| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TISSUE-SPECIFIC STEM CELLS |
Department of Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA
Key Words. Liver • Stem cell • Hepatoblast • Progenitor • Epithelial cell adhesion molecule • Neural cell adhesion molecule • Hepatocyte • Gene expression • 
-Fetoprotein
Correspondence: E. Schmelzer, Ph.D., University of North Carolina School of Medicine, Campus Box 7038, Glaxo Building Rooms 32–35, Chapel Hill, North Carolina 27599, USA. Telephone: 919-966-0346; Fax: 919-966-6112; e-mail: eva_schmelzer{at}med.unc.edu or Professor L.M. Reid, Ph.D., University of North Carolina School of Medicine, Campus Box 7038, Glaxo Building Rooms 32–35, Chapel Hill, North Carolina 27599, USA. Telephone: 919-966-0347; Fax: 919-966-6112; e-mail: stemcell{at}med.unc.edu
Received on January 18, 2006;
accepted for publication on April 12, 2006.
First published online in STEM CELLS EXPRESS April 20, 2006.
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
-fetoprotein (AFP) and adult liver-specific proteins. By contrast, hepatoblasts, the dominant cell population in fetal and neonatal livers, decline in numbers with age and are found as <0.1% of normal adult livers. They express high levels of AFP, elevated levels of albumin, low levels of expression of adult liver-specific proteins, low levels of CK19, and a loss of NCAM and CLDN-3. Mature hepatocytes lack expression altogether of EpCAM, NCAM, AFP, CLDN-3, cytokeratin 19, and have acquired the well-known adult-specific profile that includes expression of high levels of albumin, cytochrome P4503A4, connexins, phosphoenolpyruvate carboxykinase, and transferrin. Thus, hepatic stem cells have a unique stem cell phenotype, whereas hepatoblasts have low levels of expression of both stem cell genes and genes expressed in high levels in mature hepatocytes.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
The ratio of hepatic stem cells to hepatoblasts changes depending on developmental stage, with the hepatoblasts being dominant in fetal and neonatal livers and with few if any mature parenchymal cells [4]. In addition, the fetal liver is the primary site of hematopoiesis, consisting of up to 60% erythrocytes at certain developmental stages [7]. By contrast, in pediatric and adult human livers, the hepatic stem cells are the dominant pluripotent progenitor (0.3%–0.7%); the hepatoblasts are few (<0.1%), and the majority (>98%) of the parenchymal cells are diploid and polyploid hepatocytes and biliary epithelia (Schmelzer et al., manuscript submitted for publication). These findings are summarized in a recent review [4].
Although the identity of hepatic stem cells and hepatoblasts in human livers has been described recently, our knowledge is incomplete on the gene and protein expression profiles of these pluripotent progenitors and whether the profiles change during development. We have shown that epithelial cell adhesion molecule (EpCAM), a 34–40-kDa transmembrane glycoprotein [8] first described by Spurr et al. [9] and cloned by Strnad et al. [10] and Szala et al. [11] is expressed by hepatic stem cells, hepatoblasts, and committed progenitors but not by mature hepatocytes [4] (Schmelzer et al., manuscript submitted for publication; Bruce et al., manuscript submitted for publication). Thus, sorting for EpCAM results in only progenitor cells but in distinct ratios of hepatic stem cells to hepatoblasts, depending upon whether the tissue is fetal, neonatal, or adult. In these studies, we analyzed the gene expression profiles in hepatic stem cells and hepatoblasts isolated from fetal, neonatal, pediatric, and adult livers and compared these profiles to those of mature hepatocytes.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
Fetal Liver Cell Isolation
Fetal livers were obtained from Advanced Biological Resources (Alameda, CA) and were of developmental stages between 16 and 20 weeks of gestation. Nonhepatic tissue was removed, and livers were minced by scalpel and digested at 37°C by 0.06% collagenase (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in RPMI 1640 (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 0.03% deoxyribonuclease (w/v), 0.1% (w/v) bovine serum albumin fatty acid-free, 30 nM selenium (Sigma-Aldrich), and antibiotic-antimycotic mixture (Gibco). Hematopoietic cells and nonparenchymal cells were separated from the parenchymal cell fraction by slow-speed centrifugations (30g for 5 minutes in 40 ml) until the pellet showed minimal contamination with red blood cells. Cells in suspension were filtered through a 70-µm nylon mesh, and flow through was applied on a Ficoll-Paque (Amersham Biosciences-GE Healthcare, Piscataway, NJ, http://www.amersham.biosciences.com) gradient (cells in a 5-ml suspension applied on 5 ml of 84% Ficoll, placed on 5 ml of 100% Ficoll) and centrifuged for 30 minutes at 4°C at 980g. Cells forming an interphase were collected and washed once in RPMI 1640 supplemented with 0.1% bovine serum albumin fatty acid-free (w/v), 30 nM selenium, 0.054% niacinamide (w/v), insulin (0.135 USP units/ml), bovine apo-transferrin (10 µg/ml), free fatty acid mixture (2.36 µM palmitic acid, 0.21 µM palmitoleic acid, 0.88 µM stearic acid, 1.02 µM oleic acid, 2.71 µM linoleic acid, 0.43 µM linolenic acid), 1 µM M hydrocortizone, 50 µM ß-mercaptoethanol (Sigma-Aldrich), 2 mM L-glutamine, and antibiotic-antimycotic mixture (Gibco) and kept on ice for further handling.
Cell Sorting
Cell sorting for EpCAM+ cells was done using magnetic-activated cell sorting (MACS) according to manufacturer's instructions (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com). Cells were incubated in the dark at 4°C with fluorescein isothiocyanate (FITC)-conjugated antibody (Miltenyi Biotec) for 20 minutes at a concentration of 50 µl for 50 x 106 total cells in 500 µl of buffer (phosphate-buffered saline [PBS]) containing 0.5% bovine serum albumin and 2 mM EDTA (Sigma-Aldrich). EpCAM+ cells were labeled with magnetic beads using an anti-FITC Multisort Kit (Miltenyi Biotec) and selected by Midi- or MiniMACS columns and separation units (Miltenyi Biotec). All incubation and selection steps were performed on ice or at 4°C with addition of 10% accutase (Innovative Cell Technologies, San Diego, CA) to prevent aggregation of cells.
Cell Culture
Cells obtained by Ficoll gradient selection were placed in culture on plastic or collagen III (6.25 µg/cm2) (Sigma-Aldrich)-coated plastic vessels. Cultivation was done in supplemented RPMI 1640 medium (see cell isolation) with an initial 24-hour phase of 10% fetal bovine serum addition. After 24 hours, cells were cultured serum-free, and medium was changed every 4 days. Colonies that formed were picked at 10 and 20 days and collected on ice for further analyses.
Cytospins
Following enzymatic digestion of fetal livers, freshly isolated cells were centrifuged at 600g for 5 minutes at 4°C. Cells were then resuspended in PBS and 4% paraformaldehyde (1:1). Following fixation for 10 minutes at room temperature, cells were centrifuged as previously described. The supernatant was aspirated, and the cells were then blocked in PBS containing 2% Triton X-100 (Sigma-Aldrich), 10% goat serum (Gibco), and 2% cold water fish gelatin (Sigma-Aldrich). Cells were incubated for 1 hour at room temperature and then centrifuged as described above. Primary and secondary antibodies were incubated for 60 minutes at room temperature. Cells were washed twice in PBS plus 2% Triton, centrifuged, resuspended in blocking buffer, and cytospun at 800 rpm for 3 minutes using precoated cytoslides (Shandon Anatomical Pathology, Pittsburgh, PA). Cells were mounted in aqueous glycerol gelatin mounting medium (Sigma-Aldrich) and coverslipped. Images were visualized using a Leica SP2 AOBS laser scanning confocal microscope that was controlled by Leica SP2 TCS software (Leica, Bannockburn, IL, http://www.leica.com). Negative controls were performed by omitting primary antibody from the protocol and revealed no staining (data not shown).
SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analyses
Total proteins were extracted from sorted cells or from cultured cells by washing cells once with ice-cold PBS and resuspending cell pellets in ice-cold protein lysis buffer (1% Triton X-100, 10 mM TrisHCl, 150 mM NaCl, 1% protease inhibitor cocktail [Sigma-Aldrich]). Cell suspensions were spun down at 
20,000g for 5 minutes at 4°C, and supernatants were frozen in aliquots for further analysis at –20°C.
Denaturing SDS-polyacrylamide gel electrophoresis and Western blots were done as described previously [12, 13] using a Mini-Protean-3-Cell and Mini-Trans-Blot Transfer Cell (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Nitrocellulose membranes were blocked in Odyssey (LI-COR, Lincoln, NE, http://www.licor.com) blocking buffer/PBS (1:1) for 1 hour, and incubated with primary and appropriate secondary antibody in blocking buffer/PBS + 0.1% Tween-20 (Sigma-Aldrich). Membranes were washed four times for 5 minutes with PBS + 0.1% Tween-20 after each incubation with antibody. Dried membranes were scanned using an Odyssey infrared scanner (LI-COR). Quantitative protein expression was done by loading protein standards of known concentration simultaneously on the gel, creating a standard curve that was applied to calculate unknown protein concentrations in samples using the Odyssey version 1.2 software (LI-COR).
Standards for quantitative protein analyzes were as follows: albumin (Research Diagnostics, Flanders, NJ, http://www.researchd.com), -fetoprotein (AFP) (Fitzgerald Industries International, Concord, MA, http://www.fitzgerald-fii.com), cytokeratin 19 (CK19) (Research Diagnostics), and transferrin (Fitzgerald Industries International).
Primary antibodies were albumin, AFP (Sigma-Aldrich), CK19 (Cymbus Biotechnology, Hampshire, U.K.), transferrin (Rockland, Gilbertsville, PA), and CLDN-3 (Abcam, Cambridge, MA, http://www.abcam.com). Secondary antibodies were Alexa Fluor 680 (Western blots) and AF568 (cytospins) polyclonal goat anti-rabbit IgG (H+L) and Alexa Fluor 680 (Western blots) and AF488 (Cytospins) polyclonal goat anti-mouse IgG (H+L) (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com).
Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction
Gene expression was analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Gene-specific mRNAs were created as followed: total RNA from livers was extracted using the RNeasy kit (Qiagen, Valencia, CA, http://www1.qiagen.com) and reverse-transcribed by Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and oligo-(dT)(12–18) primer (Gene Link, Hawthorne, NY). cDNA was used as template in conventional PCR with gene-specific primers (for sequences, see Table 1) from which the forward primer possessed a 5' overhang for T7-promotor sequence (5'-gac tcg taa tac gac tca cta tag gg). This amplified gene-specific DNA was used for in vitro transcription with T7-RNA polymerase (Promega, Madison, WI, http://www.promega.com), generating gene-specific RNA(with an additional 5'-ggg included by T7-RNA polymerase) used as standards in quantitative real-time RT-PCR using gene-specific primers without the 5' overhang; standard ranges were linear from 1 to 108 templates. Primers were selected on different exons wherever possible. Quantitative real-time RT-PCR was done in the LightCycler instrument using the LightCycler RNA Master SYBR Green I kit (Roche). RNA from samples was extracted using RNeasy mini kit (Qiagen).
|
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
|
|
|
|
|
|
-1-antitrypsin) were not expressed by either hepatic stem cells or hepatoblasts. Although variations between different donors in gene expression profiles could be observed (variations caused not only by individual patient history but influenced by shipping and handling of the donor organ) we were able to manifest distinct trends in gene expression patterns, clearly showing different profiles for hepatic stem cells and hepatoblasts.
Expression Profiles of Hepatocytes from Livers of Donors of Different Ages
Gene expression in hepatocytes was compared with that in hepatic stem cells and is summarized in Table 4 by calculating ratios of the data from EpCAM+ versus EpCAM–-sorted cells. Gene expression data from hepatocytes from postnatal livers proved quite variable due to variation caused by organ handling/shipping (necrosis caused by time delay until processing) and donor history (drug administration, nicotine use, obesity, etc.). Nevertheless, a pattern that proved consistent was the absence of expression of the genes defining the hepatic stem cells (e.g., NCAM, CK19, c-kit, and CLDN-3) or hepatoblasts (e.g., AFP) and high levels of expression of classic liver-specific genes (cytochrome P4503A4 and transferrin).
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
The ability to isolate and purify specific subpopulations of human liver cells such as the human hepatic stem cells has been made possible only recently by using immunoselection for a defined antigen such as EpCAM, a transmembrane glycoprotein described by de Boer [14] and found now to be expressed by hepatic progenitors but not hepatocytes [4] (E. Schmelzer, L. Zhang, A. Melhem, N.G. Moss, E. Wauthier, R.E. McClelland, A. Bruce, H. Yao, W.S. Turner, N. Cheng, M.E. Furth, and L.M. Reid, manuscript submitted for publication; A. Bruce, L. Zhang, J.W. Ludlow, E. Schmelzer, A. Melhem, M. Kulik, L.M. Reid, and M.E. Furth, manuscript submitted for publication). Subdivision of the hepatic progenitor subpopulations into hepatic stem cells versus hepatoblasts can also be done by culture selection conditions that give rise to clonigenically expanding stem cell colonies (E. Schmelzer, L. Zhang, A. Melhem, N.G. Moss, E. Wauthier, R.E. McClelland, A. Bruce, H. Yao, W.S. Turner, N. Cheng, M.E. Furth, and L.M. Reid, manuscript submitted for publication). The only comparable findings by others are those of Tanimizu et al. [15, 16], who have shown isolation of hepatoblasts from murine and rodent livers by Dlk/Pref-1, a membrane protein with six extracellular epidermal growth factor-like repeats [17, 18].
By immunohistochemical analyses, we have been able to assign EpCAM expression in nonpathologic human livers to hepatic stem cells and hepatoblasts in all developmental stages (i.e., fetal, neonatal, pediatric, and adult livers). Transplantation into the livers of mice of EpCAM+-sorted cells gave rise to human liver tissue expressing human-specific liver proteins (E. Schmelzer, L. Zhang, A. Melhem, N.G. Moss, E. Wauthier, R.E. McClelland, A. Bruce, H. Yao, W.S. Turner, N. Cheng, M.E. Furth, and L.M. Reid, manuscript submitted for publication; A. Bruce, L. Zhang, J.W. Ludlow, E. Schmelzer, A. Melhem, M. Kulik, L.M. Reid, and M.E. Furth, manuscript submitted for publication). Sorting for EpCAM+ cells from pediatric and adult livers resulted in a population mainly comprised of hepatic stem cells, whereas those from fetal and neonatal livers resulted in predominantly hepatoblasts.
We show here that hepatic stem cells from livers at all developmental stages show highly similar expression profiles, defined below as the stem cell phenotype. Phenotypic characterization of gene expression at mRNA and protein levels in pluripotent human hepatic progenitors versus mature hepatocytes indicated two patterns, a stem cell phenotype and a liver-specific phenotype; a gradient from minimal levels was observed in hepatoblasts to maximal levels found in hepatocytes. The stem cell phenotype comprised expression of EpCAM, NCAM, CK19, c-kit, CLDN-3, and weak levels of albumin but no expression of AFP or adult liver-specific proteins such as transferrin, connexins, PEPCK, DPP4, or P450s. The expression of both hepatic (albumin) and biliary (CK19) markers reflects the bipotential nature of the hepatic stem cell toward both lineages of the liver, the hepatic and biliary, which are characterized by albumin and CK19 expression, respectively. In studies published elsewhere, the hepatic stem cells also express components of the Hedgehog signaling pathway, including Indian and Sonic Hedgehog and its receptor, Patched [19].
The hepatocyte phenotype comprised expression of classic liver-specific genes such as albumin, connexins, transferrin, PEPCK, DPP4, and P450s and loss of expression of the genes typifying stem cells (including EpCAM, NCAM, CK19, c-kit, and CLDN-3). Hepatoblasts proved to be cells in an intermediate position with lowered levels of the stem cell genes and with activation but low levels of adult liver-specific genes and with the unique, defining feature of AFP.
A unique and novel hepatic stem cell surface marker is CLDN-3. Together with occludins, claudins form tight junction proteins [20] that act as a barrier for the passage of ions and molecules through the paracellular pathway and to the movement of proteins and lipids between the apical and the basolateral domains of the plasma membrane. Claudins comprise a family of more than 20 members (reviewed in ref. [21]). Morita et al. [20] demonstrated that CLDN-3 is expressed in mouse lung, liver, kidney, and testis and that the mouse gut shows lineage dependent expression of CLDN-3; liver sections show staining for CLDN-3 in bile canaliculi regions [20]. After partial hepatectomy in rats, CLDN-3 is evident periportally and in sites where regenerative responses are initiated [22]. Combining our results with these previous findings, CLDN-3 is a candidate surface molecule expressed by hepatic stem cells and providing a marker in addition to NCAM, permitting selection uniquely for stem cells.
In summary, this study suggests that the pluripotent hepatic progenitors are evident in both fetal and postnatal livers and remain stable in their phenotypes throughout life, particularly the hepatic stem cells, which persist in relatively constant numbers in livers at all donor ages. The primary changes with age are in the numbers of hepatoblasts that are the dominant cell type in fetal and neonatal livers and then decline in numbers to comprise <0.1% of the parenchyma in normal adult livers. Hepatoblast numbers appear to wax and wane with need for regenerative responses, and the only conditions under which they are observed in high numbers other than in fetal development are in diseased states such as cirrhosis [23]. Their responses suggest parallels with transit-amplifying cells in other tissues.
|
DISCLOSURES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  N. Gago, V. Perez-Lopez, J. P. Sanz-Jaka, P. Cormenzana, I. Eizaguirre, A. Bernad, and A. Izeta Age-Dependent Depletion of Human Skin-Derived Progenitor Cells Stem Cells, May 1, 2009; 27(5): 1164 - 1172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Brezillon, D. Kremsdorf, and M. C. Weiss Cell therapy for the diseased liver: from stem cell biology to novel models for hepatotropic human pathogens Dis. Model. Mech., September 1, 2008; 1(2-3): 113 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Chiao, M. Elazar, Y. Xing, A. Xiong, M. Kmet, M. T. Millan, J. S. Glenn, W. H. Wong, and J. Baker Isolation and Transcriptional Profiling of Purified Hepatic Cells Derived from Human Embryonic Stem Cells Stem Cells, August 1, 2008; 26(8): 2032 - 2041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. S. Turner, C. Seagle, J. A. Galanko, O. Favorov, G. D. Prestwich, J. M. Macdonald, and L. M. Reid Nuclear Magnetic Resonance Metabolomic Footprinting of Human Hepatic Stem Cells and Hepatoblasts Cultured in Hyaluronan-Matrix Hydrogels Stem Cells, June 1, 2008; 26(6): 1547 - 1555. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Dvir-Ginzberg, T. Elkayam, and S. Cohen Induced differentiation and maturation of newborn liver cells into functional hepatic tissue in macroporous alginate scaffolds FASEB J, May 1, 2008; 22(5): 1440 - 1449. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Inada, A. Follenzi, K. Cheng, M. Surana, B. Joseph, D. Benten, S. Bandi, H. Qian, and S. Gupta Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation J. Cell Sci., April 1, 2008; 121(7): 1002 - 1013. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yamashita, M. Forgues, W. Wang, J. W. Kim, Q. Ye, H. Jia, A. Budhu, K. A. Zanetti, Y. Chen, L.-X. Qin, et al. EpCAM and {alpha}-Fetoprotein Expression Defines Novel Prognostic Subtypes of Hepatocellular Carcinoma Cancer Res., March 1, 2008; 68(5): 1451 - 1461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Duan, A. Catana, Y. Meng, N. Yamamoto, S. He, S. Gupta, S. S. Gambhir, and M. A. Zern Differentiation and Enrichment of Hepatocyte-Like Cells from Human Embryonic Stem Cells In Vitro and In Vivo Stem Cells, December 1, 2007; 25(12): 3058 - 3068. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kubota, H.-l. Yao, and L. M. Reid Identification and Characterization of Vitamin A-Storing Cells in Fetal Liver: Implications for Functional Importance of Hepatic Stellate Cells in Liver Development and Hematopoiesis Stem Cells, September 1, 2007; 25(9): 2339 - 2349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Schmelzer, L. Zhang, A. Bruce, E. Wauthier, J. Ludlow, H.-l. Yao, N. Moss, A. Melhem, R. McClelland, W. Turner, et al. Human hepatic stem cells from fetal and postnatal donors J. Exp. Med., August 6, 2007; 204(8): 1973 - 1987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Duret, S. Gerbal-Chaloin, J. Ramos, J.-M. Fabre, E. Jacquet, F. Navarro, P. Blanc, A. Sa-Cunha, P. Maurel, and M. Daujat-Chavanieu Isolation, Characterization, and Differentiation to Hepatocyte-Like Cells of Nonparenchymal Epithelial Cells from Adult Human Liver Stem Cells, July 1, 2007; 25(7): 1779 - 1790. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Herrera, S. Bruno, S. Buttiglieri, C. Tetta, S. Gatti, M. C. Deregibus, B. Bussolati, and G. Camussi Isolation and Characterization of a Stem Cell Population from Adult Human Liver Stem Cells, December 1, 2006; 24(12): 2840 - 2850. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
