| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TISSUE-SPECIFIC STEM CELLS |
Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, USA
Key Words. Perivascular cells • Bone marrow stromal cells • Multidifferentiation
Correspondence: Rocky S. Tuan, Ph.D., Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, 50 South Dr., Room 1503, BMSC 8022, National Institutes of Health, Bethesda, Maryland 20892-8022, USA. Telephone: 301-451-6854; Fax: 301-435-8017; e-mail: tuanr{at}mail.nih.gov
Received November 13, 2006;
accepted for publication February 13, 2007.
First published online in STEM CELLS EXPRESS March 1, 2007.
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Disclosure of potential conflicts of interest is found at the end of this article.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Sarugaser et al. postulated that the MSC population of the Wharton's Jelly matrix is located close to the vasculature of the cord and specifically isolated these cells, which they called human umbilical cord perivascular cells (HUCPVCs) [4]. Their work provided an initial characterization of HUCPVCs with respect to their nonhematopoietic phenotypic profile and capacity to generate colonies of fibroblastic and osteogenic cells. HUCPVCs were found to have a colony forming unit-fibroblast (CFU-F) frequency of about 1:300 and a population doubling time of 20 hours by passage 2, resulting in significant cell expansion and producing over 1010 HUCPVCs from 2–5 x 106 cells after 30 days of culture. Interestingly, these cells, which are major histocompatibility complex (MHC) class II negative, not only express both an immunoprivileged and immunomodulatory phenotype [9], but their MHC class I expression levels can also be manipulated [4], making them a potential cell source for MSC-based therapies. In addition, HUCPVCs represent a noncontroversial source of primitive mesenchymal progenitor cells that can be harvested after birth, cryogenically stored, thawed, and expanded for therapeutic uses.
The present work focused on directly comparing the proliferative and multilineage differentiation capacity of HUCPVCs to that of adult human BMSCs. Flow cytometry showed a higher expression of CD146, a marker expressed on both BMSCs and dental pulp MSCs [10], on HUCPVCs relative to BMSCs. Immunoselection for CD146 showed that CD146+ HUCPVCs had the capacity to differentiate down the osteogenic, adipogenic, and chondrogenic lineages. In addition, using standard transfection protocols, HUCPVCs were found to be amenable to gene transfection. Finally, we used gene array analysis to demonstrate the expression of components of Wnt signaling pathways, which are important in the regulation of adult MSC activities in HUCPVCs. These findings further support the potential utility of HUCPVCs for cell-based therapeutic applications.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
Phenotypic Analysis
Standard flow cytometry techniques were used to determine the cell surface epitope profile (CD49e, CD90, CD146, CD31, CD117, CD45, CD34, and Stro-1) of HUCPVCs and BMSCs. Briefly, HUCPVCs and BMSCs were incubated with saturating concentrations (1:70) of conjugated mouse monoclonal antibodies to human CD49e-phycoerythrin (PE), CD90-allophycocyanin (APC), CD146-PE, CD31, CD117-PE, CD34-PE, and CD45-PE (all antibodies were purchased from BD Biosciences, San Diego, http://www.bdbiosciences.com). Cells were incubated for 1 hour at 4°C. Stro-1 antibody detection involved incubating the cells in 200 µl of saturating concentrations of mouse IgM monoclonal antibody Stro-1 (American Type Culture Collection, Manassas, VA, http://www.atcc.org) for 30 minutes at 4°C. Prior to antibody labeling, the cells were preincubated with 1% human, goat, and mouse sera for 10 minutes. Subsequently, to block nonspecific binding, the cells were washed twice with phosphate-buffered saline (PBS) containing 2% FBS and then incubated with PE-conjugated rat anti-mouse IgM monoclonal secondary antibody (BD Biosciences). All cell suspensions were washed twice with PBS and resuspended in PBS + 2% FBS for analysis on a flow cytometer (FACSCalibur; BD Biosciences) using the CellQuest software. Positive staining was defined as the fluorescence emission that exceeded levels obtained by more than 99% of cells from the population stained with the corresponding negative controls. The isotype (negative) controls used in these studies were IgG1-PE and IgG1-APC (BD Biosciences).
Cell Proliferation Assays
HUCPVCs and BMSCs were plated at 3 x 103 cells per cm2 in 12-well plates in basal medium (10% FBS in DMEM). Medium was refreshed every 3 days. On days 3, 7, 14, and 20, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay was performed according to manufacturer's protocol (Promega, Madison, WI, http://www.promega.com).
Multilineage Differentiation Assays
Osteogenesis. Cells were grown in osteogenic growth medium (10 nM dexamethasone [DEX], 5 mM β-glycerophosphate, 50 µg/ml ascorbic acid [AA], and 10 nM 1,25-dihydroxy vitamin D3) (all reagents purchased from Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). On day 21, cultures were stained for alkaline phosphatase (ALP; Sigma) activity and mineralization was assessed by von Kossa staining (2% silver nitrate; Sigma) after 5 weeks. Quantification of ALP activity was determined by adding 4 mg/ml p-nitrophenyl phosphate (Sigma) in an alkaline buffer solution (221 Alkaline Buffer Solution; Sigma) into each well, followed by incubation for 15 minutes at 37°C. The reaction was terminated with 0.05 N NaOH, and ALP activity was estimated spectrophotometrically on the basis of A405, determined using a microplate reader.
Chondrogenesis. Cells were grown as high-density pellets (2.5 x 105 cells) for 21 days in serum-free medium (DMEM, ITS Premix [BD Biosciences], 50 µg/ml AA, 40 µg/ml L-proline, 100 µg/ml sodium pyruvate, 0.1 µM DEX) with and without 10 ng/ml recombinant human transforming growth factor (TGF)-β3 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com). On day 21, pellets were prepared for histology to detect sulfated glycosaminoglycan (sGAG) (Alcian Blue staining) and collagen content (picrosirius red staining). sGAG content was quantified using the Blyscan sGAG assay (Accurate Chemical & Scientific Corp., Wesbury, NY, http://www.accuratechemical.com).
Adipogenesis. Cells were cultured as a monolayer in the presence of adipogenic supplements (1 µM DEX, 1 µg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine; Sigma). On day 21, cultures were stained with oil red O stain (Sigma), and dye content was quantified by isopropanol elution and spectrophotometry.
Histology of Human Umbilical Cord
Human umbilical cord cryosections were generously prepared and provided by Jane Ennis (University of Toronto, Canada). Sections were stained with Harris hematoxylin (Sigma) and counterstained in aqueous eosin (Sigma) (H&E). Sections were also stained with CD146-PE or with an isotype control (IgG1-PE; BD Biosciences) following the methods described above and counterstained with Hoechst nuclear dye. Immunostained sections were observed with epifluorescence optics (Leitz, Heerbrugg, Switzerland).
Fluorescence-Activated Cell Sorting
HUCPVCs were incubated with saturating concentrations (1:100 dilution) of conjugated mouse IgG1,
anti-human CD146e-PE (BD Biosciences). Both the CD146-negative and CD146-positive cells were sorted on a MoFlo Cell Sorter (Dako, Glostrup, Denmark, http://www.dako.com) at a rate of 2,000–3,000 cells per second at 10.5 psi to 98% purity. The sorted populations were cultured under osteogenic, adipogenic, and chondrogenic conditions.
Transfection Efficiency
To assess the efficiency of commonly used transfection protocols, HUCPVCs were transfected with DsRed (Clontech, Palo Alto, CA, http://www.clontech.com) by means of FuGENE 6 Transfection Reagent (Roche Diagnostics, Basel, Switzerland, http://www.rocheapplied-science.com), Effectene Transfection Reagent (Qiagen, Hilden, Germany, http://www.qiagen.com), and nucleofection (amaxa biosystems, Gaithersburg, MD, http://www.amaxa.com). The total mass of DNA to volume of FuGENE reagent used was 2:3 in the FuGENE protocol. Effectene transfection was performed according to the manufacturer's protocol for adherent cells. Nucleofection with amaxa reagent was according to the methods described by Haleem-Smith et al. [11]. Briefly, 5 x 105 cells were incubated with 2 µg of DsRed cDNA and resuspended in 100 µl of nucleofection reagent and subject to electroporation using the manufacturer's protocol (amaxa). After 4 days post-transfection, the expression of DsRed was assessed by fluorescence microscopy and quantified by flow cytometry. To monitor the long-term stability of exogenous gene expression, positive DsRed expression was determined using flow cytometry on days 4, 7, 14, and 21 post-transfection. As control, BMSCs were similarly transfected.
Wnt Signaling Pathway
Expression of Wnt Signaling Components. To compare the Wnt signaling pathway molecular profile between HUCPVCs and BMSCs, RNA was harvested from both HUCPVCs and BMSCs (passage 3, n = 2) and analyzed, following manufacturer's recommended protocol, on an Oligo GEArray Human Wnt Signaling Pathway Microarray (catalogue number: OHS-043; SuperArray Bioscience Corporation, Frederick, MD, http://www.superarray.com).
Canonical Wnt Responsive Reporter Assay. An optimized version of the TOP-FLASH luciferase reporter plasmid (pLG3-OT) (kindly provided by Dr. Bert Vogelstein, Johns Hopkins University) was used to assay canonical Wnt-responsive transcriptional activity in HUCPVCs relative to BMSCs. Cells were cotransfected by nucleofection (amaxa) with expression constructs of Wnt3a and Wnt5a and empty vector (control) (all purchased from Upstate, Charlottesville, VA, http://www.upstate.com) and Renilla luciferase plasmid (1 µg) under the control of the cytomegalovirus promoter (Promega) and TOP-FLASH luciferase plasmid (2 µg). Luciferase activity was assayed using the Dual Luciferase Assay System according to the manufacturer's protocol (Promega) and reporter luciferase activity normalized to that of Renilla luciferase.
Data Analysis
Data are presented as mean ± SD for at least three independent experiments (different patient samples) performed in triplicate, unless stated otherwise. Statistical significance is determined by analysis of variance and set at p < .05.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
|
|
|
Expression of CD146 in Human Umbilical Cord
H&E staining of human UC revealed areas containing high and low cell densities (Fig. 5A), which correlated with nuclear dye staining (Fig. 5B). Specifically, H&E staining showed a clear demarcation of the vessel lumen, vessel wall (VW), and perivascular region (PVR) (Fig. 5A). Incubation with CD146 and nuclear staining showed that CD146 was highly prevalent in the PVR region with diffuse staining in the VW region (Fig. 5C). CD146+ cells were not readily detected in the bulk Wharton's Jelly region from these cord samples.
|
Transfection Efficiency
To assess transfection capacity, HUCPVCs and BMSCs were transfected with a DsRed plasmid construct using an electroporation-based method (amaxa) and two different types of standard liposomal transfection methods (FuGENE and Effectene). After 4 days post-transfection, fluorescence microscopy showed similar levels of DsRed positive cells from HUCPVCs and BMSCs transfected by nucleofection (Fig. 6A, 6B), whereas slightly different numbers of DsRed positive cells were seen from FuGENE and Effectene transfected HUCPVCs and BMSCs (Fig. 6C–6F). Flow cytometry quantified the relative difference in transfection efficiency between the different methods for both HUCPVCs and BMSCs (Fig. 6G). In general, nucleofection resulted in 
50% cell death but high transfection efficiency, whereas transfection with FuGENE and Effectene resulted in 90% cell viability but lower transfection efficiency. Interestingly, HUCPVCs demonstrated higher transfection efficiencies with FuGENE and Effectene compared with BMSCs (Fig. 6G).
|
Wnt Signaling Pathway Profile of HUCPVCs
As a first screen to assess biological and functional similarities between HUCPVCs and BMSCs at the molecular level, HUCPVCs and BMSCs were compared using a Wnt pathway focused oligo gene array (SuperArray Biosciences) designed to profile the expression of genes involved in and downstream of Wnt signaling. The Wnt signaling pathway is represented on the array by genes encoding the 19 glycosylated extracellular signaling molecules of the Wnt family, cell-surface receptors serving as ligands of Wnt genes (e.g., Frizzled receptors), intracellular signaling molecules and target genes involved in the cell cycle, and growth regulation and proliferation, regulators of the Wnt signaling pathway and competitive Wnt binding antagonists, and genes involved in protein modification, as a result of kinase and phosphatase activity and ubiquitination, downstream of Wnt signaling. Analysis of the gene arrays (Fig. 7A, 7B) was performed using the manufacturer's computer software to determine differential gene expression of Wnts and Frizzled in HUCPVCs and BMSCs (Fig. 7C, 7D). The results revealed that HUCPVCs expressed lower levels of Wnt1, Wnt5a, and Wnt5B (Fig. 7C) and lower levels of Fz1, Fz5, and Fz7 but a higher level of Fz2 (Fig. 7D), relative to BMSCs. There were no significant differences between HUCPVCs and BMSCs in the gene expression of all the other Wnts and Frizzled receptors (data not shown). We also observed that DKK1 and PITX2 were overexpressed (Fig. 7E), whereas a number of downstream Wnt signaling molecules were underexpressed, in HUCPVCs relative to BMSCs (Fig. 7F).
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
The nonhematopoietic nature of HUCPVCs is suggested by their lack of cell surface expression of CD45 [3, 4]. Our analysis of a variety of cell surface markers on HUCPVCs relative to BMSCs confirmed the nonhematopoietic phenotype of HUCPVCs and revealed a similar epitope profile to BMSCs, including Stro-1, a well accepted MSC marker [12] (Fig. 1A). However, the expression of CD146 was most notably elevated in HUCPVCs relative to BMSCs, both as a harvested cell population from the perivascular region as well as by direct immunostaining of umbilical cord (Fig. 5C). CD146 is characteristically expressed on circulating endothelial cells, which also express CD31, a classic endothelial marker [13]. However, the expression of CD146 on circulating endothelial cells is rarely found in the blood circulation of healthy individuals, but rather is present in a number of diseased conditions, which include inflammatory, immune, infectious, neoplastic, and cardiovascular disease [14], and has thus been considered a useful marker of cells applicable for therapeutic neovascularization and vascular repair [15]. We confirmed by coimmunostaining of CD146 with CD31 that CD146+ HUCPVCs are not endothelial in origin due to the lack of CD31 expression. Our results, in part, corroborate with those of Shi et al., who demonstrated that Stro-1+ BMSCs express CD146, and that this population could give rise to CFU-Fs [10]. Our work, however, extends upon these observations and shows that a CD146+-sorted HUCPVC population has the capacity to differentiate down the osteogenic, chondrogenic, and adipogenic mesenchymal lineages. Taken together, these results infer a potential novel role of HUCPVCs to circulate in biological fluid, similar to CD146+ endothelial cells, and participate in the repair of diseased and/or damaged tissue. In future studies, the capacity of HUCPVCs to transit biological fluid and "home" to organs of the body will be examined in appropriate animal models.
HUCPVCs demonstrated a greater proliferative capacity when compared with BMSCs, especially after 7 days of culture, as shown by a significant difference in cumulative population doublings (Fig. 2A). Interestingly, after 20 days, HUCPVCs did not experience contact-inhibited cell growth, unlike BMSCs, which failed to continue to proliferate after reaching 100% surface confluence. HUCPVCs continued to grow by cell multilayering and formed aggregates overlying a layer of confluent cells. These cell aggregates contained live cells as determined by trypan blue exclusion, and when transferred to a new tissue culture flask, these cell aggregates demonstrated the capacity to plate out and proliferate to colonize the culture substrate (data not shown). Using a Cancer Pathway Finder Gene Array (SuperArray Biosciences), we have ruled out the possible transformation to a cancer phenotype of HUCPVCs generated from these aggregates (data not shown). However, we are currently examining the changes in the cell surface membrane expression of a variety of adhesion molecules to identify the molecular mechanism involved in this phenomenon.
Our results also showed that HUCPVCs, as a population, have multilineage differentiation capacity. Importantly, HUCPVCs undergo osteogenesis at a faster rate than BMSCs, based on the increase in ALP positive cells (Fig. 3A), and after 5 weeks of culture under osteogenic conditions, HUCPVCs generated a greater extent of mineralization than BMSCs, including bone nodule generation (Fig. 3B). This difference may be attributed to the fact that the initial cell isolate of HUCPVCs has a higher frequency of osteoprogenitor cells [4] than bone marrow-derived BMSCs [16]. Nevertheless, this finding may render HUCPVCs a readily available source of cells for orthopaedic and tissue engineering strategies, which require suitable cells for abundant matrix synthesis [17]. Furthermore, cartilaginous pellets derived from HUCPVCs displayed an extracellular matrix that stained positive for sGAG and collagen, similar to that seen in cultures of BMSCs. As such, HUCPVCs may also be considered a viable cell source for adult human cartilage repair [18]. Studies are underway to evaluate the mechanical properties of the elaborated chondrogenic matrix according to the methods described by Mauck et al. [19].
Cell-based strategies may inevitably involve the introduction of transgenes into the donor cell population and, thus, in order to evaluate HUCPVCs as a candidate cell source for a variety of gene-based strategies, we studied the transfectability of HUCPVCs using conventional and established transfection methods for mammalian cells. FuGENE and Effectene transfection reagents were chosen, as they represent nontoxic methods (high cell viability) of introducing DNA into cells [20], whereas nucleofection using the amaxa system provides high transfection efficiency but lower cell viability [11]. Nucleofection resulted in the highest transfection efficiency for both HUCPVCs and BMSCs (>50% of DsRed expression) but the highest cell death, as determined by propidium iodide staining, (50%) relative to FuGENE and Effectene (<2%) (data not shown). Interestingly, HUCPVCs were also transfectable using FuGENE (18%), whereas BMSCs showed marginal DsRed expression with FuGENE (Fig. 6). The results observed for BMSCs with FuGENE are consistent with those reported by others [21]. The ability to introduce exogenous DNA into HUCPVCs using conventional methods, which are straightforward and allow for high-throughput, renders HUCPVCs a promising MSC candidate source of cells for gene therapy approaches, similar to BMSCs [22, 23]. However, despite the higher transfection efficiency with nucleofection, HUCPVCs lost DsRed expression at a faster rate compared with BMSCs (Fig. 5H), suggesting a possibly different mode of transgene incorporation; the higher proliferative rate observed in HUCPVCs relative to BMSCs may also interfere with long-term transgene stability. Viral methods of transgene incorporation may thus be a more effective approach to achieve long-term transgene incorporation in HUCPVCs [24]. However, for short-term studies, HUCPVCs represent a useful cell type for the introduction of transgenes using conventional methods, resulting in high cell viabilities.
To further examine the biological characteristics of HUCPVCs, we employed a gene array approach to compare the Wnt signaling pathway gene expression profiles of HUCPVCs and BMSCs. We and others have shown that the Wnt signaling pathways play a critical role in the maintenance and osteogenic potential of bone marrow-derived BMSCs [25, 26] and may be modulated to enhance bone repair in a variety of human diseases, such as osteoporosis and osteosarcoma. Our results showed that HUCPVCs express genes of Wnt signaling pathways, and the profile was generally similar to that of BMSCs except for several over- and underexpressed genes, such as DKK1/Fz2 and Wnt1/Fz1, respectively (Fig. 7). The functional significance of these differences is currently being studied. Importantly, HUCPVCs demonstrated the capacity to transduce canonical Wnt signaling in response to Wnt3a, a canonical Wnt, which was blocked when Wnt5a, a noncanonical Wnt, was transfected into the cells (Fig. 7G). Taken together, these findings reveal that HUCPVCs and BMSCs share common Wnt signaling pathways and, thus, the tools and reagents used to study Wnt signaling pathways in other cell systems, including BMSCs, can be applied to HUCPVCs.
In summary, our work demonstrates that HUCPVCs represent a nonhematopoietic, nonendothelial cell population with the capacity to proliferate and differentiate down the osteogenic, chondrogenic, and adipogenic lineages. The results from our direct comparison of HUCPVCs to BMSCs further support HUCPVCs as a candidate MSC population for cell-based therapeutic strategies.
|
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. L. Troyer and M. L. Weiss Concise Review: Wharton's Jelly-Derived Cells Are a Primitive Stromal Cell Population Stem Cells, March 1, 2008; 26(3): 591 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Karahuseyinoglu, C. Kocaefe, D. Balci, E. Erdemli, and A. Can Functional Structure of Adipocytes Differentiated from Human Umbilical Cord Stroma-Derived Stem Cells Stem Cells, March 1, 2008; 26(3): 682 - 691. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wu, M. W. Lensch, J. Wylie-Sears, G. Q. Daley, and J. Bischoff Hemogenic Endothelial Progenitor Cells Isolated from Human Umbilical Cord Blood Stem Cells, November 1, 2007; 25(11): 2770 - 2776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Can and S. Karahuseyinoglu Concise Review: Human Umbilical Cord Stroma with Regard to the Source of Fetus-Derived Stem Cells Stem Cells, November 1, 2007; 25(11): 2886 - 2895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Park, K. H. Jung, S. H. Kim, K. S. Kim, M. R. Choi, Y. Kim, and Y. G. Chai Functional Expression of Ion Channels in Mesenchymal Stem Cells Derived from Umbilical Cord Vein Stem Cells, August 1, 2007; 25(8): 2044 - 2052. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
