of Fibroblastoid Morphology from BM, UCB, and AT
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At an initial plating density of 1 x 106 cells per cm2, both BM-and AT-derived fibroblastoid cells formed a monolayer 4¨C5 days after initial plating and were termed FACs. In contrast to BM or AT, UCB-derived fibroblastoid cells formed CFU-F and could be detected first 2¨C4 weeks after plating when applying the same initial plating density. In contrast to UCB, for the generation of CFU-F from BM and AT a lower initial plating density was necessary. The number of CFU-F calculated at the basis of 1 x 106 initially plated cells was highest for AT (557 ¡À 673), followed by BM (83 ¡À 61); it was lowest for UCB (0.002 ¡À 0.004) (p # _8 Y, E7 w5 n3 c4 A  e

The success rate of isolating FACs and CFU-F from both BM (n = 21) and AT (n = 18) was 100%. In contrast, the success rate in UCB (n = 59) was only 29% from all units processed. The rate could be enhanced to 63% by FCS precoating and by taking into account only units of optimal quality . However, no differences concerning the morphology of the adherent cells derived from the three tissues were obvious (Fig. 1A¨C1C).
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Expansion Characteristi
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