Mol Cell Biochem (2015) 401:155–164 DOI 10.1007/s11010-014-2303-0
The effect of extended passaging on the phenotype and osteogenic potential of human umbilical cord mesenchymal stem cells Zhe Shi • Liang Zhao • Gengtao Qiu Ruixuan He • Michael S. Detamore
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Received: 31 July 2014 / Accepted: 10 December 2014 / Published online: 3 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Retaining biological characteristics in the extended passaging is crucial for human umbilical cord mesenchymal stem cells (hUCMSCs) in tissue engineering. We aimed to assess morphology, viability, MSC marker expression, and osteogenic activity of hUCSMCs after extended passaging. Passages 4 (P4) and 16 (P16) hUCMSCs displayed similar morphology and viability. The flow cytometry results showed that CD73, CD90, and CD105 were highly expressed at P1–P16. CD166 expression decreased progressively from 90 % at P2 to 61.5 % at P5 (p \ 0.05), followed by stable expression through P16. Results from calcium deposition alkaline phosphatase activity and RT-PCR assay showed that both P4 and P16 hUCMSCs differentiated down an osteogenic lineage, with no significant difference in osteogenic capacity (p \ 0.05).
Liang Zhao, Zhe Shi and Gengtao Qiu have contributed equally to this work. Z. Shi Department of Orthopaedic Surgery, The Third Affilliated Hospital, Southern Medical University, Guangzhou, China L. Zhao (&) G. Qiu R. He Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Ave, Guangzhou 510515, China e-mail: [email protected] G. Qiu Department of Orthopaedic Surgery, ShunDe First People Hospital, Shunde, Foshan, China M. S. Detamore (&) Department of Chemical & Petroleum Engineering, University of Kansas, 4132 Learned Hall, 1530 W 15th Street, Lawrence, KS 66045, USA e-mail: [email protected]
High-passage UMCSCs maintained stable expression of MSC CD markers as well as stable osteogenic activity. hUCMSCs may thus be suitable for tissue engineering and regenerative medicine applications. Keywords Mesenchymal stem cell Human umbilical cord Proliferation Osteogenic differentiation CD166
Introduction Stem-cell-based tissue engineering has the potential to revolutionize medicine by facilitating the regeneration of damaged and diseased tissues [1–4]. Mesenchymal stem cells (MSCs) have potential applications in bone tissue engineering, as they exhibit osteogenic characteristics and differentiation properties [5]. Common MSC sources include bone marrow [6, 7], umbilical cord blood [6], periosteum [8], synovium [9], and adipose tissue [10, 11]. Bone marrow-derived mesenchymal stem cells (BMSCs) are by far the most commonly used in bone tissue engineering. However, previous studies have concluded that in vitro expansion of hBMSCs may be limited due to the loss of differentiation potential with increased passaging [12, 13]. In addition, a mice study showed that the growth potential of BMSC is reversely correlated to the age of the bone marrow donor [14]. This limitation necessitates the identification of other cell sources that can be easily isolated, expanded, and have characteristics and differentiation properties similar to those of MSCs. Human umbilical cord mesenchymal stromal cells (hUCMSCs), derived from the Wharton’s Jelly of umbilical cords, appear to bear multi-potential mesenchymal stem cell characteristics. These cells can differentiate into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells
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[15–24]. The use of hUCMSCs poses several major advantages: (1) Umbilical cords are medical waste discarded after birth and can therefore be collected at a low cost; (2) Because many babies are born each year, hUCMSCs are a virtually inexhaustible stem cell source; (3) hUCMSCs can be collected non-invasively, unlike the harvesting procedure required for BMSCs; (4) hUCMSCs can be collected without the ethical controversies surrounding human embryonic stem cells (hESCs); (5) hUCMSCs are a primitive MSC population that exhibit a high degree of plasticity and developmental flexibility; and (6) hUCMSCs thus do not appear to cause immune-rejection responses in vivo [16, 19–21, 25–29]. These advantages make hUCMSCs a highly desirable stem cell source. However, the value of these cells for tissue engineering depends on their capacity to retain their phenotype and differentiation ability over many passages in culture. Study on equine cells reported UCMSCs proliferating in culture twice as long as BMSCs before senescence [30]. In line with that study, Scheers et al., recently demonstrated that extensive passing of hUCMSC still possessed a stable growth and hepatic differentiation potential as compared to the early-passage hUCMSCs [31]. However, the osteogenic differentiation potential of extensive passing of hUCMSC still remains to be further investigated. Accordingly, this study aimed to investigate the influence of extended monolayer expansion on the phenotype, viability, and osteogenic differentiation of hUCMSCs. Over 16 passages, we assessed the morphology, viability, MSC marker expression, and osteogenic activity of these cells to determine their stability through extended passaging, a feature required of cells suitable for tissue engineering.
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consisting of low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG), 10 % MSC-qualified fetal bovine serum (FBS), and 1 % penicillin/streptomycin (PS) (Invitrogen, Carlsbad, CA). Cells were plated in cell culture flasks at 3,000 cells/cm2 (passage 0). Non-adherent cells were rinsed off 3 days after plating. When the attached hUCMSCs reached confluence, they were then trypsinized using 0.05 % trypsin–EDTA (Invitrogen) and expanded (passage 1). Culture medium was changed every 2 days, and cells were passaged at a ratio of approximately 1:4 at each passage thereafter. hUCMSC cultures that were passaged 4 or less times were designated ‘early passage,’ while those passaged 16 times were designated ‘late passage.’ MTT cell proliferation assay P1 to P16 hUCMSCs were plated onto 24-well plates (104 cells/well) (38). MTT assays were performed 1, 3, and 7 days after plating using an MTT cell proliferation assay kit according to the manufacturer’s protocol (Invitrogen). In brief, culture medium containing 0.25 mg/mL MTT was added to each well, and cells were further incubated at 37 °C for 20 min. The specimens were homogenized, suspended, and analyzed at 540 nm (Fluoroskan ascent, Thermo Electron Corporation, Waltham, MA). Colony-forming unit assays
Materials and methods
To assess the frequency of colony-forming unit fibroblasts (CFU-Fs), a limiting dilution assay was performed on P1 to P16 hUCMSCs. Freshly isolated cells (100/sample) were suspended in complete culture medium and placed in a 60 mm2 sterile culture dish. The cells were incubated for 7 days followed by staining with 3 % crystal violet in methanol for 10 min. All visible colonies were counted.
Isolation and culture hUCMSCs
Flow cytometric analysis of hUCMSCs
hUCMSCs were harvested following our previously described method [18], with IRB approval (KU-Lawrence no.15402, KU Medical Center no. 10951) and informed consent. Six umbilical cords (three males and three females; mean length, 18 ± 5 cm) were obtained from the University of Kansas Medical Center and were processed within 24 h. Briefly, each umbilical cord was cut into 5 cm pieces and rinsed with sterile phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) to remove as many blood cells as possible. Blood vessels were removed from each piece after incising the cord lengthwise. The cells making up the umbilical cord stroma were isolated following treatment with collagenase type I. Isolated hUCMSCs were cultured in complete culture medium
At each passage, hUCMSCs were characterized using flow cytometry according to the expression of surface antigens specific to the mesenchymal stem cell lineage (CD13, CD29, CD44, CD49e, CD73, CD90, CD105, CD166) and hematopoietic surface markers (CD34, CD45). All supplies were purchased from BD Biosciences (San Jose, CA), except CD73 and CD105 (eBioscience, San Diego, CA). In brief, approximately 0.5 9 106 cells per vial were used for staining. Nonspecific binding was blocked by incubation in staining buffer (PBS with 2 % FBS) for 15 min on ice. Cells treated with a single-label antigen were incubated 20 min on ice. Mouse isotype antigens served as the control. Samples were analyzed using a FACscan (Becton– Dickinson, San Jose, CA).
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Osteogenic differentiation P4 and P16 hUCMSCs were cultured in osteogenic medium. Approximately 5 9 103 cells/cm2 were seeded into each well of 12-well tissue-culture plates. hUCMSCs were incubated in complete culture medium for 1 day. The medium was then replaced with StemPro osteogenesis differentiation kit medium containing osteogenesis differentiation basal medium and an osteogenic supplement (Invitrogen, Cat. No. A10072-01). The medium was changed every 2 days for 21 days.
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Sequence Detection System. TaqMan gene expression assay kits (Applied Biosystems), including two predesigned specific primers and probes, were used to measure the transcript levels of the following genes: collagen type I (CI, Hs00164004), osteocalcin (OC, Hs01587813), Runx2 (Hs00231692), bone sialoprotein (BSP, Hs00173720), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905). The relative expression level of each target gene was evaluated using the 2-DDCt method (Livak and Schmittgen 2001) on days 1, 7, 14, and 21 using samples in complete culture medium on 1 day as the calibrator (n = 5).
Biochemical assays and histological staining Data analysis Osteogenic groups were assayed for calcium deposition and alkaline phosphatase (ALP) activity on days 1, 7, 14, and 21. Calcium content was measured using the orthocresolphthalein complexone (OCPC) method (after Brugge and Jansen 2002). Briefly, the specimens were homogenized and suspended in 1 N acetic acid overnight. Digestion solution (10 lL) and assay reagent (300 lL) were mixed and incubated for 10 min at room temperature and then read at 575 nm (Fluoroskan ascent, Thermo Electron Corporation, Waltham, MA). Normal control serum (Stanbio, Boerne TX) containing a colorimetric p-nitrophenyl phosphate assay kit (Stanbio) was used to measure alkaline phosphatase (ALP) activity according to the manufacturer’s instructions, with a known concentration of ALP as the standard. Each sample was processed through 2 freeze-thaw cycles (-70 °C and room temperature, 45 min each) to rupture the cell membranes and release the proteins and DNA from the cells. To each well of a culture plate, 500-lL ALP substrate reagent and 100-lL cell lysate (samples and controls) were added, followed by gentle mixing and incubation for 60 s at 37 °C. Plates were read at 405 nm every 20 min for 3 h. Each sample was normalized to its DNA content. Histological staining was performed using alizarin red staining (Sigma) for mineralization on day 21 [27, 32, 33]. In brief, the cells were washed twice with PBS, fixed for 30 min with 10 % paraformaldehyde, and washed with PBS. Cells were stained with alizarin red for 30 s to 5 min, and the reaction was observed microscopically. Quantitative real-time reverse transcription polymerase chain reaction The total RNA was extracted using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). mRNA was reverse-transcribed into cDNA using a HighCapacity cDNA Archive kit (Applied Biosystems, Foster city, CA). Quantitative real-time RT-PCR reactions were performed using an Applied Biosystems 7500 Fast
All data are expressed as mean ± standard deviation and were analyzed by ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test when significance was detected by ANOVA. Differences between groups were considered significant for p \ 0.05.
Results Growth characteristics of hUCMSCs Early and late passages of hUCMSCs were compared to assess their stability in culture. Cells from early and late passages displayed a similar characteristic MSC spindleshape morphology, with directionality and regularity (Fig. 1a–c). The viability of hUCMSCs was measured using the MTT cell viability assay. MTT assays showed that the viability of early and late hUCMSCs was similar (p [ 0.1) (Fig. 1d). In addition, there were no significant differences (p [ 0.1) in viability from P1 to P16 (at 7 days) (Fig. 1e). Clonogenic capacity was investigated using the CFU-F assay. Early and late hUCMSCs had similar growth kinetics (p [ 0.1) (Fig. 1f). No significant differences in CFU-Fs from P1 to P16 were observed (p [ 0.1) (Fig. 1g). Together, these data indicate passaging of hUCMSCs did not alter their growth characteristics. Phenotypic profiles of hUCMSCs The stability of hUCMSC characteristics over the course of extended expansion from P1 to P16 was determined using flow cytometry. hUCMSCs were negative for the hematopoietic markers CD34 and CD45 but were positive for the mesenchymal stem cell surface markers CD29, CD49e, CD73, CD90, CD105, and CD166. P1 hUCMSCs expressed high levels of MSC surface markers CD73 (96.5 ± 2.2), CD90 (93.5 ± 3.5), CD105 (99.1 ± 2.5), and CD166 (90.5 ± 4.8 %). Expression of these markers
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Fig. 1 Morphology and proliferation of hUCMSCs 3 days after plating in complete culture medium (mean ± standard deviation; n = 6). a P0 hUCMSCs, scale bar = 100 lm. b P4 hUCMSCs, scale bar = 100 lm. c P16 hUCMSCs, scale bar = 100 lm. d MTT assay
of P4 and P16 hUCMSCs. e MTT assay analysis-histogram plots of hUCMSCs from P1 to P16, measured at 7 days. f Number of CFU-F colonies with P4 and P16 hUCMSCs. g Analysis-histogram plots of CFU-F colonies of hUCMSCs from P1 to P16
remained high throughout the whole expansion period as shown in Fig. 2, with the exception of CD166. At P16, hUCMSCs expressed CD73 (96.5 ± 2.4), CD90 (93.5 ± 2.7), CD105 (99.4 ± 0.5), and CD166 (61.5 ± 2.5 %). A progressive decrease in CD166
expression over the course of the early passages was observed, from 95 % at P2 to 58 % at P5 (p \ 0.05), followed by stable expression through P16 (Figs. 2, 3). With the exception of CD166 expression, the phenotype of hUCMSCs was stable throughout passaging.
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Fig. 2 Surface phenotypic characterization of hUCMSCs from a representative sample of P1 and P16. hUCMSCs were positive for CD29, CD44, CD49e, CD73, CD90, CD105, and CD166
Osteogenic differentiation of hUCMSCs The osteogenic potential of hUCMSCs early and late in the course of passaging was compared. P4 and P16 hUCMSCs both differentiated down an osteogenic lineage after 21 days. The degree of osteogenesis did not differ significantly between P4 and P16 hUCMSCs. The appearance of
alizarin-red-positive nodules was similar between P4 and P16 hUCMSCs (Fig. 4a, b). The activity of ALP in hUCMSCs undergoing osteogenic differentiation was also similar between these cells. ALP activity increased as much as 18-fold over controls in P4 hUCMSCs and 17-fold in P16 hUCMSCs on day 14 (Fig. 4c). There was no significant difference in ALP activity between P4 and P16
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160 Fig. 3 Flow cytometry analysis-histogram plots of surface marker expression from P1 to P16 (mean ± standard deviation; n = 6). A high proportion of hUCMSCs expressed CD29, CD44, CD49e, CD73, CD90 and CD105. These high expression levels persisted throughout the expansion period
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hUCMSCs (p [ 0.1). A peak 13-fold increase in calcium deposition was observed in both P4 and P16 hUCMSCs on day 21(Fig. 4d), with no significant difference between P4 and P16 hUCMSCs (p [ 0.1). qRT-PCR was used to compare mRNA expression levels of osteogenic genes. Results show that in P4 hUCMSCs on day 21, CI increased
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4.4-fold, OC increased 4.5-fold, Runx2 increased 3.2-fold, and BSP increased 2.2-fold (Fig. 5). In P16 hUCMSCs on day 21 days, CI increased 4.1-fold, OC increased 4.5-fold, Runx2 increased 3.1-fold, and BSP increased 2.1-fold. There was no significant difference in the expression of any of these markers between P4 and P16 hUCMSCs (p [ 0.1).
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Fig. 4 Osteogenic potential of early and late passage hUCMSCs. a, b Representative images of P4 and P16 hUCMSCs under osteogenic culture for 21 days. Alizarin Red staining-positive nodules were typically observed in both P4 and P16 hUCMSCs. a Alizarin Red staining with P4 hUCMSCs. b Alizarin Red staining with P16
hUCMSCs. c, d Biochemical assay data are presented as the mean ± standard deviation; n = 4. Control hUCMSCs were cultured in complete culture medium. c ALP activity peaked at 14 days and slightly decreased at 21 days. d Calcium content. Scale bar = 500 lm
Discussion
Parts of our results are consistent with previous studies [31, 35, 36]. In addition, we demonstrated more evidences supporting that late passages still possessed the osteogenic differentiation potential in the present study. Under osteogenic differentiation conditions, hUCMSCs from both early and late passages were able to differentiate toward an osteogenic lineage. The ability of HUCMSCs to retain the osteogenic differentiation potential in late passages is promising for future bone tissue engineering applications. In evaluating the suitability of mesenchymal stem cells for medical applications, one must consider their availability and potential to yield a reasonable number of viable cells [37–41]. Accordingly, one of the main goals of this study was to evaluate the viability of hUCMSCs during extended passaging before the induction of osteogenic differentiation. One of the striking features of hUCMSCs observed in a previous study was their capacity to expand cell numbers almost 300-fold over seven passages [27]. In the present study, we observed no loss of viability with passaging. In addition, over the course of 15 passages, hUCMSCs maintained a seemingly homogeneous morphology, appearing as slender spindle-shape cells with a narrow cytoplasm
The current study provides evidence that hUCMSCs maintain their phenotype over multiple passages. The expression of CD cell surface markers except CD166 remained virtually unchanged between early and late passages of hUCMSCs, and P4 and P16 hUCMSCs exhibited similar osteogenic activity. In previous studies, cultured hUCMSCs were successfully guided toward osteogenic differentiation [16, 27, 34]. Wharton’s jelly extract could efficiently slow down the senescence and improve the differentiation ability of late passages of UCMSCs [35]. Umbilical cord tissue pieces derived UCMSCs could mention the proliferation profile, cell cycle distribution and MSC surface markers expression until P10 [36]. In the present study, hUCMSCs were investigated for the influence of cell expansion on the phenotype, proliferative capacity, and osteogenic potential. We observed that hUCMSCs maintained their MSC-like phenotype and viability, after extended passaging. hUCMSCs from both early and late passages exhibited high expression of surface markers associated with BMSCs.
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(Fig. 1). MTT and CFU-F assays showed that the growth dynamics of hUCMSCs did not differ significantly with expansion. Flow cytometric analysis in this study revealed that hUCMSCs expressed matrix receptors (CD44), integrin markers (CD29), and MSC markers (CD73, CD90, CD105 and CD166) but no hematopoietic cell markers (CD34, CD45) (Fig. 2). This observation is consistent with the results of previous studies [20, 42]. We also observed that the high level of CD73, CD90, and CD105 expression did not change significantly during extended passaging. These results suggest that the hUCMSCs characteristics were relatively stable during monolayer expansion during the tested passages. To our knowledge, this is the first study to report the effects of extended passaging on CD166 expression in hUCMSCs. The number of cells expressing CD166 gradually decreased from 90 % at P2 to 61.5 % at P5, where it stabilized through P16 (Fig. 3). CD166 is an adhesion molecule that promotes cell–cell aggregation through the actin cytoskeleton [43]. In stem cells, this transmembrane protein is known to be involved in osteogenic differentiation [44]. The decrease we observed in CD166 expression did not
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Fig. 5 Quantitative relative gene expression profile of hUCMSCs cultured with osteogenic medium on days 7, 14, and 21 (mean ± standard deviation; n = 5). a collagen type I(CI) b Osteocalcin (OC) c Runx2 d Bone sialoprotein (BSP)
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prevent osteogenic activity in early or late passages. However, we did not include passages 0–3 hUCMSCs in osteogenic differentiation, leaving uncertainty as to whether decreased CD166 expression decreased the osteogenic potential of hUCMSCs below that of passage 4 cells. Future studies that investigate the osteogenic differentiation potential of P1–P3 hUCMSCs and block CD166 with neutralizing antibody during osteogenic differentiation should be conducted to address the role of CD166 in the osteogenesis of hUCMSC. In vitro osteogenic differentiation of hUCMSCs has been demonstrated previously [16, 27, 34]. Furthermore, hUCMSCs have been shown to undergo a greater extent of mineralization than BMSCs [15], although this may not hold true in 3D tissue culture. In the present study, both P4 and P16 hUCMSCs showed some degree of osteogenic differentiation over a 3 week period, as evidenced by ALP activity, calcium quantification, histology, and bone marker gene expression (CI, OC, Runx2, and BSP). This evidence of hUCMSC osteogenic capacity is consistent with reports from previous studies [16, 27, 34]. Interestingly, late passage hUCMSCs retained the same osteogenic capacity as early passage hUCMSCs. In contrast, several previous
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studies have shown that BMSCs that had undergone extended expansion exhibited decreased differentiation potentials and lower proliferation rates [44–46]. The difference in our results suggests that hUCMSCs are earlierstage cells than adult MSCs, conferring yet another advantage over BMSCs. Although hUCMSCs appear to share some characteristics with MSCs, further studies are needed to understand the functional nature of their multi-potential capacity. The encouraging results of the current study give reason to examine whether adipogenic and chondrogenic potential are also retained over extended passages. In addition, in vivo studies are needed to study potential applications such as bone tissue regeneration. There are still some limitations in this study. In the qPCR experiments, only one reference gene (GAPDH) was used, which may lead to misleading results. More reference genes should be used according to the quantitative real-time PCR experiments (MIQE) guidelines [47]. Second, we did not assess the oncogenecity of hUCMSC at extended passage. However, an in vivo study by Wang et al., demonstrated that although with genomic alterations, P30 hUCMSCs cannot form tumors after subcutaneously transplanting in the immunodeficient mice [48]. These limitations should be addressed in the future study.
Conclusion hUCMSCs maintained their phenotype, proliferation, and osteogenic capacity after multiple passages. hUCMSCs may thus have the potential to be expanded by several orders of magnitude for bone regeneration. Such a capacity would pave the way for technologies that could use hUCMSCs harvested at birth to provide large cell numbers for therapies later in life. Acknowledgments We gratefully acknowledge Prof. Zhengliang Chen at the Southern Medical University for FCM assistance and useful discussions. This study was supported by National Natural Science Foundation of China 31328008 (LZ), 31100695(LZ) Natural Science Foundation of Guangdong s20130010014253 (LZ), Guangdong Provincial Science and Technology Project 2012B010200024 (LZ) and Guangzhou Science and Technology Project 2012027(LZ). Conflict of Interest
None declared.
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The effect of extended passaging on the phenotype and osteogenic potential of human umbilical cord mesenchymal stem cells Zhe Shi • Liang Zhao • Gengtao Qiu Ruixuan He • Michael S. Detamore
•
Received: 31 July 2014 / Accepted: 10 December 2014 / Published online: 3 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Retaining biological characteristics in the extended passaging is crucial for human umbilical cord mesenchymal stem cells (hUCMSCs) in tissue engineering. We aimed to assess morphology, viability, MSC marker expression, and osteogenic activity of hUCSMCs after extended passaging. Passages 4 (P4) and 16 (P16) hUCMSCs displayed similar morphology and viability. The flow cytometry results showed that CD73, CD90, and CD105 were highly expressed at P1–P16. CD166 expression decreased progressively from 90 % at P2 to 61.5 % at P5 (p \ 0.05), followed by stable expression through P16. Results from calcium deposition alkaline phosphatase activity and RT-PCR assay showed that both P4 and P16 hUCMSCs differentiated down an osteogenic lineage, with no significant difference in osteogenic capacity (p \ 0.05).
Liang Zhao, Zhe Shi and Gengtao Qiu have contributed equally to this work. Z. Shi Department of Orthopaedic Surgery, The Third Affilliated Hospital, Southern Medical University, Guangzhou, China L. Zhao (&) G. Qiu R. He Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Ave, Guangzhou 510515, China e-mail: [email protected] G. Qiu Department of Orthopaedic Surgery, ShunDe First People Hospital, Shunde, Foshan, China M. S. Detamore (&) Department of Chemical & Petroleum Engineering, University of Kansas, 4132 Learned Hall, 1530 W 15th Street, Lawrence, KS 66045, USA e-mail: [email protected]
High-passage UMCSCs maintained stable expression of MSC CD markers as well as stable osteogenic activity. hUCMSCs may thus be suitable for tissue engineering and regenerative medicine applications. Keywords Mesenchymal stem cell Human umbilical cord Proliferation Osteogenic differentiation CD166
Introduction Stem-cell-based tissue engineering has the potential to revolutionize medicine by facilitating the regeneration of damaged and diseased tissues [1–4]. Mesenchymal stem cells (MSCs) have potential applications in bone tissue engineering, as they exhibit osteogenic characteristics and differentiation properties [5]. Common MSC sources include bone marrow [6, 7], umbilical cord blood [6], periosteum [8], synovium [9], and adipose tissue [10, 11]. Bone marrow-derived mesenchymal stem cells (BMSCs) are by far the most commonly used in bone tissue engineering. However, previous studies have concluded that in vitro expansion of hBMSCs may be limited due to the loss of differentiation potential with increased passaging [12, 13]. In addition, a mice study showed that the growth potential of BMSC is reversely correlated to the age of the bone marrow donor [14]. This limitation necessitates the identification of other cell sources that can be easily isolated, expanded, and have characteristics and differentiation properties similar to those of MSCs. Human umbilical cord mesenchymal stromal cells (hUCMSCs), derived from the Wharton’s Jelly of umbilical cords, appear to bear multi-potential mesenchymal stem cell characteristics. These cells can differentiate into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells
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[15–24]. The use of hUCMSCs poses several major advantages: (1) Umbilical cords are medical waste discarded after birth and can therefore be collected at a low cost; (2) Because many babies are born each year, hUCMSCs are a virtually inexhaustible stem cell source; (3) hUCMSCs can be collected non-invasively, unlike the harvesting procedure required for BMSCs; (4) hUCMSCs can be collected without the ethical controversies surrounding human embryonic stem cells (hESCs); (5) hUCMSCs are a primitive MSC population that exhibit a high degree of plasticity and developmental flexibility; and (6) hUCMSCs thus do not appear to cause immune-rejection responses in vivo [16, 19–21, 25–29]. These advantages make hUCMSCs a highly desirable stem cell source. However, the value of these cells for tissue engineering depends on their capacity to retain their phenotype and differentiation ability over many passages in culture. Study on equine cells reported UCMSCs proliferating in culture twice as long as BMSCs before senescence [30]. In line with that study, Scheers et al., recently demonstrated that extensive passing of hUCMSC still possessed a stable growth and hepatic differentiation potential as compared to the early-passage hUCMSCs [31]. However, the osteogenic differentiation potential of extensive passing of hUCMSC still remains to be further investigated. Accordingly, this study aimed to investigate the influence of extended monolayer expansion on the phenotype, viability, and osteogenic differentiation of hUCMSCs. Over 16 passages, we assessed the morphology, viability, MSC marker expression, and osteogenic activity of these cells to determine their stability through extended passaging, a feature required of cells suitable for tissue engineering.
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consisting of low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG), 10 % MSC-qualified fetal bovine serum (FBS), and 1 % penicillin/streptomycin (PS) (Invitrogen, Carlsbad, CA). Cells were plated in cell culture flasks at 3,000 cells/cm2 (passage 0). Non-adherent cells were rinsed off 3 days after plating. When the attached hUCMSCs reached confluence, they were then trypsinized using 0.05 % trypsin–EDTA (Invitrogen) and expanded (passage 1). Culture medium was changed every 2 days, and cells were passaged at a ratio of approximately 1:4 at each passage thereafter. hUCMSC cultures that were passaged 4 or less times were designated ‘early passage,’ while those passaged 16 times were designated ‘late passage.’ MTT cell proliferation assay P1 to P16 hUCMSCs were plated onto 24-well plates (104 cells/well) (38). MTT assays were performed 1, 3, and 7 days after plating using an MTT cell proliferation assay kit according to the manufacturer’s protocol (Invitrogen). In brief, culture medium containing 0.25 mg/mL MTT was added to each well, and cells were further incubated at 37 °C for 20 min. The specimens were homogenized, suspended, and analyzed at 540 nm (Fluoroskan ascent, Thermo Electron Corporation, Waltham, MA). Colony-forming unit assays
Materials and methods
To assess the frequency of colony-forming unit fibroblasts (CFU-Fs), a limiting dilution assay was performed on P1 to P16 hUCMSCs. Freshly isolated cells (100/sample) were suspended in complete culture medium and placed in a 60 mm2 sterile culture dish. The cells were incubated for 7 days followed by staining with 3 % crystal violet in methanol for 10 min. All visible colonies were counted.
Isolation and culture hUCMSCs
Flow cytometric analysis of hUCMSCs
hUCMSCs were harvested following our previously described method [18], with IRB approval (KU-Lawrence no.15402, KU Medical Center no. 10951) and informed consent. Six umbilical cords (three males and three females; mean length, 18 ± 5 cm) were obtained from the University of Kansas Medical Center and were processed within 24 h. Briefly, each umbilical cord was cut into 5 cm pieces and rinsed with sterile phosphate-buffered saline (PBS) (Sigma, St. Louis, MO) to remove as many blood cells as possible. Blood vessels were removed from each piece after incising the cord lengthwise. The cells making up the umbilical cord stroma were isolated following treatment with collagenase type I. Isolated hUCMSCs were cultured in complete culture medium
At each passage, hUCMSCs were characterized using flow cytometry according to the expression of surface antigens specific to the mesenchymal stem cell lineage (CD13, CD29, CD44, CD49e, CD73, CD90, CD105, CD166) and hematopoietic surface markers (CD34, CD45). All supplies were purchased from BD Biosciences (San Jose, CA), except CD73 and CD105 (eBioscience, San Diego, CA). In brief, approximately 0.5 9 106 cells per vial were used for staining. Nonspecific binding was blocked by incubation in staining buffer (PBS with 2 % FBS) for 15 min on ice. Cells treated with a single-label antigen were incubated 20 min on ice. Mouse isotype antigens served as the control. Samples were analyzed using a FACscan (Becton– Dickinson, San Jose, CA).
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Osteogenic differentiation P4 and P16 hUCMSCs were cultured in osteogenic medium. Approximately 5 9 103 cells/cm2 were seeded into each well of 12-well tissue-culture plates. hUCMSCs were incubated in complete culture medium for 1 day. The medium was then replaced with StemPro osteogenesis differentiation kit medium containing osteogenesis differentiation basal medium and an osteogenic supplement (Invitrogen, Cat. No. A10072-01). The medium was changed every 2 days for 21 days.
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Sequence Detection System. TaqMan gene expression assay kits (Applied Biosystems), including two predesigned specific primers and probes, were used to measure the transcript levels of the following genes: collagen type I (CI, Hs00164004), osteocalcin (OC, Hs01587813), Runx2 (Hs00231692), bone sialoprotein (BSP, Hs00173720), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905). The relative expression level of each target gene was evaluated using the 2-DDCt method (Livak and Schmittgen 2001) on days 1, 7, 14, and 21 using samples in complete culture medium on 1 day as the calibrator (n = 5).
Biochemical assays and histological staining Data analysis Osteogenic groups were assayed for calcium deposition and alkaline phosphatase (ALP) activity on days 1, 7, 14, and 21. Calcium content was measured using the orthocresolphthalein complexone (OCPC) method (after Brugge and Jansen 2002). Briefly, the specimens were homogenized and suspended in 1 N acetic acid overnight. Digestion solution (10 lL) and assay reagent (300 lL) were mixed and incubated for 10 min at room temperature and then read at 575 nm (Fluoroskan ascent, Thermo Electron Corporation, Waltham, MA). Normal control serum (Stanbio, Boerne TX) containing a colorimetric p-nitrophenyl phosphate assay kit (Stanbio) was used to measure alkaline phosphatase (ALP) activity according to the manufacturer’s instructions, with a known concentration of ALP as the standard. Each sample was processed through 2 freeze-thaw cycles (-70 °C and room temperature, 45 min each) to rupture the cell membranes and release the proteins and DNA from the cells. To each well of a culture plate, 500-lL ALP substrate reagent and 100-lL cell lysate (samples and controls) were added, followed by gentle mixing and incubation for 60 s at 37 °C. Plates were read at 405 nm every 20 min for 3 h. Each sample was normalized to its DNA content. Histological staining was performed using alizarin red staining (Sigma) for mineralization on day 21 [27, 32, 33]. In brief, the cells were washed twice with PBS, fixed for 30 min with 10 % paraformaldehyde, and washed with PBS. Cells were stained with alizarin red for 30 s to 5 min, and the reaction was observed microscopically. Quantitative real-time reverse transcription polymerase chain reaction The total RNA was extracted using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). mRNA was reverse-transcribed into cDNA using a HighCapacity cDNA Archive kit (Applied Biosystems, Foster city, CA). Quantitative real-time RT-PCR reactions were performed using an Applied Biosystems 7500 Fast
All data are expressed as mean ± standard deviation and were analyzed by ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test when significance was detected by ANOVA. Differences between groups were considered significant for p \ 0.05.
Results Growth characteristics of hUCMSCs Early and late passages of hUCMSCs were compared to assess their stability in culture. Cells from early and late passages displayed a similar characteristic MSC spindleshape morphology, with directionality and regularity (Fig. 1a–c). The viability of hUCMSCs was measured using the MTT cell viability assay. MTT assays showed that the viability of early and late hUCMSCs was similar (p [ 0.1) (Fig. 1d). In addition, there were no significant differences (p [ 0.1) in viability from P1 to P16 (at 7 days) (Fig. 1e). Clonogenic capacity was investigated using the CFU-F assay. Early and late hUCMSCs had similar growth kinetics (p [ 0.1) (Fig. 1f). No significant differences in CFU-Fs from P1 to P16 were observed (p [ 0.1) (Fig. 1g). Together, these data indicate passaging of hUCMSCs did not alter their growth characteristics. Phenotypic profiles of hUCMSCs The stability of hUCMSC characteristics over the course of extended expansion from P1 to P16 was determined using flow cytometry. hUCMSCs were negative for the hematopoietic markers CD34 and CD45 but were positive for the mesenchymal stem cell surface markers CD29, CD49e, CD73, CD90, CD105, and CD166. P1 hUCMSCs expressed high levels of MSC surface markers CD73 (96.5 ± 2.2), CD90 (93.5 ± 3.5), CD105 (99.1 ± 2.5), and CD166 (90.5 ± 4.8 %). Expression of these markers
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remained high throughout the whole expansion period as shown in Fig. 2, with the exception of CD166. At P16, hUCMSCs expressed CD73 (96.5 ± 2.4), CD90 (93.5 ± 2.7), CD105 (99.4 ± 0.5), and CD166 (61.5 ± 2.5 %). A progressive decrease in CD166
expression over the course of the early passages was observed, from 95 % at P2 to 58 % at P5 (p \ 0.05), followed by stable expression through P16 (Figs. 2, 3). With the exception of CD166 expression, the phenotype of hUCMSCs was stable throughout passaging.
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Osteogenic differentiation of hUCMSCs The osteogenic potential of hUCMSCs early and late in the course of passaging was compared. P4 and P16 hUCMSCs both differentiated down an osteogenic lineage after 21 days. The degree of osteogenesis did not differ significantly between P4 and P16 hUCMSCs. The appearance of
alizarin-red-positive nodules was similar between P4 and P16 hUCMSCs (Fig. 4a, b). The activity of ALP in hUCMSCs undergoing osteogenic differentiation was also similar between these cells. ALP activity increased as much as 18-fold over controls in P4 hUCMSCs and 17-fold in P16 hUCMSCs on day 14 (Fig. 4c). There was no significant difference in ALP activity between P4 and P16
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160 Fig. 3 Flow cytometry analysis-histogram plots of surface marker expression from P1 to P16 (mean ± standard deviation; n = 6). A high proportion of hUCMSCs expressed CD29, CD44, CD49e, CD73, CD90 and CD105. These high expression levels persisted throughout the expansion period
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hUCMSCs (p [ 0.1). A peak 13-fold increase in calcium deposition was observed in both P4 and P16 hUCMSCs on day 21(Fig. 4d), with no significant difference between P4 and P16 hUCMSCs (p [ 0.1). qRT-PCR was used to compare mRNA expression levels of osteogenic genes. Results show that in P4 hUCMSCs on day 21, CI increased
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4.4-fold, OC increased 4.5-fold, Runx2 increased 3.2-fold, and BSP increased 2.2-fold (Fig. 5). In P16 hUCMSCs on day 21 days, CI increased 4.1-fold, OC increased 4.5-fold, Runx2 increased 3.1-fold, and BSP increased 2.1-fold. There was no significant difference in the expression of any of these markers between P4 and P16 hUCMSCs (p [ 0.1).
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hUCMSCs. c, d Biochemical assay data are presented as the mean ± standard deviation; n = 4. Control hUCMSCs were cultured in complete culture medium. c ALP activity peaked at 14 days and slightly decreased at 21 days. d Calcium content. Scale bar = 500 lm
Discussion
Parts of our results are consistent with previous studies [31, 35, 36]. In addition, we demonstrated more evidences supporting that late passages still possessed the osteogenic differentiation potential in the present study. Under osteogenic differentiation conditions, hUCMSCs from both early and late passages were able to differentiate toward an osteogenic lineage. The ability of HUCMSCs to retain the osteogenic differentiation potential in late passages is promising for future bone tissue engineering applications. In evaluating the suitability of mesenchymal stem cells for medical applications, one must consider their availability and potential to yield a reasonable number of viable cells [37–41]. Accordingly, one of the main goals of this study was to evaluate the viability of hUCMSCs during extended passaging before the induction of osteogenic differentiation. One of the striking features of hUCMSCs observed in a previous study was their capacity to expand cell numbers almost 300-fold over seven passages [27]. In the present study, we observed no loss of viability with passaging. In addition, over the course of 15 passages, hUCMSCs maintained a seemingly homogeneous morphology, appearing as slender spindle-shape cells with a narrow cytoplasm
The current study provides evidence that hUCMSCs maintain their phenotype over multiple passages. The expression of CD cell surface markers except CD166 remained virtually unchanged between early and late passages of hUCMSCs, and P4 and P16 hUCMSCs exhibited similar osteogenic activity. In previous studies, cultured hUCMSCs were successfully guided toward osteogenic differentiation [16, 27, 34]. Wharton’s jelly extract could efficiently slow down the senescence and improve the differentiation ability of late passages of UCMSCs [35]. Umbilical cord tissue pieces derived UCMSCs could mention the proliferation profile, cell cycle distribution and MSC surface markers expression until P10 [36]. In the present study, hUCMSCs were investigated for the influence of cell expansion on the phenotype, proliferative capacity, and osteogenic potential. We observed that hUCMSCs maintained their MSC-like phenotype and viability, after extended passaging. hUCMSCs from both early and late passages exhibited high expression of surface markers associated with BMSCs.
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(Fig. 1). MTT and CFU-F assays showed that the growth dynamics of hUCMSCs did not differ significantly with expansion. Flow cytometric analysis in this study revealed that hUCMSCs expressed matrix receptors (CD44), integrin markers (CD29), and MSC markers (CD73, CD90, CD105 and CD166) but no hematopoietic cell markers (CD34, CD45) (Fig. 2). This observation is consistent with the results of previous studies [20, 42]. We also observed that the high level of CD73, CD90, and CD105 expression did not change significantly during extended passaging. These results suggest that the hUCMSCs characteristics were relatively stable during monolayer expansion during the tested passages. To our knowledge, this is the first study to report the effects of extended passaging on CD166 expression in hUCMSCs. The number of cells expressing CD166 gradually decreased from 90 % at P2 to 61.5 % at P5, where it stabilized through P16 (Fig. 3). CD166 is an adhesion molecule that promotes cell–cell aggregation through the actin cytoskeleton [43]. In stem cells, this transmembrane protein is known to be involved in osteogenic differentiation [44]. The decrease we observed in CD166 expression did not
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prevent osteogenic activity in early or late passages. However, we did not include passages 0–3 hUCMSCs in osteogenic differentiation, leaving uncertainty as to whether decreased CD166 expression decreased the osteogenic potential of hUCMSCs below that of passage 4 cells. Future studies that investigate the osteogenic differentiation potential of P1–P3 hUCMSCs and block CD166 with neutralizing antibody during osteogenic differentiation should be conducted to address the role of CD166 in the osteogenesis of hUCMSC. In vitro osteogenic differentiation of hUCMSCs has been demonstrated previously [16, 27, 34]. Furthermore, hUCMSCs have been shown to undergo a greater extent of mineralization than BMSCs [15], although this may not hold true in 3D tissue culture. In the present study, both P4 and P16 hUCMSCs showed some degree of osteogenic differentiation over a 3 week period, as evidenced by ALP activity, calcium quantification, histology, and bone marker gene expression (CI, OC, Runx2, and BSP). This evidence of hUCMSC osteogenic capacity is consistent with reports from previous studies [16, 27, 34]. Interestingly, late passage hUCMSCs retained the same osteogenic capacity as early passage hUCMSCs. In contrast, several previous
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studies have shown that BMSCs that had undergone extended expansion exhibited decreased differentiation potentials and lower proliferation rates [44–46]. The difference in our results suggests that hUCMSCs are earlierstage cells than adult MSCs, conferring yet another advantage over BMSCs. Although hUCMSCs appear to share some characteristics with MSCs, further studies are needed to understand the functional nature of their multi-potential capacity. The encouraging results of the current study give reason to examine whether adipogenic and chondrogenic potential are also retained over extended passages. In addition, in vivo studies are needed to study potential applications such as bone tissue regeneration. There are still some limitations in this study. In the qPCR experiments, only one reference gene (GAPDH) was used, which may lead to misleading results. More reference genes should be used according to the quantitative real-time PCR experiments (MIQE) guidelines [47]. Second, we did not assess the oncogenecity of hUCMSC at extended passage. However, an in vivo study by Wang et al., demonstrated that although with genomic alterations, P30 hUCMSCs cannot form tumors after subcutaneously transplanting in the immunodeficient mice [48]. These limitations should be addressed in the future study.
Conclusion hUCMSCs maintained their phenotype, proliferation, and osteogenic capacity after multiple passages. hUCMSCs may thus have the potential to be expanded by several orders of magnitude for bone regeneration. Such a capacity would pave the way for technologies that could use hUCMSCs harvested at birth to provide large cell numbers for therapies later in life. Acknowledgments We gratefully acknowledge Prof. Zhengliang Chen at the Southern Medical University for FCM assistance and useful discussions. This study was supported by National Natural Science Foundation of China 31328008 (LZ), 31100695(LZ) Natural Science Foundation of Guangdong s20130010014253 (LZ), Guangdong Provincial Science and Technology Project 2012B010200024 (LZ) and Guangzhou Science and Technology Project 2012027(LZ). Conflict of Interest
None declared.
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