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Expanding autologous multipotent mesenchymal bone marrow stromal cells Jan E. Brinchmann Cell Therapy, Institute of Immunology, Rikshospitalet Medical Centre, Oslo, Norway Received 6 February 2007; received in revised form 10 April 2007; accepted 8 May 2007 Available online 8 June 2007 Abstract In this brief review, I will start by redefining the cells: mesenchymal stem cells should now be called multipotent mesenchymal stromal cells, with the same acronym (MSC). I will review the role of MSC as immunosuppressive and immunoprotected cells, and then go on to describe how cell culture strategies may depend on the desired functionality of MSC. In order to expand MSC in vitro, the cells have to be cultured as adherent cells on plastic surfaces. To obtain the best cell culture conditions, a number of methodological decisions have to be made. Special considerations are necessary if the cells are to be used for the treatment of patients. © 2007 Elsevier B.V. All rights reserved. Keywords: Mesenchymal stem cells; Multipotent mesenchymal stromal cells; Immunosuppressive cells; Immunoprotected cells; In vitro cell culture 1. Introduction Mesenchymal stem cells (MSC) were first studied by Alexander Friedenstein, who isolated bone-forming progen- itor cells from rat bone marrow [1]. The term MSC was coined by Mark Pittenger, who in 1999 demonstrated how MSC from bone marrow (BM-MSC) could be cultured in vitro with a stable phenotype through many population doublings, and that they could be induced to differentiate into adipocytic, chondrocytic and osteocytic lineages in vitro [2]. The presence of multipotent MSC in adipose tissue (AT- MSC) was first demonstrated by Zuk et al. [3]. We subse- quently demonstrated how purified, uncultured AT-MSC could be isolated and characterized at the phenotype and transcriptome level, and identified the changes in gene expression induced by in vitro culture of these cells [4]. The minimal criteria for defining MSC have now been defined by The International Society for Cellular Therapy (ISCT) [5]. They are: MSC should be plastic-adherent when maintained in standard culture conditions. MSC should express CD105, CD73 and CD90, lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA DR surface molecules. MSC must differentiate to osteoblasts, adipocytes and chondrocytes in vitro. However, this population of plastic adherent cells is heterogenous, normally does not express telomerase and reaches senscence at 30 40 population doublings in vitro [6]. These are some of the main reasons why, as a result of discussions on terminology within the international stem cell research community, ISCT in 2005 suggested that the name be changed to multipotent me- senchymal stromal cells, but that the acronym (MSC) be maintained [7]. 2. Immunological aspects of MSC It has been known for some years that MSC have immu- nosuppressive properties in vitro. This has been determined by adding MSC to T-cells stimulated by mitogens, antigens and alloantigens in vitro, and an effect has also been demonstrated in vivo in a skin graft model, and in a murine model of auto- immune encephalomyelitis [813]. The molecular mechan- isms responsible for this effect have not been fully revealed, but direct contact between the MSC and the T cell does not Journal of the Neurological Sciences 265 (2008) 127 130 www.elsevier.com/locate/jns Tel.: +47 23 07 37 66; fax: +47 23 07 38 22. E-mail address: [email protected]. 0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2007.05.006

Expanding autologous multipotent mesenchymal bone marrow stromal cells

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Expanding autologous multipotent mesenchymalbone marrow stromal cells

Jan E. Brinchmann ⁎

Cell Therapy, Institute of Immunology, Rikshospitalet Medical Centre, Oslo, Norway

Received 6 February 2007; received in revised form 10 April 2007; accepted 8 May 2007Available online 8 June 2007

Abstract

In this brief review, I will start by redefining the cells: mesenchymal stem cells should now be called multipotent mesenchymal stromalcells, with the same acronym (MSC). I will review the role of MSC as immunosuppressive and immunoprotected cells, and then go on todescribe how cell culture strategies may depend on the desired functionality of MSC. In order to expand MSC in vitro, the cells have to becultured as adherent cells on plastic surfaces. To obtain the best cell culture conditions, a number of methodological decisions have to bemade. Special considerations are necessary if the cells are to be used for the treatment of patients.© 2007 Elsevier B.V. All rights reserved.

Keywords: Mesenchymal stem cells; Multipotent mesenchymal stromal cells; Immunosuppressive cells; Immunoprotected cells; In vitro cell culture

1. Introduction

Mesenchymal stem cells (MSC) were first studied byAlexander Friedenstein, who isolated bone-forming progen-itor cells from rat bone marrow [1]. The term MSC wascoined by Mark Pittenger, who in 1999 demonstrated howMSC from bone marrow (BM-MSC) could be cultured invitro with a stable phenotype through many populationdoublings, and that they could be induced to differentiate intoadipocytic, chondrocytic and osteocytic lineages in vitro [2].The presence of multipotent MSC in adipose tissue (AT-MSC) was first demonstrated by Zuk et al. [3]. We subse-quently demonstrated how purified, uncultured AT-MSCcould be isolated and characterized at the phenotype andtranscriptome level, and identified the changes in geneexpression induced by in vitro culture of these cells [4]. Theminimal criteria for defining MSC have now been defined byThe International Society for Cellular Therapy (ISCT) [5].They are: MSC should be plastic-adherent when maintainedin standard culture conditions. MSC should express CD105,

CD73 and CD90, lack expression of CD45, CD34, CD14 orCD11b, CD79α or CD19 and HLA DR surface molecules.MSC must differentiate to osteoblasts, adipocytes andchondrocytes in vitro. However, this population of plasticadherent cells is heterogenous, normally does not expresstelomerase and reaches senscence at 30 – 40 populationdoublings in vitro [6]. These are some of the main reasonswhy, as a result of discussions on terminology within theinternational stem cell research community, ISCT in 2005suggested that the name be changed to multipotent me-senchymal stromal cells, but that the acronym (MSC) bemaintained [7].

2. Immunological aspects of MSC

It has been known for some years that MSC have immu-nosuppressive properties in vitro. This has been determined byadding MSC to T-cells stimulated by mitogens, antigens andalloantigens in vitro, and an effect has also been demonstratedin vivo in a skin graft model, and in a murine model of auto-immune encephalomyelitis [8–13]. The molecular mechan-isms responsible for this effect have not been fully revealed,but direct contact between the MSC and the T cell does not

Journal of the Neurological Sciences 265 (2008) 127–130www.elsevier.com/locate/jns

⁎ Tel.: +47 23 07 37 66; fax: +47 23 07 38 22.E-mail address: [email protected].

0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jns.2007.05.006

seem to be required. A number of candidates for the role as theimmunosuppressive factor secreted by MSC have beenproposed, including a combination of hepatocyte growthfactor and transforming growth factor α [8], indoleamine-2,3-deoxygenase (IDO) associated with the conversion oftryptophane to kynurenine [14] and prostaglandin E2 [15].Mechanistically, coculture with MSC has been shown toinduce an irreversible cell cycle arrest in activated Tcells [16],while the effect of IDO may be apoptosis of the T cellsmediated by kynurenine [17]. Interactions have also beendescribed betweenMSC and other cells of the immune systemin vitro [18]. However, the relevance of these immunosup-pressive effects of MSC in vivo is not entirely certain [19].

In vitro experiments have suggested that MSCmay escapedetection and removal by alloreactive T cells [12]. This may,in part, be due to the fact that MSC express MCH class I, butnot class II and other immunostimulatory molecules.However, even after induced expression of MHC class IImolecules by interferon γ in vitro, Tcell allogeneic responsesto MSC could still be shown to be absent [20]. Theimmunogenicity and long term survival of MSC in vivo hasnot yet been determined. The amazing effect of injection ofhaploidentical MSC on the life threatening graft versus hostdisease (GVHD) in a young boy suggests thatMSC survive inan allogeneic environment at least long enough to exert animmunosuppressive effect on allostimulated Tcells [21]. Thefact that a second injection of MSC from the same donorshowed an equally good effect might suggest that these MSCdid not induce a memory immune response, but the patientwas heavily immunosuppressed, which makes the interpre-tation of these results uncertain [21]. In a murine model,allogeneic MSC could be shown to be rejected some weeksafter implantation, and more quickly after reimplantation intothe same host, suggesting immunological mechanisms withmemory [22].

3. Two-dimensional culture of BM-MSC

The precursor frequency of cells able to adhere, spread andform MSC colonies is no more than 1:1000 in mononuclearcells from bone marrow (BM-MNC) [23]. Thus, the numberof cell divisions required to obtain a large number of MSCmay be considerable. Obviously, if small BM aspirates arecollected, the cells need to undergo more populationdoublings in order to reach a certain cell number. As MSCreach senescence after 30 – 40 population doublings in vitro,it is possible that the collection of a small BM aspirate maylimit the ability of the MSC to proliferate in vivo.

Traditionally, bone marrow mononuclear cells are used toseed plastic surfaces for 2D culture of MSC. However, it ispossible to seed the unseparated bone marrow after lysis of theerythrocytes. This may be advantageous when only smallaspirates are available. It is possible that seeding of unse-parated bone marrow may include cells in the culture whichwould have been lost in the separation process. For MSC, theseeding surface is traditionally uncoated plastic. The brand of

tissue culture flask chosenmay be of importance for the rate ofexpansion of the MSC [24]. The seeding density is alsoimportant: the lower the seeding density, the greater the cellnumber per seeded cell after a given time [24–26]. However,low seeding densities require very large surfaces in order toobtain large numbers of cells for therapeutic purposes. Unlessmultilayer cell factories are used, the seeding densitymay haveto be determined by the number of cells required for a par-ticular application and the number of flasks that it is practical tohandle.

The choice of medium is also of importance for the endresult of the culture [24]. A serum free alternative hasrecently been described, but whether this allows long termexpansion of large numbers of cells remains uncertain [27].Thus, serum must be added to supplement most media.Traditionally, fetal bovine serum (FBS) has been used aftercareful selection of the best batch. However, certain risksassociated with the use of FBS has stimulated a search forother sources of serum. The transmission of variantCreutzfeld–Jacob disease through ingestion of bovinematerial created awareness of the possibility of FBS carryingprions or other zoonotic agents, but the risk of transmissionof diseases via FBS is considered to be small [28]. A greaterrisk associated with the use of MSC expanded in FBS seemsto be the immunogenicity of FBS proteins [29]. Indeed, MSCtransplantation failure has been noted as a consequence ofimmune attack on FBS proteins carried by transplanted cellsexpanded in FBS [30]. Clearly, a human source of serumwould be advantageous. We tested a commercially availablepool of human sera, but found that this did not support theproliferation of MSC [23]. Autologous serum did supportMSC proliferation, however, with a more rapid rate of cellexpansion and with senescence occurring later in these cells[23]. Comparison between cells expanded in FBS and auto-logous serum suggested that FBS may act as a differentiationinducing factor. Also, the gene expression in the cellsexpanded in autologous serum was remarkably stable overmany passages. The use of autologous serum for expansionof large numbers of cells requires a large amount of serum –200 ml is required to obtain 108 cells – and this may be aproblem in some cell therapy protocols. In addition, it is notyet known if the serum from ill patients is able to supportMSC proliferation as well as the serum from healthy donors.Human platelet lysate may represent a more robust source ofhuman proteins [31]. This is made as a large pool of plateletlysates mixed with fresh frozen plasma from several donors.Large pools should ensure minimal batch-to-batch variation,but the factors present in pools of allogeneic serum, whichled to their failure to support MSC proliferation, willpresumably be present also in pools of fresh frozen plasma.Human platelet lysate may prove to be the best source ofhuman proteins, but experiments are required to ensure thatthe cytokines present in the lysate do not induce undue dif-ferentiation in the MSC.

One final factor to consider in the design of optimalculture conditions for MSC is the concentration of gases in

128 J.E. Brinchmann / Journal of the Neurological Sciences 265 (2008) 127–130

the incubator. These cells are not adapted to the oxygenconcentrations in room air, and they are used to higher CO2

concentrations than the 5% normally provided in mostincubator atmospheres. The impact of reducing the pO2 andincreasing the pCO2 in the incubator atmosphere is notknown for human MSC, but similar cells, multipotent adultprogenitor cells from bone marrow, were recently shown tohave a higher proliferative potential, a more undifferentiatedprofile of gene expression and greater ability to differentiateto many lineages of cells in a swine model [32].

Like most other cells adapted to a life in close proximityto extracellular matrix (ECM), MSC depend on ECM signalsfor survival. In vitro, adhesion to plastic substitutes foradhesion to ECM. When their adhesion to plastic isinterrupted, MSC will die by a process called anoikis [33].The time frame for anoikis to occur in in vitro expandedMSC is not known, but cell death is likely to appear aftersome hours to a few days. By this time the cells must havehomed to the site of damage, where surrounding tissue mayprovide survival signals, or else they are likely to die.

4. Special applications of MSC and their culturerequirements

In order to be rapidly expanded in numbers, MSC must becultured adherent to plastic or coated plastic surfaces. However,for some applications, special culture conditions may berequired. In order to use MSC to make bone, the cells must beplaced in some form of scaffold, perhaps in combination withcytokines [34]. To induce chondrogenesis in MSC, the cellsneed to be placed in a chondrogenesis induction medium con-taining cytokines, and to be established in three-dimensionslcultures. These may be pellet cultures [2] or other forms ofscaffolds [35]. However, in many applications where MSC areused to regenerate tissues, the cells are used directly afterexpansion in two-dimensional (2D) culture,with the expectationthat cues delivered from the tissue to be regenerated will inducethe correct differentiation pathway in the MSC. As a case inpoint, some murine MSC transplanted into the lateral ventriclesof the neonatal mouse brain could be shown to exhibit mor-phological and immunohistochemical characteristics of astro-cytes [36].

5. Quality control

As with all cells expanded under the regulations of GoodManufacturing Practice, microbiological testing should be per-formed on the bonemarrow sample, and then at regular intervalsduring the cell culture. If the cells are to be transplantedimmediately after in vitro culture, the results from the bacterialcultures performed on supernatant from the last change ofmedium must be relied upon for documentation of the sterilityof the cells. The results from the microbiological testing per-formed when the cultures are concluded will only be availableafter the transplantation. In our laboratory, we remove anti-biotics from the cell culture after the first week. At this time any

contaminatingmicroorganism sensitive to the antibiotics will bekilled, and if non-sensitive microorganisms are present, con-tinued presence of antibiotics may possibly suppress, but notremove these contaminants. There is the added advantage thatallergic responses to antibiotics will not occur.

Flow cytometric examination of the cells should beperformed during the last passage of the cells. To fulfil theformal criteria for definition of MSC antibodies specific forCD105, CD73, CD90, CD45, CD34, CD14 or CD11b,CD79α or CD19 and HLA DR surface molecules should beincluded. Finally, the karyotype of cells to be transplantedshoud be investigated. If cells are to be cryopreserved prior totransplantation, time may allow this examination to beperformed before clinical use of the cells. If fresh cells are tobe transplanted, one may choose to perform karyotyping onearly passage cells, or one may decide to examine cells from aselection of donors in order at least to determine the prevalenceof karyotypic changes present in MSC cultured in that par-ticular laboratory. To date, no malignancies have been des-cribed in humans transplanted with MSC, but karyotypicchanges have been observed in humanMSC expanded in vitro(Podesta, M. Safety issues in clinical-grade MSC expansion.Presentation at the EHA Scientific Workshop on Biology andClinical Applications of Mesenchymal Stem Cells).

6. Conclusions

MSC have been known for decades, but scientific focus onthese cells have only recently become intense. To date, twoclinical applications are considered for MSC: they may beused for their immunosuppressive properties, or for theirpotential role in tissue regeneration. In either case, largenumbers of cells are required. In order to proliferate in vitro,the cells need to be seeded on plastic surfaces. A number ofmethodological choices have to be made in order to providethe best conditions for in vitro expansion of these cells. Thechoices are described in this review, but data are not availableyet to clearly describe how one culture procedure is betterthan the others.

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