Specimen paper: This sSpecimen paper shows how a manuscript that neededs much revision to get it in shape so thatfor

it to have has a much greater chance of being accepted by a journal after review. This shows many changes that members of the BioMedES staff have to make from the original. To study this specimen, you will need to open up this document up in Word, and then from the under the main menu select Review and go to Track Cchanges. This has a submenu that will give you four choices, of which Final showing markup gives you all the changes made. The final document can be seen under Final. The original document and the changes can also be viewed in it. The authors have kindly allowed us to use this paper as an example. For the sake of anonimity , names have been changed. Hhumanized substitutes for animal sera in human mesenchymal stem cell culture and differentiation
J Orbetaa*, F Kantob* and P Gilberta#

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Address of Institute to which JO and PG belong, and bAddress of FK

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# Corresponding Author authors email: fll.kavitha@yahoo.comp.gilbert@yahoo.com *Both authors contributed equally

ABSTRACT Abstract Xenogenic substances used in The use of aanimal sera in cell culture is are a major deterrent for using such cells in cellular therapy owing because they areto potentially hazardous, necessitating contamination with xenogenic agents. This necessitates the development of humanized supplements to overcome such this problems. This study investigates the use of two human-derived supplements in human mesenchymal stem cell (hMSCs) culture, namely human platelet lysate (hPL) and umbilical cord blood serum (UCBS), in human mesenchymal stem cell (hMSCs) culture. Properties ( including growth kinetics and differentiation potential) of hMSCs cultured in either of these supplements including growth kinetics and the differentiation potential were better than those cultured in animal sera-supplemented media. This is also the first report concerningNew data on hepatogenic differentiation of hMSCs cultured in UCBSsupplemented media is also presented. Development of humanized alternatives to FBS could be revolutionizeary clinical cellular therapeutic strategies in terms ofif these results can be extrapolating extrapolated results from the in- vitro culturing and expansion ed / expandedof hMSCs to direct clinical cellular therapeutic strategies. [This is an unstructured abstract, as required by Cell Biology International; other journals may require structured abstracts: background methods - results conclusion.] Keywords: Umbilical Cord Blood Serum, Hepatogenic differentiation, Mesenchymal Stem Cells, FBSalternatives, Human Platelet Lysate. [Note use of capital letter for the beginning of each entry; for some journals, the lower case is used.]

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Abbreviations: hPL, human platelet lysate; [and more added in the style just given leave out wellused abbreviations, e.g. RNA, DNA]
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1. INTRODUCTION Introduction [Subheadings follow a particular pattern.] A phenomenal amount of research has been done on aAdult human bone marrow-derived mesenchymal stem cells (hMSCs) have been subjected to a phenomenal amount of research concerning their potential as a tool in regenerative medicine. However, the transition of these therapeutic strategies to the actual clinical scenarios has been impeded by various factors such as including the conventional use addition of animal sera as to the mediamedia supplements inof regular hMSC -culture practicess. In addition to their nonconformity to good clinical practice (GCP), the utilizationuse of animal sera (typically such as fetal bovine serum, (FBS) has economic, ethical and scientific drawbacks, including cytotoxicity of uncharacterized factors and the possibility of contamination with xenogeneic proteins (Spees et al., 2004)*[1]. *[Reader: this is called the Harvard reference system for citations in theused in the text,
with the list at the end of the article given in alphabetical order. A citation in the form [1] uses the Vancouver system. CBI uses the former, and so each numbered text reference below shouldhas to be changed accordingly, which has not been done any further in this manuscript, nor has the reference list been prepared alphabetically.

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These matters concernraise thes question as to whether FBS should can be adopted for the in -vitro expansion of hMSCs before possible use in regenerative medicine; if not, as appears to be the case,. This necessitates the development of suitable alternatives to FBS that are animal -protein-free and completely safe for therapeutic applicationsuse is needed. Chemically-defined serum-free media provides a controlled, consistent environments for cell growth, but the optimization of the concentration of individual supplements to the culture media, such as human serum albumin, that are added to the culture media is a tedious task and expensive. Serum has been reported tocan protect cells from cytotoxic agents by unknown mechanisms and to act as a buffering agent [2]. Therefore, the choice of serum remains a critical factor in the culture of hMSCs. Consequently, this has led to the search for suitable alternatives nonxenogenic preparations from human sources with properties similar to FBS that are not xenogenic. Human serum is a potential alternative to FBS for in animal cell culture. Autologous human serum has been shown to beis equivalent to 10% FBS in terms of supporting the proliferation and differentiation capacity of hMSCs, and could be utilized used for clinicalin therapy [3, 4]. Pooled human AB serum has been used for to supporting in -vitro expansion of hMSCs; this avoids issues associated with batch variability of the serum [5]. Other human-derived alternatives, such as human platelet lysate (hPL) and umbilical cord blood serum (UCBS), have demonstratedare efficientcy in terms of in vitro growth of hMSCs. UCBS is an efficient andalso a cost-effective alternative to FBS that is collected from cord blood, and is a biological waste product that is generally discarded after parturition. The placenta and the blood present therein previously nourished the developing fetus and are enriched with in several growth promoting factors. Therefore, UCBS is a potential human-derived substitute for FBS in hMSC culture, and hMSCs cultured with UCBS exhibit a higher proliferationproliferation more rapidly potential than those grown with FBS [6]. These results demonstrate that UCBS could be used as an effective substitute for FBS for developing clinically useful protocols requiring the culture of hMSCs. Platelet lysates/ releasates (a new word?) are anotheralso promising alternative to FBS. Subcellular fractions of platelets contain numerous bioactive molecules, including growth factors, such as platelet-derived growth factor (PDGF),
2

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The authors start each section on a new page; this is usually unnecessary.

nerve growth factor (NGF) and transforming growth factor (TGF), growth factors, adhesive proteins, coagulation factors, mitogens, protease inhibitors and proteoglycans that have the potential tohelp support the growth and viability of many a number of animal cell lines [7]. Cultures supplemented with hPL need to be subcultured have significantly higher expansion ratesquicker than cells cultures givend with FBS [8]. hMSCs grown in media supplemented with hPL exhibited anproliferated more rapidly enhanced proliferative ability in media supplemented with hPL and retained the capacity to differentiate while remaining immunosilent. Therefore, hPL is a potential candidate for the in- vitro expansion of stem cells for regenerative medicine [9]. In this research,A we present a comparative study concerning of the ability of hMSCs to proliferate and differentiate when grown in media supplemented with FBS, hPL and UCBS is herein presented, and will also deal. This study is the first report concerning with the the use of UCBS replacement of FBS in to supporting the hepatogenic differentiation of hMSCs when used as a replacement for FBS in hMSC culture.

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Mmaterials and methods 2.11. Preparation of Umbilical Cord Blood Serum Surplus umbilical cord blood collected for routine screening tests was used after obtainingwith the informed consent from patients*.
*If there is an ethical issue involved, generally consent of the patients plus a note that the research was approved by the appropriate board of the institution involved is required (see below).

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UThe umbilical cord blood without anticoagulants was incubated at room temperature for 3 h without anticoagulants, allowing the blood to clot and the cells to settle. SThe serum was separated by centrifugation at 1800 rpm for 20 min. The UCBS obtained was heat-inactivated, aliquoted and stored at -80C. 2. 2 Preparation of human Platelet LysatehPL Surplus platelet-rich plasma (PRP) past expiry date was collected from the blood bank and the cell number estimated by counting on a Neubauer Chamber. The PRP was centrifuged at 1200 rpm for 10 min, the. The plasma supernatant was discarded, and the platelet pelletconcentrate was diluted in with Dulbeccos Phosphate Buffered Saline (PBS) (Himedia)/ aAutologous plasma was used to 9 adjust theto obtain a final cell count of to 6 x 10 cellsplatelets/ml. This platelet-enriched fraction o was stored in aliquots at -80 C until userequired, at which time. Before use, the tubes were centrifuged at 13,200 rpm for 5 min to remove debris. 2.3. Isolation and culture of bBone mMarrow-derived Mesenchymal Stem CellshMSCs Bone marrow aspirates in heparin-containing syringes were obtained from patients undergoing cardiac surgery at the Frontier Lifeline Hospital, Chennai, after obtaining informed consent and Institutional Ethics Committee clearance.
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Bone Marrow Mononuclear Ccells (MNCs) were isolated using the Ficoll-Hypaque (Himedia) 3 (d= 1.077 g/cm ) gradient technique and plated on high-glucose Dulbeccos modified Eagles medium (DMEM) containing 4500 mg/L glucose, 4.00 mM L-glutamine, 4500 mg/L glucose, 110 mg/L sodium pyruvate (Hyclone), and 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin (antibiotic-antimycotic solution - Himedia). The medium was supplemented with filter-sterilized 10% FBS (PAN Biotech 0.2 m sterile filtered), or 5% hPL or 5% UCBS. The cell cultures were maintained at 37C in a humidified 5% CO 2 in air incubator. Non-adherent cells were removed after three 3 days and the medium replaced. Adherent hMSCs grew as CFU-Fs and were regularly passaged upon becomingwhen 80-90% confluent. The cells were passaged by after adding 0.25% trypsin-EDTA (Himedia).
Three points in the above: (1) except for the number 1, numbers can should usually be given as 2, 3, 4, and so on, rather than as two, three, four, etc. (And (2) when commercial preparations are used, the companies supplying them should be mentioned and there location (e.g. Sigma, St Louis, MO, USA; see just below). A number of abbreviations have appeared that could have been given under the abstract.

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2.4. RT-PCR Analysis

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RNA eExtraction RNA was isolated from hMSCs cultured in 10% FBS, 5% hPL or 5% UCBS for analysis of stem cell surface marker analysis. Briefly, cells were lysed with 1 ml Tri-reagent (Sigma-Aldrich, city?), mixed with 200 l chloroform and centrifuged at 13,200 rpm for 10 min. The aqueous layer was transferred to a fresh microfuge tubes and, treated with 500 l isopropanol, and. The tubes were ce cenntrifuged at 13,200 rpm for 20 min. The RNA-containing pellet containing RNA was washed with 500 l 70% ethanol, dried and resuspended in 50 l diethyl pyrocarbonate (DEPC) water. Generation of cDNA cDNA was generated using H-Minus M-MuLV reverse transcriptase (Fermentas) as per the manufacturers instructions. A mixture of RNA (10ng) prepared by the aforementioned above procedure, 5 g oligo(dT)18 and 12.5 l of DEPC-treated H2O was heated to 65C for 5 min and and transferredcooled to 4C. To this reaction mixture, 5X 5x reaction buffer, Ribolock RNAse inhibitor, 10 mM dNTP mix and Revert Aid H-Minus M-MuLV reverse transcriptase were added. The and the mixture was treated heated at to 42C for 60 min and. The transcriptase activityon was terminated by treating raising the temperature tothe mixture at 70C for 10 min. The prepared cDNA was stored at -20C. PCR aAmplification of hMSC sSurface mMarkers The isolated The cDNA isolated by the above procedure was used as a template for a PCR reactiontreatment, using CD 73 specific forward and reverse primers. The amplified PCR product was subjected to electrophoresedis on a 2% agarose gel for visualization. 2.5. Colony- forming unit-fibroblast (CFU-F) assay Briefly, equal numbers of isolated MNCs were plated on 35 mm diameter Petri dishes (NUNC) at a 5 2 concentration of 10 cells/cm in DMEM -supplemented with FBS, UCBS or hPL. After in-vitro culture for 14 days, the cells were stained with 0.5% cCrystal vViolet (in methanol) for 5 min, washed with PBS and dried. The number of colonies with diameters > 1 mm was enumerated in for each experimental condition.
Note that consistency is very important; for example the authors have used in vitro, in-vitro, in vitro, and in-vitro at different places throughout this text. There is no need for italics now for Latin words, and so in vitro sshould have been used throughoutconsistently.

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2.6. Growth cCurves To assess the growth pattern and kinetics of hMSCs in the presence of FBS, hPL or UCBS, 2 hMSCs (P2) were seeded at a concentration of 800 cells/cm in 24-well plates (NUNC), and cell. The number of cells was number counted over a two2- week period. 2.7. Hepatogenic dDifferentiation Hepatogenic differentiation was induced in 70% confluent P2-MSCs in 6-well culture plates (NUNC). The hMSCs wereby culturing themed in hepatogenic induction medium containing DMEM-low glucose (1000 mg/L glucose, 4.00 mM L-glutamine, 110 mg/L sodium pyruvate) supplemented with 20 ng/mL hepatocyte growth factor (HGF), 10 mM nicotinamide, 0.1M dexamethasone, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin, and their respective medium supplement, i.e. either - 10% FBS or 5% UCBS. The control group was maintained in DMEM highglucose containing 4.00 mM L-glutamine, 4500 mg/L glucose, 110 mg/L sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin, with either 10% FBS or 5% UCBS.
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The medium was replaced with fresh medium containing the respective serum every four 4 days. Media from the wells were collected at specific intervals and stored at -20C for before urea quantificationmeasurement. 2.8 . Glycogen sStaining For of hHepatocytes On day 22 after of hepatogenic induction, cells in the hepatogenic group (and the control groups) were subjected tostained with periodic acid- Schiff staining reagent for the presence of glycogen accumulation in the cells, a unique functional feature of hepatocytes. The media were removed from the wells and the cells were rinsed three 3 times with PBS, then fixed with 4% paraformaldehyde for 30 min, and oxidized with in 1% periodic acid for 10 min. The wells were rinsed with water and treatedbefore with Schiff's base was added for 10 min. After thoroughly rinsing with water, the cells were counterstained with hematoxylin for 5 min. Hematoxylin waswas After rinsing in washed off with water, and the cells were dried and examinedbefore observing under an by Inverted pPhasecContrast mMicroscopey (Olympus CKX41). 2.9. Urea Assay Cell culture media collected on days 14, 16, 19 and 21 from the hepatogenic group and control groups (as a negative control) were tested for urea by the glutathione kinetic method [10] and the optical densities were measured at 492 nm. Fresh DMEM high-glucose containing 4.00 mM L-glutamine, 4500 mg/L glucose, 110 mg/L sodium pyruvate 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin, 10% FBS/ 5% UCBS and hepatogenic media with respective serum supplements were taken as day 0 samples for the control and hepatogenic groups, respectively, and were assayed to quantify the initial level of urea present.

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2.10. Adipogenic dDifferentiation Adipogenesis was induced on 70% confluent P2-MSCs by culturing in adipogenic medium containing 500 M 3-isobutyl-1-methylxanthine (IBMX) and 1M dexamethasone in DMEM high-glucose containing 4.00 mM L-glutamine, 4500 mg/L glucose, 110 mg/L sodium pyruvate and 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin, with either 10% FBS or 5% UCBS. After four 4 days, induction was arrested and the cells were maintained in DMEM high-glucose containing 4.00 mM L-glutamine, 4500 mg/L glucose, 110 mg/L sodium pyruvate and 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin, with the respective serum supplement. After 48 h, the medium was replaced with adipogenic induction medium and the pulsing method of induction was repeated. The cells were regularly observed examined under anby inverted phase- contrast microscopye for the presence of oil globules.

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Note reference to the use of DMEM with similar supplements this could have been simplified by referring to it is some specific way, removing the need for the details being given each time (not done in this revision). If the results are going to require statistical analysis, the tests applied should be added here as a further subsection (2.11).

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3. RESULTS Results I)3.1 hMSCMesenchymal Stem Cell Culture and eExpansion in different mMedia sSupplements Mesenchymal stem cellshMSC [if abbreviations have been used before, keep using them instead of the full phrase as in the original document here] were isolated from human adult bone marrow and cultured separately in three 3 different culture -media supplements, FBS, hPL and UCBS. The hMSCs seeded at a 5 2 density of 10 cells/cm in 10% FBS-containing medium presented with had typical fibroblast-like morphology and reached 90% confluence at P 0 in 28 days. After three 3 passages, the RNA isolated from the cells was used to screen for expression of the MSC surface marker, CD 73. Its eExpression of CD 73 confirmed that the cellsy were are indeed stem cells [Figure 1]. CD 73 was expressed in cells grown in hPL and UCBS, providing evidenceindicating that these culture supplements did not alter the expression of stem cellthis markers . The molecular characteristics of cells grown in 5% UCBS or 5% hPL were similar to those hMSCs grown in 10% FBS; they showed MSC surface markers, such as CD73, and presented with typical morphology [Figure 1]. hMSCs cultured in UCBS supplemented medium appeared longer and more spindle-shaped than those grown in FBS cultured medium. The morphological and proliferative properties of hMSCs grown in UCBS were retained through multiple many passages, and possessed high colony-forming and replicative properties, even at later passages, compared to FBScultured cells. On average, the approximate time taken for hMSCs to reach confluence when MNCs MSCs were seeded 5 2 at a cell density of 10 cells/cm under in 10% FBS culture conditionsmedium was 28 days. Faster growth rates were observedoccurred in 5% UCBS and 5% hPL media, the cells forming tightly packed colonies and that becamereaching confluent byce in 22 days, after when being seeded at the same density as those grown in FBS. This enhanced rateproliferative potential was validated confirmed by growth curve kinetics and CColony Forming Unit-Fibroblast (CFU-F) assays. The growth curves of hMSCs grown in DMEM containing 10% FBS, 5% hPL and 5% UCBS were compared . Cells grown ingrew faster in DMEM supplemented with 5% UCBS or or 5% hPL-supplemented media demonstrated faster growth than the in10% FBS medium-cultured cells [Figure 2]. The capacity of hMSCs cultured in 5% UCBS or 5% hPL media to form colonies in-vitro was compared to those grown in 10% FBS in the CFU-F assay. The number of cells capable of forming colonies 14 days after plating the mononuclear cells in the culture dish was highest within 5% UCBS- mediumculture conditions, followed by 5% hPL-supplemented medium. The heightened increased colonogenic activity of the human supplements on the CFU-F-forming efficiency of hMSCs was evident fromced the by larger colony sizes in hPL and UCBS-supplemented cells, while whereas the CFU-F frequency and colony sizes were significantly less smaller in FBS cultured hMSCs. Colonies of hMSCs grown in hPL and UCBS were larger and more numerous than those grown in the presence of UCBS. Therefore, UCBS and hPL support better in vitro growth and proliferation of hMSCs in-vitro. II)3.2 Differentiation pPotential of hMSCs cultured in UCBS-media. A: Analysiszing of the potential of UCBS as a rReplacement for FBS in . hepatocyte differentiation Observation of eEpithelioid cCells in hHepatogenic-I-induced cCells Morphological changes in MSCs in the presence of either UCBS or FBS were noted upon hepatogenic induction of the cells. MSCs cultured under both conditions demonstrated a conspicuous morphological change from the typical elongated MSC structure to small, round, epithelioid structures clustered in small groups from approximately ~day 14 onwards [Figure 3]. The number of epithelioid cells was significantly higher in the UCBS -culturesd wells than in FBS cultures conditions. Cells in the control group without no hepatogenic induction factors exhibited had fibroblast morphology and greater cell density. The hepatocyte-like cells formed were subjected to functional assays includingstained with periodic acid-Schiffs staining (to check indicatefor glycogen accumulation) and urea production , both showed features exclusive to hepatocytes.

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 Periodic aAcid Schiff's sStaining after hepatogenic induction

On day 22 after hepatogenic induction, the cells were analyzed for glycogen accumulation using periodic acid-Schiff (PAS) staining. The cells of the hepatocyte differentiation-induced group exhibited showedmagenta staining in the cytoplasm demonstrating the accumulation of glycogen, whereas. n Unon-induced controlsells showed had no cytoplasmic glycogen accumulation. More cells stained with PAS in the UCBS group than in the FBS group. Within the hepatocyte differentiationinduced group, differentiated cells under the influence ofin UCBS medium had visibly more stained regions than the FBS- cultured cells [Figure 4].
Urea Quantification

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Urea production, a functional indication of hepatogenic differentiation, was monitored by measuring secreted urea levels in the cell culture media. Urea productionIt increased approximately around 12 days after induction and was seen to increased consistently thereafter [Figure 4]. Cells grown in UCBS produced more urea from day 15 onwards than those grown in FBS, thereby validating successful hepatogenic differentiation of the cells grown in UCBS supplemented medium. 3.3 adipocytic Adipocyte differentiation; accumulation of lipids in induced hMSCs
Accumulation of lipids in induced hMSCs

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hMSCs grown in UCBS and FBS were monitored for lipid accumulation seven 7 days after adipocyte induction. Oil globules were noted in the UCBS cultured hMSCs, although they were less dense than in cells cultured in FBS supplemented medium [Figure 5]. This is in agreement with an earlier report concerning repressed adipogenesis of MSCs in the presence of UCBS [11]. 4. Discussion The quest for a comparatively safer and more efficient alternative to fetal bovine serum for stem cell culture has been ongoing. HThe human-derived supplements UCBS and hPL have beenare preferred to other substitutes for in hMSC culture. These media additives provide pure, therapeutic-quality hMSCs, preserving vital specific properties, such as characteristic MSC -surface marker expression, multi-lineage differentiation potential, characteristic immunomodulatory properties [12], and self-renewal capacity across many passages, and also induce high proliferative and colonogenic activity [8]. In this study we report onWe have shown the preservation of typical MSC features, including cell morphology, cell surface markers and differentiation potential in hMSCs grown in UCBS or hPL-supplemented media,. The results demonstratinge the efficiency of these growth supplements in supporting the in vitro culture and expansion of adult human bone marrow-derived MSCs. In addition, we presentNew the first evidence shows that UCBS-supplemented medium supports the in vitro hepatogenic differentiation of hMSCs invitro.

hMSCs grown in these supplements express molecular markers, such as CD73. Moreover, the increased growth and colonogenic activity of hMSCs induced by unidentified factors in these alternative media supplements were apparent from the shorter doubling rates, higher frequency of colonogenic cells and larger colony size. In agreement with previous reports, colonies grown in hPLsupplemented medium were densely packed with small spindle-shaped cells, in contrast to the loosely connected cells in FBS cultures [13], and the colonies comprised had more cells in cultures containing each of the human supplements. The unique factors present in platelets that have an enhanced mitogenic effect on hMSCs remain unknown, but it has been reported that the heterogeneous platelet alpha granules contain either pro- or anti-angiogenic factors;. t The composition of these factors released by platelets depends on the mode of activation employed to release the granules [ 14]. The distinct growth-stimulating effects of UCBS on human bone marrow MSCs may be attributed to the numerous growth factors present in the nutrient-rich umbilical cord blood and placental blood that nourishes the developing fetus during gestation. Therefore, these growth supplements provide a
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unique and favorable micro-environment for the in vitro culture of clinical-grade hMSCs. In this investigation, weWe also tested the capacity of UCBS to support the multi-lineage differentiation of hMSCs, particularly the adipogenic and hepatogenic lineages. Previous studies have demonstratedshow that the in vitro differentiation of hMSCs into adipocytes, chondrocytes, osteocytes [12] and hepatocytes [15] is retained in the presence of hPL. It has been reported that UCBS -cultured hMSCs undergo adipogenic differentiation [11], and there have been reports of biased differentiation of hMSCs into osteocytes in the presence of hUCBS has been reported [16], but the results are not inconclusive. We also show theThis is the first report novel illustrating finding ofthe hepatogenic differentiation capacity of hMSCs grown in UCBS-supplemented culture. Differentiation into a hepatogenic lineage was induced in FBS-cultured and UCBS-cultured MSCs by adding hepatogenic induction medium. On hepatogenicAfter induction, changes in MSC morphology were observed microscopically;: the cells shed their characteristic spindle-shaped morphology and transformed into polygonal cells of epitheloid morphology. The various functional changes induced by the hepatogenic induction medium in the hMSCs were noticeable from day 15, when there was a surge in urea production by the cells. This feature was more significant in cells cultured in the presence of UCBS than those grown in FBS.
Note: the authors here are repeating the findings given in the Results section. This is unnecessary; discussion of them is required herein this section; so this. Much of this section might be considerably reduced. by doing sot.

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Another indication of hepatocellular function is the production and storage of glycogen in the cells. herefore be cut. This is viewed microscopically by the periodic acid-Schiff staining method, where glycogen in the cells retains a magenta stain. The PAS stain was more prominent in hepatocytes differentiated from hMSCs cultured in the presence of UCBS, suggesting enhanced hepatogenic differentiation. There was also adipogenic differentiation of UCBS-supplemented MSCs, although there were fewer oil globules than in FBS-supplemented MSCs. This observation is in agreement with an earlier report demonstrating repressed adipogenesis when MSCs were cultured in medium containing UCBS [11]. Further investigations into the underlying mechanisms responsible for heightened hepatogenic and osteogenic differentiation [16] and suppressed adipogenesis [11] in the presence of UCBS, and stringent characterization of the unique causative factors in the serum, are required to explain definitively more explicitly these shifts in differentiation patterns in MSCs grown in UCBSsupplemented medium. TheseMore studies are require needed to be carried out to optimize the consistency of the serum and to ratify it its use as a safe and efficient alternative to FBS alternative.
ThCONCLUSIONS The discovery of hepatogenic differentiation of hMSCs in UCBS-conditioned medium may have helped to bring research closer to xeno-free -MSC therapy for treating liver diseases. Despite the commencement of clinical trials using hepatocytes obtained from FBS-cultured MSCs [17], human-derived alternatives such as UCBS should be seriously considered for their allogenicity, up-regulation of growth and hepatogenic differentiation capacities in hMSCs. However, extensive in -vivo studies are essential to elucidate fully the functional characteristics of hepatocytes cultured in FBS-free medium conditioned with UCBS. TIn conclusion, ourhe results presented in this report demonstrate that UCBS and hPL are safe humanized media additives that promote better colonogenic growth and long term expansion of hMSCs, while maintaining the multi-lineage differentiation potential of the cells. This is significant in the current era of
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cellular therapy and regenerative medicine, where there is a necessity for consistency in hMSC-culture protocols in laboratories around the world. A uniform, controlled cell culture environment would provide genomic and epigenetic stability in cultured cells, with reproducible results and minimal heterogeneity among cell populations. The discovery of human-derived alternatives that avoid the xenogenic protein contamination problems of FBS is beneficial at a pre-clinical level and should be considered. Clinical studies with FBS-cultured MSCs have been ongoing under regulatory approval, but many questions regarding the safety of these transplanted cells to a patient remain unanswered. Optimization of the bestsuited culture conditions is a formidable task. Nonetheless, hPL and UCBS have great potential as cell culture media supplements, and the clinical implications of this advancement will help accelerate the transition of research to the next phase in cellular therapeutic applications.
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AAcknowledgements: [Grant giving bodies and grant numbers should be included here.] The authors wish to thank the surgeons and doctors at Frontier Lifeline for their input and cooperation. Special thanks to Mr. Suneel Rallapalli and Mr. Dillip Kumar Bishi for their technical input during the differentiation experiments. We also wish to thank Mr. Pandian A. for help in the collection of umbilical cord blood.

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References [N.B. This would be in alphabetical order ifwhen the Harvard system had been used with text citations such as (Spees et al, 2004). Set the references out in the precise order of the journal to which you are submitting.] 1. Spees JL, Gregory CA, Singh H, et al.: Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther. 9(5), 747-756 (2004). 2. Cifrian E, Guidry A, Marquardt WW: Role of milk fractions, serum, and divalent cations in protection of mammary epithelial cells of cows against damage by Staphylococcus aureus toxins. Am J Vet Res. 57(3), 308-312 (1996). 3. Kobayashi T, Watanabe H, Yanagawa T, et al.: Motility and growth of human bone-marrow mesenchymal stem cells during ex vivo expansion in autologous serum. J Bone Joint Surg Br. 87(10), 1426-1433 (2005). 4. Stute N, Holtz K, Bubenheim M, Lange C, Blake F, Zander AR: Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use. Exp Hematol. 32(12), 1212-1225 (2004). 5. Kocaoemer A, Kern S, Klter H, Bieback K: Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells. 25(5), 12701278 (2007). 6. Shetty P, Bharucha K, Tanavde V: Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells. Cell Biol Int. 31(3), 293298 (2007). 7. Johansson L, Klinth J, Holmqvist O, Ohlson S: Platelet lysate: a replacement for fetal bovine serum in animal cell culture? Cytotechnology. 42(2), 67-74 (2003). 8. Lange C, Cakiroglu F, Spiess AN, Cappallo-Obermann H, Dierlamm J, Zander AR: Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J Cell Physiol. 213(1), 18-26 (2007). 9. Capelli C, Domenghini M, Borleri G, et al.: Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplant. 40(8), 785-791 (2007). 10. Cecil R: The Action of Urea on the Disulphide Groups of Oxidized Glutathione and Cystine. Biochem J. 49(2), 183-185 (1951). 11. Phadnis SM, Joglekar MV, Venkateshan V, Ghaskadbi SM, Hardikar AA, Bhonde RR: Human Umbilical Cord Blood Serum Promotes Growth, Proliferation, As Well As Differentiation of Human Bone Marrow-Derived Progenitor Cells. In Vitro Cell Dev Biol Anim . 42(10), 283-286 (2006). 12. Doucet C, Ernou I, Zhang Y, et al.: Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol. 205(2), 228-236 (2005). 13. Bieback K, Hecker A, Kocamer A, et al.: Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells. 27(9), 2331-2341 (2009). 14. Italiano JE Jr, Richardson JL, Patel-Hett S, et al.: Angiogenesis is regulated by a novel mechanism: Pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood. 111, 12271233 (2008). 15. Kazemnejad S, Allameh A, Gharehbaghian A, Soleimani M, Amirizadeh N, Jazayeri M: Efficient replacing of fetal bovine serum with human platelet releasate during propagation and differentiation of human bone-marrow-derived mesenchymal stem cells to functional hepatocyte-like
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cells. Vox Sang. 95(2), 149-158 (2008). 16. Jung J, Moon N, Ahn JY, et al.: Mesenchymal stromal cells expanded in human allogenic cord blood serum display higher self-renewal and enhanced osteogenic potential. Stem Cells Dev. 18(4), 559-571 (2009). 17. Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M et al.: Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Arch Iran Med. 10(4):459-66 (2007).

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Acknowledgement: The authors wish to thank the surgeons and doctors at Frontier Lifeline for their input and cooperation. Special thanks to Mr. Suneel Rallapalli and Mr. Dillip Kumar Bishi for their technical input during the differentiation experiments. We also wish to thank Mr. Pandian A. for help in the collection of umbilical cord blood.

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N.B. Figures, Tables etc are heir legends have not been covered in this specimen paper.

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