1、Accepted ManuscriptTranscriptomics of human multipotent mesenchymal stromalcells:Retrospective analysis and future prospectsNaresh Kasoju,Hui Wang,Bo Zhang,Julian George,Shan Gao,James T.Triffitt,Zhanfeng Cui,Hua YePII:S0734-9750(17)30044-7DOI:doi:10.1016/j.biotechadv.2017.04.005Reference:JBA 7121To
2、 appear in:Biotechnology AdvancesReceived date:28 October 2016Revised date:19 April 2017Accepted date:20 April 2017Please cite this article as:Naresh Kasoju,Hui Wang,Bo Zhang,Julian George,ShanGao,James T.Triffitt,Zhanfeng Cui,Hua Ye,Transcriptomics of human multipotentmesenchymal stromal cells:Retr
3、ospective analysis and future prospects.The addressfor the corresponding author was captured as affiliation for all authors.Please check ifappropriate.Jba(2017),doi:10.1016/j.biotechadv.2017.04.005This is a PDF file of an unedited manuscript that has been accepted for publication.Asa service to our
4、customers we are providing this early version of the manuscript.Themanuscript will undergo copyediting,typesetting,and review of the resulting proof beforeit is published in its final form.Please note that during the production process errors maybe discovered which could affect the content,and all l
5、egal disclaimers that apply to thejournal pertain.ACCEPTED MANUSCRIPTTitle Transcriptomics of Human Multipotent Mesenchymal Stromal Cells:Retrospective Analysis and Future Prospects Authors Naresh Kasoju a,Hui Wang a,b,Bo Zhang a,Julian George a,Shan Gao c,James T.Triffitt d,Zhanfeng Cui a,Hua Ye a,
6、*Affiliations a Institute of Biomedical Engineering,Department of Engineering Science,University of Oxford,Oxford,United Kingdom.b Department of Zoology,University of Oxford,Oxford,United Kingdom.c Suzhou Institute of Biomedical Engineering and Technology,Chinese Academy of Sciences,Suzhou,China.d N
7、uffield Department of Orthopaedics,Rheumatology and Musculoskeletal Sciences,University of Oxford,Oxford,United Kingdom.Footnotes*Corresponding author e-mail address:hua.yeeng.ox.ac.uk(H.Ye).These authors contributed equally.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTAbstract The plastic-adherent,fibrobl
8、ast-like,clonogenic cells found in the human body now defined as multipotent“Mesenchymal Stromal Cells”(MSCs)hold immense potential for cell-based therapies.Recently,research and basic knowledge of these cells has fast-tracked,both from fundamental and translational perspectives.There have been impo
9、rtant discoveries with respect to the available variety of tissue sources,the development of protocols for their easy isolation and in vitro expansion and for directed differentiation into various cell types.In addition,there has been discovery of novel abilities such as immune-modulation and the fu
10、rther development of the use of biomaterials to aid isolation,expansion and differentiation together with improved delivery to the selected optimal tissue site.However,the molecular fingerprint of MSCs in these contexts remains imprecise and inadequate.Consequently,without this crucial knowledge it
11、is difficult to achieve progress to determine with precision their practical developmental potentials.Detailed investigations on the global gene expression,or transcriptome,of MSCs could offer essential clues in this regard.In this article,we address the challenges associated with MSC transcriptome
12、studies,the paradoxes observed in published experimental results and the need for careful transcriptomic analysis.We describe the exemplary applications with various transcriptome platforms that are used to address the variation in biomarkers and the identification of differentiation processes.The e
13、volution and the potentials for adapting next-generation sequencing(NGS)technology in transcriptome analysis are discussed.Lastly,based on review of the existing understanding and published studies,we propose how NGS may be applied to promote further understanding of the biology of MSCs and their us
14、e in allied fields such as regenerative medicine.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTKeywords:Mesenchymal Stem Cells;Phenotype;Gene Expression;Stemness;Differentiation;Transcriptomics;Micro Array;Next-Generation Sequencing;Biomaterials;Regenerative Medicine Abbreviations:MSCs,multipotent mesenchym
15、al stromal cells;AD-MSCs,adipose-derived MSCs;BM-MSCs,bone marrow-derived MSCs;PL-MSCs,placenta-derived MSCs;FL-MSCs,fetal-derived MSCs;NSCs,neural stem cells;HSCs,hematopoietic stem cells;ESCs,embryonic stem cells;NGS,next-generation sequencing;RNA-seq,RNA sequencing;mRNA,messenger ribonucleic acid
16、;cDNA,complementary deoxyribonucleic acid;DEG,differentially expressed genes;FPKM,fragments per kilo base million;RPKM,reads per kilo base million;ISCT,international society for cellular therapy;TGF,transforming growth factor.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTContents 1.Introduction 2.Need for a
17、 global view of MSCs gene expression 2.1.Deciphering the identity code 2.2.Addressing the paradoxes of expansion 2.3.Understanding emerging trends in stem cell research 3.Comparative transcriptome analysis and applications to study MSCs 3.1.Identification of biomarkers and donor variations 3.2.Track
18、ing cell differentiation 3.3.Exploring disease association 3.4.Optimizing culture conditions 3.5.Health and safety aspects 4.NGS:technology advantage and applications in MSC research 4.1.Evolution of gene sequencing technologies 4.2.NGS in transcriptomics research 4.3.NGS-based MSC transcriptomics 4
19、.3.1.NGS in understanding MSC biology 4.3.2.NGS and MSCs in understanding developmental biology 4.3.3.NGS and MSCs in understanding disease conditions 4.3.4.NGS in MSC-based regenerative medicine and tissue engineering 5.Concluding remarks and future directions Acknowledgements Conflict of interest
20、References ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT1.Introduction Seminal studies involving bone marrow transplants in a patient suffering from the blood cancer condition leukaemia were first performed by Edward D.Thomas during the 1950s(Little and Storb,2002).These investigations together with those
21、by Robert A.Good in the 1960s for treatment of the non-cancer condition severe combined immunodeficiency syndrome prompted the belief that bone marrow possesses a unique pool of cells with the potential ability to cure human blood diseases(Gatti et al.,1968;Thomas et al.,1957).Subsequently,classical
22、 studies by Alexander J.Friedenstein during the 1970s and 80s,identified the non-haemopoietic cells in the marrow as adherent,fibroblast-like,clonogenic cells.These cells showed high“replicative capacity”in vitro and had the“ability to differentiate”into osteoblasts,chondrocytes,adipocytes and hemat
23、opoietic-supporting stroma in vivo(Friedenstein et al.,1970;Friedenstein et al.,1966).This unique pool of cells,were initially termed“colony forming unit-fibroblastic”but are now popularly defined as“Mesenchymal Stromal Cell”(MSCs).They were first isolated from bone marrow as 0.001-0.01%of the total
24、 nucleated cells and it was proposed that these cells were part of a wider stromal cell system of the body and that stromal fibroblastic cells were present in many other tissues(Owen,1988).However,from much definitive work it was shown that only the bone marrow contained MSCs that had the inherent c
25、apacity to differentiate into all lineage-specific component cells of the osteoblastic lineage and were termed by Friedenstein as“determined osteoprogenitor cells”.Other stromal fibroblastic cells,either from the bone marrow or from a large spectrum of other tissues,could only be induced into the os
26、teogenic lineage by specific inductive molecules and these were termed“inducible osteogenic progenitor cells”(Friedenstein,1973).Since then,other non-marrow tissues harbouring MSCs that are more easily obtainable and accessible,such as adipose and umbilical cord tissues,have been studied in more det
27、ail(Bennett et al.,1991;Fotia et al.,2015;Hass et al.,ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT2011;Minguell et al.,2001;Pierantozzi et al.,2010;Riekstina et al.,2009;Sharma and Bhonde,2015;Strioga,Marius et al.,2012).Simultaneously,novel protocols have continuously been developed to differentiate thes
28、e MSCs in vitro into lineage-specific cell types(Beresford et al.,1994;Gundle et al.,1995;Marie and Fromigu,2006;Oreffo et al.,1997;Xiong et al.,2014).The notion that MSCs of a tissue origin can differentiate into a particular set of progeny only was taken over by fascinating and controversial inves
29、tigations that introduced a phenomenon termed“trans-differentiation”to turn“brain into blood”(Bjornson et al.,1999),“blood into brain”(Mezey et al.,2000)and other cross-lineage differentiation(Krause et al.,2001).In addition,several other noteworthy investigations revealed the immune-suppressive/mod
30、ulatory/regulatory abilities of MSCs(Caplan,2007;Fiorina et al.,2009;Ren et al.,2008;Siegel et al.,2009).This led to an increased excitement about the possible potential of MSCs in treatments for various other diseases or conditions.Taken together,it was evident that a)the discovery of various tissu
31、e sources of adult stem cells,b)the development of defined protocols for their easy isolation,culture and differentiation into lineage-specific cell types,c)the design of novel strategies for induction of differentiation of these cells into a variety of cell types other than found in their tissue of
32、 origin,and d)discovery of their novel abilities,such as immune-modulatory ability,has elevated MSCs to the forefront of stem cell research activities.The fascination of these cells relates both to their fundamental nature as well as to their translational possibilities(Figure 1).However,even now MS
33、C research is still at an early phase with high potential but with many remaining questions.Major challenges include:isolation with high accuracy and viable yield,in vitro expansion without losing stemness characteristics and efficient differentiation protocols in 2D cultures(i.e.conventional two-di
34、mensional cell cultures systems)and in 3D cultures(i.e.three-ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTdimensional cell culture systems developed using biomaterials that resemble physiological environments).Detailed investigations on the global gene expression or transcriptome of MSCs could possibly off
35、er essential clues to further our understanding on MSCs.A single transcriptome is a snapshot of all the active genes at a given time.When a series of transcriptomes is obtained,comparative transcriptome analysis can be performed to detect trends of gene expression changes in response to a treatment
36、or a native variable.Gene expressions in an individual sample need to be normalized,for example to housekeeping genes,or by a mathematical formula,to enable quantitative or semi-quantitative comparison among samples for identification of DEGs due to a treatment or a biological time course.A typical
37、transcriptome approach requires the total mRNA population of a sample as the starting material.From total RNA extracts,the commonly used method is to apply oligo-dT probes for mRNA isolation followed by biochemical conversion to cDNA which will be examined by gene identification technologies such as
38、 microarray(Figure 2).Early comparative transcriptome work with MSCs demonstrated molecular similarities between human AD-MSCs and BM-MSCs(Katz et al.,2005),as well as population plasticity and biochemical heterogeneity(Phinney,2007,2009;Phinney and Isakova,2005).By comparing transcription patterns
39、of MSCs and MSC-like cells,isolated and cultured under identical conditions,the expression pattern was identified to be cell-type-specific rather than governed by isolation and culture conditions(Ulloa-Montoya et al.,2007).However,culture media with or without fetal bovine serum as a nutritional sup
40、plement affected the MSC transcriptome stability during in vitro expansion(Shahdadfar et al.,2005).Human BM-MSCs cultured in two different expansion media(BM-MSC-M1 and BM-MSC-M2)displayed changes in their gene expression profiles(Wagner,W.et al.,2006).In addition,culture duration has been found to
41、affect MSC biology;long-term in vitro ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTexpansion has altered gene expressions of adult MSCs(Izadpanah et al.,2008).Comparative transcriptome analysis also determined MSC-disease associations.For example,differential expression of genes reflected impaired clonogen
42、ic and proliferative potentials determined in BM-MSCs isolated from patients with active rheumatoid arthritis when compared with those from healthy donors(Kastrinaki et al.,2008).To this end,we believe that the detailed investigation of the transcriptome of MSCs could potentially offer invaluable so
43、lutions.High-throughput technologies such as microarray analysis do trigger much interest for transcriptomics studies in general,but the associated high costs and technical limitations remain restricting factors.With the advent of relatively cost-effective and robust technologies,such as NGS,the fie
44、ld of transcriptomics is gaining momentum.We believe,therefore,that it is time to make use of such emerging technologies to overcome the bottlenecks in MSCs research.In this article,we discuss the necessity for global gene expression analysis of MSCs.This is followed by a description of the various
45、methods and tools available for transcriptome analysis.We continue by a description of a few examples of investigations that have been carried out on this topic so far and conclude by discussing the future prospects for transcriptome analysis of the MSC system.2.Need for a global view of MSCs gene e
46、xpression 2.1.Deciphering the identity code With the overwhelming expansion of research on MSCs,there was ambiguity not only in the nomenclature but also in the identification of these cells.After defining the fibroblast-like plastic-adherent cells,regardless of the tissue of origin,as“multipotent m
47、esenchymal stromal cells”in the year 2005,the Mesenchymal and Tissue Stem Cell ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTCommittee of ISCT proposed three minimal criteria for defining the multipotent MSCs in year 2006(Dominici et al.,2006).“First,MSC must be plastic-adherent when maintained in standard
48、culture conditions.Second,MSC must express CD105,CD73 and CD90,and lack expression of CD45,CD34,CD14 or CD11b,CD79 or CD19 and HLA-DR surface molecules.Third,MSC must differentiate to osteoblasts,adipocytes and chondroblasts in vitro”(Dominici et al.,2006).Yet,numerous investigations on MSCs have re
49、ported several deviations from the ISCT minimal criteria,mainly due to isolation,anatomical origin and donor-related issues.For example,MSCs have been traditionally isolated utilizing their plastic adherence property and identified using a set of positive and negative surface markers as described by
50、 ISCT;this,however,would retain various other types of cells besides MSCs(Baustian et al.,2015;Ramakrishnan et al.,2013).Even when the contaminating cell population was eliminated by using improved isolation and enrichment procedures,heterogeneity was reported within the isolated MSC population(Wagn
