Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production Major transitions can be expected within the next few decades aiming at the reduction of pollution and global warming and at energy saving measures . For these purposes , new sustainable biorefinery concepts will be needed that will replace the traditional mineral oil-based synthesis of specialty and bulk chemicals . An important group of these chemicals are those that comprise N-functionalities . Many plant components contained in biomass rest or waste stream fractions contain these N-functionalities in proteins and free amino acids that can be used as starting materials for the synthesis of biopolymers and chemicals . This paper describes the economic and technological feasibility for cyanophycin production by fermentation of the potato waste stream Protamylasse ™ or directly in plants and its subsequent conversion to a number of N-containing bulk chemicals . Introduction Plants have the ability to use the incident sunlight for the biosynthesis of a tremendous variety of compounds that may contain a number of functionalized atoms or groups . An important group of these functionalized compounds are proteins and especially the individual amino acids that contain one or more nitrogen atoms . When starting from crude oil or naphtha , the incorporation of functionalities -LRB- e.g. , − NH2 -RRB- into derived bulk chemicals -LRB- such as 1,2-ethanediamine and 1,4-butanediamine -RRB- requires considerable amounts of energy and catalysts . However , some amino acids appear to be very suitable starting materials for highly functionalized bulk chemicals -LRB- Scott et al. 2007 -RRB- . The biorefinery concept is a rapidly emerging field of research and commercial activities aiming at the integral use of all components of agricultural crops . In addition to the main product such as starch or oil , also , other side stream fractions including protein , free amino acid , and fiber fractions have high potential for valorization . An example of such waste stream fraction , Protamylasse ™ that remains after starch and protein extraction from potato and its possible application as a substrate for microbial fermentation and production process for cyanophycin -LRB- Elbahloul et al. 2005a , b , 2006 -RRB- , will be described in detail in the current paper . Cyanophycin -LRB- multiarginyl-poly -LSB- l-aspartic acid -RSB- ; CGP , cyanophycin granule peptide -RRB- is a non-ribosomal protein-like polymer which consists of equimolar amounts of aspartic acid and arginine arranged as a poly-aspartic acid backbone to which arginine residues are linked to the β-carboxyl group of each aspartate by its α-amino group . In nature , cyanophycin is produced by most , but not all , cyanobacteria as a temporary nitrogen reserve material during the transition of cells from the exponential phase to the stationary phase . The polymerization reaction is catalyzed by only one enzyme , which is referred to as cyanophycin synthetase -LRB- CphA -RRB- . Because of the low polymer content and the slow growth of cyanobacteria resulting in only low cell densities , cyanobacteria are not suitable for large-scale production of cyanophycin . Therefore , the cphA genes from a number of cyanobacteria have been expressed in several bacteria , and , more recently , also in plants . Furthermore , the polymer isolated from recombinant strains contained lysine as an additional amino acid constituent . Now that cyanophycin can be produced in sufficient amounts by pilot scale fermentations for studying its material properties , it appears of biotechnological interest because purified cyanophycin can be chemically converted into a polymer with a reduced arginine content , which might be used like poly-aspartic acid as a biodegradable substitute for synthetic polyacrylate in various technical processes . In addition , cyanophycin might also be of interest for other applications when the hitherto unknown physical and material properties of this polymer will be revealed . On the other hand , cyanophycin is a convenient source of the constituent amino acids that may be regarded as nitrogen-functionalized precursor chemicals . In the current paper , conditions will be discussed for the technological and economic feasibility of cyanophycin production by microbial fermentation and by cyanophycin production directly in plants . The conditions for fermentative cyanophycin production will be based upon the use of cheap substrates derived from agricultural waste streams and the possible cyanophycin production simultaneously with other fermentation products like ethanol . This aspect is denoted process integration . Biorefinery and its place in the production of chemicals The depletion in fossil feedstocks , increasing oil prices and the ecological problems associated with CO2 emissions , are forcing the development of alternative resources for energy , transport fuels , and chemicals : the replacement of fossil resources with CO2 neutral biomass . Potentially , biomass may be used to replace fossil raw materials in several major applications : heat , electricity , transport fuels , chemicals , and other industrial use . Each of these groups represents about 20 % of the total fossil consumption in the industrialized countries -LRB- Oil Market Report of the International Energy Agency 2004 -RRB- . Large variations in the cost of these products at the wholesale level , based on their energy content , are evident -LRB- Table 1 -RRB- . When one considers the contribution to costs by the raw materials -LRB- expressed per GJ end product -RRB- , large differences are also seen . Heat can be produced from coal for around 3 $ / GJ due to utilizing inexpensive feedstocks with high conversion efficiency -LRB- about 100 % -RRB- , while the raw material costs for electricity is double -LRB- 6 $ / GJ -RRB- due to a conversion yield of about 50 % . Most notable is the high raw material costs for chemicals . Here , expensive raw materials -LRB- oil -RRB- are used with low -LRB- er -RRB- conversion yields -LRB- Sanders et al. 2005 , 2007 -RRB- . Table 1Different applications and contributions of biomassContributionIntegral cost prices -LRB- $ / GJ end product -RRB- Raw material cost fossil -LRB- $ / GJ -RRB- Percentage of total energy in the Netherlands -LRB- 3.000 PJ -RRB- consumed per application -LRB- % -RRB- Heat43 -LRB- Coal -RRB- ± 20Electricity226 -LRB- Coal -RRB- ± 20Transport fuel108 -LRB- Oil -RRB- ± 20Average bulk chemicals7530 -LRB- Oil -RRB- ± 20Rest of industry ± 20 To obtain a good net income for biomass , an effective biorefinery system is required for the separation of the harvested crop into fractions for use in -LRB- several of -RRB- these applications . These may be used directly as the desired product or undergo conversion by chemical , enzymatic , and/or microbial means to obtain other products . Biorefinery systems are well established for a number of crops . For example , soybeans are the raw materials for large biorefineries to produce oil -LRB- for biofuels -RRB- , proteins and valuable nutraceuticals . Less well explored is the use of biomass to make industrial chemicals . Effort to produce chemicals with constant quality and performance -LRB- such as lactic acid -RRB- has been addressed , but has mainly focused on the use of carbohydrates as raw materials and use of biotechnology for conversion . However , for effective biorefinery approaches , other biomass fractions should also be considered for the production of chemicals . It is anticipated that the substitution of petrochemical transportation fuels with biofuels will rise significantly in the coming years . This means that a rise in the production of biodiesel will lead to large volumes of glycerol as a residual stream , and indeed , some companies are already investigating the use of glycerol to produce chemicals . While the awareness of large volumes of glycerol from biofuel production is apparent , one should not overlook other waste streams . Indeed , from biofuel production , an immense concomitant waste stream of protein will also be generated . Sources of proteins and amino acids are not limited to those generated from biofuel production , but also from other industries such as potato starch production . For example , during AVEBE 's processing of potatoes for starch extraction , the main waste stream is Protamylasse ™ , which mainly contains sugars , organic acids , proteins , and free amino acids and has currently no major market use . Some of the amino acids present in such sources could be very suitable raw materials for preparing -LRB- highly -RRB- functionalized chemicals traditionally prepared by the petrochemical industry -LRB- Sanders et al. 2007 -RRB- . Generally , the conversion of crude oil products utilizes primary products -LRB- ethylene , etc. -RRB- , and their conversion to materials or -LRB- functional -RRB- chemicals makes use of co-reagents such as ammonia and various process steps to introduce functionalities such as − NH2 . Conversely , many products found in biomass , such as proteins and amino acids , often contain these functionalities . Therefore , it is attractive to exploit this to bypass the use , and preparation , of co-reagents as well as eliminate various process steps by utilizing suitable biomass-based precursors for the production of chemicals . Thus , the production of chemicals from biomass takes advantage of the biomass structure in a more efficient way than the production of fuels or electricity alone and can potentially save more fossil energy than producing energy alone -LRB- Scott et al. 2007 -RRB- . When used in combination with environmentally sound production and processing techniques across the whole biomass production chain , i.e. , from cultivation and harvest , its -LRB- pre -RRB- treatment and conversion to products , the use of biomass is considered a sustainable alternative to conventional feedstocks , which is reflected by sound economic advantages in both raw material and investment costs . General introduction on NRPs and especially cyanophycin Cyanophycin -LRB- also referred to as CGP , cyanophycin granule polypeptide -RRB- that belongs to the family of bacterial poly-amino acids together with poly-γ-glutamic acid and poly-ɛ-lysine , was discovered in 1887 by Borzi -LRB- 1887 -RRB- during microscopic studies of cyanobacteria and was later found in all groups of cyanobacteria -LRB- Oppermann-Sanio and Steinbüchel 2002 -RRB- . The cyanophycin molecule structure is related to that of poly -LRB- aspartic acid -RRB- s , but , unlike synthetic poly-aspartic acid , it is a comb-like polymer with α-amino-α-carboxy-linked l-aspartic acid residues representing the poly -LRB- α-l-aspartic acid -RRB- backbone and l-arginine residues bound to the β-carboxylic groups of aspartic acids -LRB- Simon and Weathers 1976 ; for recent review , see Obst and Steinbüchel 2004 ; Fig. 1 -RRB- . Cyanophycin is synthesized by most , but not all , cyanobacteria as a temporary nitrogen reserve material during the transition of cells from the exponential phase to the stationary phase -LRB- Mackerras et al. 1990 -RRB- . At neutral pH and physiological ionic strength , cyanophycin is insoluble and deposited in the cytoplasm as membraneless granules -LRB- Lawry and Simon 1982 -RRB- . Fig. 1Chemical structure of the cyanophycin monomer Cyanophycin isolated from Cyanobacteria is highly polydisperse and shows a molecular weight range of 25 -- 100 kDa as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to a polymerization degree of 90 -- 400 -LRB- Simon 1971 ; Simon and Weathers 1976 -RRB- . Cyanophycin is a transiently accumulated storage compound which is synthesized under conditions of low temperature or low light intensity . Its accumulation can be artificially enhanced by the addition of chloramphenicol as an inhibitor of ribosomal protein biosynthesis -LRB- Simon 1973 -RRB- . Cyanophycin plays an important role in the conservation of nitrogen , carbon , and energy and , as indicated by its biosynthesis in presence of chloramphenicol , is non-ribosomally synthesized by CphA . Cyanophycin is accumulated in the cytoplasm of cyanobacteria as membraneless granules -LRB- Allen and Weathers 1980 -RRB- in the early stationary growth phase -LRB- Mackerras et al. 1990 ; Liotenberg et al. 1996 -RRB- . When growth is resumed , for example due to a change in cultivation conditions , cyanophycin is reutilized by the cells -LRB- Mackerras et al. 1990 -RRB- . Krehenbrink et al. -LRB- 2002 -RRB- and Ziegler et al. -LRB- 2002 -RRB- showed that cyanophycin occurs even in heterotrophic bacteria like Acinetobacter sp. and Desulfitobacterium hafniense and therefore confirmed the wide distribution of this biopolymer and its function in nature as a general storage compound . Cyanophycin is of biotechnological interest because the purified polymer can be chemically converted into a polymer with reduced arginine content -LRB- Joentgen et al. 1998 -RRB- , which might be used like poly-aspartic acid as a biodegradable substitute for synthetic polyacrylate in various technical processes . In addition , cyanophycin might also be of interest for other applications if the unknown physical and material properties of this polymer are revealed . Because of the low polymer content and the slow growth of cyanobacteria resulting in only low cell densities , cyanobacteria are not suitable for large-scale production of cyanophycin -LRB- Schwamborn 1998 -RRB- , and sufficient amounts of cyanophycin were hitherto not available . The polymerization reaction is catalyzed by only one enzyme , which is referred to as CphA -LRB- Ziegler et al. 1998 -RRB- . The cphA genes from Anabaena variabilis ATCC 29413 , Anabaena sp. strain PCC7120 , Synechocystis sp. strain PCC6803 , Synechocystis sp. strain PCC6308 , Synechococcus elongatus , Synechococcus sp. strain MA19 and others were cloned and expressed in Escherichia coli -LRB- Aboulmagd et al. 2000 ; Berg et al. 2000 ; Hai et al. 1999 ; Oppermann-Sanio et al. 1999 ; Ziegler et al. 1998 -RRB- . More recently , heterologous expression of cphA was also demonstrated at a small scale in recombinant strains of Ralstonia eutropha , Corynebacterium glutamicum , and Pseudomonas putida -LRB- Aboulmagd et al. 2001 -RRB- . Whereas in cyanobacteria the molecular mass of the polymer strands ranged from 25 to 100 kDa -LRB- Simon 1976 -RRB- , the polymer from recombinant strains harboring cphA as well as in vitro-synthesized polymer exhibited a much lower range -LRB- 25 to 30 kDa -RRB- and polydispersity . Furthermore , it was found that the polymer isolated from recombinant strains contained lysine as an additional amino acid constituent -LRB- Aboulmagd et al. 2001 ; Ziegler et al. 1998 -RRB- . Recently , the results of a detailed in silico analysis of the occurrence of enzymes involved in cyanophycin metabolism was published -LRB- Füser and Steinbüchel 2007 -RRB- . Recently , the earlier postulated instability of recombinant E. coli strains employed for cyanophycin production was also confirmed in both DH1 and DH5α . This instability may be caused by loss of the plasmid during fermentation . However , as cyanophycin production continues in cultures that rapidly appear to loose the ampicillin resistance employed for selection and plasmid maintenance , other explanations are also under consideration such as competition for Arg and Asp by both cyanophycin and the ampicillin resistance protein and the theoretical possibility that the ampicillin resistance protein could be trapped into the cyanophycin granule or at least be made inaccessible to the ampicillin . Due to the wide knowledge of its metabolism and available genetic tools , E. coli is one of the most commonly used bacterial hosts for the production of recombinant proteins -LRB- Lee 1996 -RRB- . Several expression systems have been developed for technical-scale production of recombinant proteins in E. coli based on the regulated trp , lac , or lambda PL promoter -LRB- Hannig and Makrides 1998 -RRB- . The cultivation of recombinant E. coli strains harboring cphA from Synechocystis sp. strain PCC6803 at the 500-l scale for the production of cyanophycin has been described -LRB- Frey et al. 2002 -RRB- . As the previously described method for the purification of cyanophycin -LRB- Simon and Weathers 1976 -RRB- is not applicable to a large scale , a simplified method for isolation of the polymer at the technical scale was elaborated . Biosynthesis of cyanophycin was extensively studied in the 1970s by Simon and coworkers -LRB- Simon 1971 , 1976 ; Simon and Weathers 1976 -RRB- . Later , this led to the identification of cyanophycin synthetase enzymes and the encoding genes -LRB- cphA -RRB- in various organisms -LRB- Ziegler et al. 1998 ; Aboulmagd et al. 2000 ; Berg et al. 2000 ; Hai et al. 2002 -RRB- . Subsequently , the enzymes involved in the degradation of cyanophycin by intracellular cyanophcyinases of cyanobacteria -LRB- cphB -RRB- and extracellular depolymerases , like hydrolase -LRB- cphE -RRB- and cyanophycinase -LRB- cphI -RRB- genes , were identified -LRB- Obst et al. 2002 , 2004 ; Obst and Steinbüchel 2004 -RRB- . Elbahloul et al. -LRB- 2005a , b -RRB- have found that inactivation of the cyanophycinase gene in Acinetobacter resulted in significantly less cyanophycin accumulation than the wild type presumably due to a shortage of cyanophycin primer molecules . On the contrary , cyanophycin is highly resistant against hydrolytic cleavage by proteases such as trypsin , pronase , pepsin , carboxypeptidases B , carboxypeptidase C , and leucin-aminopeptidase , and cyanophycin is also resistant against arginases -LRB- Simon and Weathers 1976 -RRB- . Because the cyanophycin synthetase genes -LRB- cphA -RRB- of many cyanobacteria , and recently , also of other microorganisms were identified , cloned , and heterologously expressed in other bacteria -LRB- Aboulmagd et al. 2001 -RRB- conferring the ability to produce comparably large amounts of cyanophycin -LRB- up to 50 % of CDW -RRB- in a much shorter period of time -LRB- 1 -- 2 days -RRB- as compared to cyanobacteria -LRB- about 4 weeks -RRB- and cyanophycin production was demonstrated at the 30 - to 500-l scale -LRB- Aboulmagd et al. 2001 ; Frey et al. 2002 ; Voß and Steinbüchel 2006 -RRB- , the interest in cyanophycin as a potential raw material has constantly increased over the last few years . Improvement of fermentation conditions , feeding regimes , and the possibility to produce cyanophycin now by the employment of many genetically engineered bacteria with industrial relevance like R. eutropha , C. glutamicum , or P. putida -LRB- Aboulmagd et al. 2001 -RRB- in complex but also in defined media make it appear likely that further improvement of cyanophycin production in bacteria will be achieved during future studies . These studies may include elementary mode analyses -LRB- Diniz et al. 2006 -RRB- or more conventional approaches involving experimental design using fermenter arrays and principal component analyses . In conclusion , large-scale fermentation processes for cyanophycin production and downstream processing are available for a number of different microorganisms able to grow in different substrates , including the potato waste stream Protamylasse ™ , and also , low cyanophycin yields were reported in plants . Production and economic aspects of fermentative cyanophycin production An important contribution to sustainability can be made by the use of a considerable plant waste stream for the production of renewable , biodegradable , and biocompatible polymers and/or valuable chemicals that are now produced on large scale from petroleum . Some of the polymer classes to be developed may be expected to replace some existing mineral petroleum-based polymers as soon as competitive production prices can be obtained and/or supporting measures will be taken to promote the use of renewable resources . On the other hand , completely novel types of biopolymers may be developed for completely novel applications . AVEBE , located in the northern part of The Netherlands , is the largest potato-starch-producing company in the world involved in extraction , processing , and sales of starch and starch-derived products . During processing of potatoes for starch extraction , the main waste stream is Protamylasse ™ . Annually , AVEBE produces about 120,000 m3 of Protamylasse ™ containing about 70,000 tons of dry matter , mainly consisting of sugars -LRB- 14,000 tons -RRB- , organic acids -LRB- 13,300 tons -RRB- , proteins and free amino acids -LRB- 18,000 tons -RRB- . The amino acids arginine and aspartic acid amount to about 1,000 tons each and lysine to about 700 tons . Currently , there is no proper outlet for this Protamylasse ™ other than low value epandage -LRB- e.g. , by Bos Agra-Service , NL in which the salts present , such as potassium , are used as fertilizer -RRB- , whereas rough calculation shows that the total intrinsic gross value of the valuable components in the Protamylasse ™ is about 45 million euros . A number of research objectives is now in progress to add substantial value to the entire potato starch production chain -LRB- and thus its economical feasibility -RRB- . Protamylasse ™ may be considered a model for other agricultural waste streams , such as grass juice and beet residue . It is not certain whether concentrations of all medium components in the Protamylasse ™ will be optimal to sustain microbial growth and cyanophycin production , and it is anticipated that additional medium components need to be identified and/or tailor-made production strains constructed . This will require a detailed analysis of Protamylasse ™ components before and after the cyanophycin production phase . To calculate the economic feasibility of cyanophycin production using Protamylasse ™ , several aspects need to be included as follows : Necessary steps from potato starch and protein extraction , yielding Protamylasse ™ , to the final purified product cyanophycin -LRB- which is considered an intermediate product for derived polymer types and N-containing bulk chemicals -RRB- include : shipment to a fermentation plant , dilution to 5 -- 6 % -LRB- v/v -RRB- using tap water , removal of potato particles by filtration , disposal -LRB- or alternative use -RRB- of particle fraction -LRB- 60 % DM -RRB- , loading of step up fermenters -LSB- to allow an microbial inoculum concentration of 10 % -LRB- v/v -RRB- per step ; e.g. , 10 → 100 → 1,000 → 10,000 → 100,000 l -RSB- , sterilization , cooling , addition of ampicillin , inoculation , -LRB- batch -RRB- fermentation , addition of acid and/or base for pH control , cell harvesting and concentration , disposal or recycling of spent Protamylasse ™ , cell disruption -LRB- not for E. coli -RRB- , cyanophycin extraction using pH 2 , neutralization , cyanophycin crystallization , precipitation , purification , and storage.Yearly Protamylasse ™ supply , 120,000 m3 ; 70,000 tons -LRB- 60 % DM -RRB- Protamylasse ™ dry matter composition : amino acids -LRB- 18,000 tons -RRB- , sugars -LRB- 14,000 tons -RRB- , organic acids -LRB- 13,300 tons -RRB- , ash -LRB- 22,200 tons -RRB- . For further details on the composition , see Elbahloul et al. -LRB- 2005a , b -RRB- . Small-scale fermentation process data : Protamylasse ™ concentration , 5 -- 6 % -LRB- v/v -RRB- ; fermenter volume , 25 l ; strain E. coli DH1 -LRB- pMa/c5 -914 : : cphA -RRB- ; temperature 37 °C ; maximal OD and cyanophycin yield reached after 15 h ; optimal pH 7.5 -- 8.0 ; biomass yield 5 -- 10 g/l -LRB- CDW -RRB- ; cyanophycin content , 25 % -LRB- w/w -RRB- ; cyanophycin composition , Asp , 50 % ; Arg , 45 % ; Lys , 5 % ; E. coli occasionally -LRB- 5 -- 10 % -RRB- incorporates lysine in stead of arginine ; poly -LRB- Asp -- Arg -RRB- is non-soluble in water at neutral pH ; poly -LRB- Asp -- Arg -- Lys -RRB- is soluble in water . Assuming that most of the solid particles will be removed from the Protamylasse ™ by filtration , a yearly amount of 48,000 m3 of Protamylasse ™ liquid juice will become available , which is used at a 5 % dilution during fermentation , thus , providing a yearly amount of 960,000 m3 of diluted fermentation broth . This volume would be enough to run 9,600 × 100 m3 fermenter volumes . Further assuming a 1 week 's run time -LRB- including cleaning , sterilization , fermentation , and harvest -RRB- a park of , e.g. , 185 × 100 m3 or preferably 20 × 1,000 m3 fermenter units -LRB- step up units included -RRB- could be operated continuously . With the current E. coli biomass yield of 5 g/l -LRB- CDM -RRB- with a cyanophycin content of 25 % -LRB- w/w DM -RRB- , this would yield a yearly amount of 1,200 tons of purified cyanophycin . At an estimated market price of $ 1,000 per ton cyanophycin , the Protamylasse ™ juice fraction -LRB- 40 % v/v -RRB- would yield a yearly income of only 1.2 million euros . However , by only increasing the amount of microbial biomass from the actual 5 g/l -LRB- CDM , E. coli -RRB- to a realistic value of 100 g/l -LRB- CDM , S. cerevisiae -RRB- with the same cyanophycin content of 25 % -LRB- w/w DM -RRB- , this yearly income could be raised to 24 million euros . If within the same fermentation run via so-called process integration , in addition to cyanophycin , ethanol also -LSB- yielding from 960,000 m3 at 5 % -LRB- v/v -RRB- about 48,000 m3 ethanol and an additional yearly income of 27.4 million euros at $ 2.8 per US gallon , $ 0.57 per liter -RRB- could be produced using semi-aerobic fermentation with S. cerevisiae , this yearly income could be further raised to about 50 million euros . The Protamylasse ™ particle fraction can , in principle , also be used for cyanophycin and ethanol production . Preliminary calculations using the model of Golden Grain Energy , LLC -LRB- http://sec.edgar-online.com/ 2004/06/14 / 0001104659-04-016859 / Section7.asp -RRB- suggest that the break-even point could roughly be reached within about 3 -- 5 years . Further process improvements may be obtainable and are necessary to make this process economically feasible . These obtainable figures provide estimates as to the investments and costs that will be necessary to start a cyanophycin production fermentation facility . However , before fermentative cyanophycin production on industrial scale can be started , a number of bottlenecks should be overcome . Table 2 lists current bottlenecks and proposed measures for optimal and economically feasible cyanophycin production . Table 2Economical and technological bottlenecks and proposed measuresBottleneckProposed measure -LRB- s -RRB- Investments , including costs for fermentation and downstream processing equipmentThe calculation provided here suggests that these may be acceptableCosts for the production of cyanophycin , cyanophycin-derived products and for downstream processing of biomassConstruction of a sufficiently productive microbial strain to convert or simply utilize constituents of plant waste streams like Protamylasse ™ and to incorporate these compounds , presumably amino acids , into the cyanophycin polymer chain during cyanophycin biosynthesisPhenotypic instability of E. coli production strains used until now , DH1 and DH5α , containing plasmid pMa/c5 -914 : : cphA6803Construction of stable strains with integrated copies of the cyanophycinsynthesis genesLow biomass yields of the E. coli strains usedSince not all components present in the current source of Protamylasse ™ may have the proper concentration for current laboratory strain -LRB- s -RRB- , an optimization may require the addition of substrates other than Protamylasse ™ , for example other plant waste streams . Sufficient provision of amino acids like arginine should be ensured during the production phaseOptimization of microbial biomass formationBy using yeasts as alternative production organisms biomass yields could be increased to 100 g/l CDM for S. cerevisiae -LSB- factor 20 × -RSB- or 150 g/l CDM for Pichia pastoris -LSB- factor 30 × -RSB- if in Protamylasse ™ the same yields can be obtained as in dedicated growth mediaSub-optimal fermentation processesFermentation technology and feeding regimes have to be developed for optimum amino acid utilization or biosynthesis from Protamylasse ™ or other plant waste streamsGeneration of valuable side stream particle fraction of Protamylasse ™ Alternative use of the side stream particle fraction of Protamylasse ™ , e.g. by using cyanophycin producing filamentous fungiCo-production with , e.g. , ethanolWhen using S. cerevisiae as the production organisms and -LRB- semi - -RRB- anaerobic fermentation both cyanophycin and ethanol could possibly be produced during the same runCosts for cyanophycin extractionDevelopment of alternative cheap cyanophycin extraction methods using , e.g. , hydro-cyclone equipment for the non-soluble fractionCost-efficient production of cyanophycin in plantsThe transfer of the bacterial cyanophycin synthetase gene -LRB- cphA -RRB- into eukaryotic hosts , mostly plants and its effective expression in suitable organs or cell compartments is a major step -LRB- see below -RRB- Efficacy of downstream processingDownstream processing has to be adapted and optimized for cyanophycin or cyanophycin derivatives containing biomass , which will be either bacterial cells or eukaryotic -LRB- mostly plant -RRB- cells or tissuesLack of insight in possible modifications of cyanophycin , their impact on cyanophycin properties and market potentialThe diverse possibilities to modify the cyanophycin molecule chemically or enzymatically has to be exhaustingly explored to identify all potential key applications for cyanophycin-derived products and to find the most suitable products with regard to market potential and the possibility of their commercializationLack of knowledge concerning properties of known cyanophycin synthetases and their genetic engineeringThe possibility to modify the active sites of the cyanophycin synthetases in order to change its substrate specificity and to allow the production of cyanophycin derivatives has to be determinedInsufficient insight in all possible applications for cyanophycin as a polymer or as a starting material for chemical synthesesThe exploitation of cyanophycins and cyanophycin-derived molecules as substitutes for well established industrial products or as renewable raw materials has to be determined precisely Cyanophycin production in plants Transgenic plants can be utilized to produce renewable resources for industrial purposes in a CO2-neutral , environmentally acceptable , and competitive way . Poly-3-hydroxybutyrate -LRB- PHB -RRB- was the first plastic-like compound produced in plants -LRB- Poirier et al. 1992 -RRB- , followed by , e.g. , poly-3-hydroxyalkanoate -LRB- PHA ; Poirier 2002 -RRB- and medium chain-length PHA in potato -LRB- Romano et al. 2003 , 2005 -RRB- and showed the feasibility to produce biopolymers in plants . Recently , it has been shown that it is also possible to produce cyanophycin in plants -LRB- Neumann et al. 2005 -RRB- . For this , the Thermosynechococcus elongatus BP-1 cyanophycin synthetase gene was expressed constitutively under a 35S promoter in tobacco and potato plants . It was shown that approximately 1.14 and 0.24 % dry weight could be accumulated in the cytosol of tobacco and potato leaves , respectively . The size -LRB- 35 kDa -RRB- , amino acid composition -LRB- Asp/Arg/Lys = 1:1.05:0.1 -RRB- , and structure of the plant-produced polymer was similar to that produced in transgenic E. coli expressing the same gene ; however , the amount and molecular weight of the cyanophycin produced in plants was much lower than that observed in bacteria -LRB- up to 50 % dry weight and 125 kDa in bacteria -RRB- . The experiments have provided proof of concept for the potential of producing cyanophycin in plants . Production of the cyanophycin biopolymer in potato is of high interest to the potato starch industry . Production in this plant does not require any additional infrastructure . After processing of the potatoes , cyanophycin can be isolated from the Protamylasse ™ . However , for commercial application , the efficiency of cyanophycin accumulation in potato has to be significantly improved . Neumann et al. -LRB- 2005 -RRB- already indicated that directing the cyanophycin synthetase into several other compartments , such as the chloroplasts , could lead to increased accumulation of cyanophycin . However , chloroplasts in the cyanophycin-producing cells differ morphologically from wild-type chloroplasts . There are fewer and smaller grana stacks , and the growth rate is slower . One of the possible explanations for these properties is depletion of amino acid resources as a result of cyanophycin production -LRB- Neumann et al. 2005 -RRB- . The pioneering experiments by Neumann et al. -LRB- 2005 -RRB- have opened up a new field for the production of cyanopycin in agricultural crops and show that more research is needed before being introduced into agricultural production -LRB- Conrad 2005 -RRB- . Additional strategies Priming cyanophycin elongation In in vitro studies , it has been shown that cyanophycin synthetase works more efficiently with a -LRB- β-Asp/Arg -RRB- 3/Arg primer . In planta production of such a primer may enhance cyanophycin biosynthesis . Cyanophycinases encoded by the chpB and chpE genes can degrade cyanophycin into arginine -- aspartic acid dipeptides -LRB- Asp -- Arg -RRB- which can not be used as primer for cyanophycin biosynthesis . The cphI gene encodes a plant-type asparaginase able to hydrolyze β-Asp/Arg bonds and that , thus , may be responsible for the last step in cyanophycin degradation . Bacterial studies have indicated that cphI expression contributes to a higher cyanophycin level . It might be possible to use a poly-Asp backbone as primer for cyanophycin biosynthesis . This peptide can be produced by ribosomal protein biosynthesis . The gene should be under the control of a low-level promoter to prevent the production of many peptides , and thus , the production of many low molecular weight polymers . Optimization of amino acid biosynthesis It has been shown that cyanophycin synthetase uses arginine and aspartic acid as its major amino acid source but that it also can incorporate lysine in the cyanophycin polymer -LRB- Berg et al. 2000 -RRB- . It is unclear how this affects the properties of the polymer . Based on the chemical composition of Protamylasse ™ and the affinity of the enzyme for arginine and lysine , it can be proposed that lysine accounts for 1.5 % of the total cyanophycin . To reduce this amount , three strategies are possible , i.e. , improve biosynthesis of arginine , reduce the level of lysine , and/or transform lysine into arginine . It is possible that the availability of substrates -LRB- Asp and Arg -RRB- in plants is limiting or off-balance . Therefore , it is important to identify the organs that have the highest concentrations of available substrates and to investigate whether the substrate supply can be enhanced by introduction of genes involved in substrate production . Comparison of economics of cyanophycin production by fermentation or in plants As for some other commodities , depending on production price , market volume and final product price -LRB- Fig. 2 -RRB- for cyanophycin specialty product applications , fermentation is the preferred production method -LRB- roughly below 20.000 tons annually -RRB- , whereas for bulk quantities of cyanophycin , the production directly in plants is preferred -LRB- above 20,000 tons -RRB- . Therefore , for the production of cyanophycin-derived bulk quantities of nitrogen-containing chemicals , plants are considered the best production organisms , whereas for specialty polymers , fermentation may be the preferred production technology . Fig. 2Cyanophycin production in planta or by fermentation . Gray square : raw material costs , filled square : fermentation costs , open square : recovery and purification costs Assuming that a typical fermentative production of a bulk product , such as lysine , citric acid or glutamic acid costs about $ 1,500 per ton and that these costs consist of : $ 500 for the raw materials , $ 500 for the fermentation process , and $ 500 for recovery and purification . The advantage of producing in plants is that both the raw material costs and the fermentation costs can almost be neglected . On the other hand , recovery costs could be much higher . For the sake of the reasoning , it is assumed that recovery costs for cyanophycin production in plants will be the same as in the case of fermented production , i.e. , $ 500 per ton . In case of a fermentation process , a typical production volume for a company would be in the order of magnitude of 100,000 tons/year . The turnover would then be 150 million euros per year -LRB- i.e. , $ 1,500 per ton × 100,000 tons/year -RRB- . In the case of production in plants , as raw material and fermentation costs are negligible , cost savings would be 100 million euros per year . This would be the maximum advantage , as this calculation does not include any costs for the production of the crop nor any additional costs for biorefining of the crop and treatment of side products . A similar reasoning would bring a maximum advantage for a product with a typical company production volume of 20,000 tons per year , such as the case with a medium-sized monomer . In this case , cost savings per ton would be higher , but as the volume is much smaller , the maximum advantage could be around 70 million euros . For specialty products with a volume of 300 tons per year -LRB- enzymes -RRB- , a maximum advantage is estimated to be in the order of 5 -- 10 million euros . For pharmaceutical production with a volume of 10 tons per year , the advantage would be around 0.5 -- 1.5 million euros , as the volume would be small and production costs would mainly be ascribed to recovery . Other plant side streams In addition to the possible exploitation of Protamylasse ™ , a large number of other plant side streams may be used for cyanophycin production , including grass juice , which remains after protein extraction or beet or cane molasses remaining after sugar extraction . It is foreseen that with the production of biodiesel and bioethanol , large volumes of side streams will become available , all containing major protein quantities . Such streams will include dried distillers grain and solubles -LRB- DDGS -RRB- from corn and wheat , press cake from palm oil and rape seed oil . These streams all have in common a very low cost price , which implies that when used as a microbial growth medium , a major contribution to fermentation process costs -LRB- about 30 % -RRB- can be eliminated . However , it is important to note that none of any waste stream -LRB- to be -RRB- considered has exactly the same chemical or element composition as the production organism and its product -LRB- s -RRB- , including , e.g. , CO2 . This implies that in each fermentation process to be developed using plant waste streams as substrates , limiting components should be supplemented . For the identification of such limiting components , fermentation test runs should be performed , preferably supported by statistic analyses such as elementary mode analysis -LRB- Diniz et al. 2006 -RRB- . Only after performing such exercises can reliable estimates of integral project costs be obtained . Cyanophycin-derived bulk chemicals Cyanophycin can be hydrolyzed to its constituent amino acids , aspartic acid , and arginine . These amino acids may be utilized directly in food and pharmaceutical applications . However , based on the chemical structure of these amino acids and the presence of functionalized nitrogen-containing groups , it is possible to anticipate their conversion to a number of industrial chemicals , including : Arginine may be converted to 1,4-butanediamine . 1,4-diaminobutane , derived from petrochemistry , is currently used as a co-monomer in the production of nylon-4,6 . The volume of production is not known , but is estimated to be in the range of 10,000 s tons per year with a value of > $ 1,600 per ton.Conversion of aspartic acid to acrylonitrile is also envisaged . Using current petrochemical technology , the current worldwide market volume of acrylonitrile is 2.7 -- 5 × 106 tons per year and a price of about $ 800 -- 1,000 per ton . Other chemicals which could be obtained from cyanophycin but are currently prepared from fossil resources include , e.g. , 1,4-butanediol and urea . The production of cyanophycin by plants will drastically reduce the cost price , potentially below $ 1,000 per ton . This will enable the production of functionalized bulk chemicals such as 1,4-diaminobutane , and possibly , also acrylonitrile . The transition from mineral oil to plant-based precursor production has a considerable impact . The use of ammonia for the incorporation of nitrogen into chemicals is very important , but also very energy-intensive . Therefore , if the incorporation of nitrogen can be realized in systems based on plant -LRB- rest -RRB- streams in the form of protein or amino acid precursors , then this will yield considerable energy savings . Cyanophycin-based biopolymers Poly-aspartic acid is derived from cyanophycin after the hydrolytic removal of arginine . This polymer has properties that are very similar to poly-acrylic acid . The cost price of this polymer can be set on 1,000 $ / ton in the cost calculations for arginine here above . As the volumes of these products will be similar , only 1,000 tons/year will be manufactured . Higher market prices might be obtained for special applications in food and/or pharmaceutical applications . This might change the cost structure of arginine in a way that market volumes might double or triple . Cyanophycin as such might have applications as a polymer . Furthermore , derivatives obtained by enzymatic/genetic and/or chemical modifications might give valuable properties . Without thorough investigations , we can not anticipate on the value of these polymers . Utilization in this area can be expected after 10 to 12 years after the beginning of the proposed research approaches for research and development . Possible cyanophycin modifications , applications for bulk chemicals and for polymers By incorporating other amino acids , different types of polymers can be made . So far , several cyanophycins have been produced in recombinant strains of E. coli up to 50 % dry weight . However , especially for health care medical and food packaging applications , E. coli may not be the best commercial production organism . Therefore , the development of alternative food-grade production organisms is also one of the objectives of the current activities . One suitable candidate may be the bakers yeast Saccharomyces cerevisiae -LRB- and others : see above -RRB- . In addition , being stable at a pH between 3 and 9 , cyanophycin can also be hydrolyzed in concentrated volumes into its pure components , arginine and aspartic acid . This would make the whole process a novel biological extraction procedure for the selected amino acids . Outlook Given the anticipated cost development for fossil energy carriers and environmental regulations , the chemical industry is facing increasing financial pressure and is thus looking for possibilities to broach new resources as a basis for polymer production . Important considerations in this search are to lower energy costs and prices of raw material and to develop cheaper and more sustainable production processes . Unlike poly-γ-glutamic acid and poly-ɛ-lysine , cyanophycin has not been commercialized yet . Cyanophycin can be broken down into the individual amino acids that can be used as building blocks in various industrial processes . Because of its homogeneous structure and composition , the cyanophycin polymer and its derivatives also appear to be good candidates as starting materials for the production of nitrogen-rich commodity products , which are based on nitrogen-rich chemicals , like , for example , nylons . For example , for poly -LRB- aspartic acid -RRB- which is the polymer backbone of cyanophycin , various applications have been developed ranging from water-softening or detergent applications to applications in the paper , building material , petroleum or leather industry , in cosmetics , as well as many dispersant applications . Cyanophycin can be chemically converted into a polymer with a reduced arginine content , which might be used like poly-aspartic acid as a biodegradable substitute for synthetic polyacrylate in various technical processes -LRB- Schwamborn 1998 -RRB- . Thus , cyanophycin may find applications in cyanophycin-derived bulk chemicals and in cyanophycin-based biopolymers . It can be expected that economical activities can be developed within the following areas , such as : fermentation industry , biopolymer production , processing , modification and product development -LRB- also for medical technology -RRB- , packaging industry , food and feed supplementation industries and , last but not least , state-of-the-art technology -LRB- which , in turn , will attract additional financial sources and economical activities -RRB- . It should be emphasized , however , that the mentioned applications are still uncertain and that these are so far only potential applications . On the one hand , this development will lead to the substitution of chemicals that are now produced at the cost of fossil raw materials , such as oil . As oil may be depleted in about 50 years , and as there seems to be a correlation of the use of fossil raw materials with climate changes , it is essential to develop alternatives . The anticipated alternatives can be produced by fermentation and , in principle , by plant production systems , and so , giving a new economic and knowledge intensive value to the fermentation industry and/or to agriculture . On the other hand , novel types of polymers will be developed that do not simply replace existing applications but that will enter novel product markets . Elements of the contents of this paper are included in a dedicated patent application -LRB- Elbahloul et al. 2006 -RRB- .

O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

biorefinery
protamylasse
cyanophycin
bulk chemicals

plant waste, rest stream
non-ribosomal
n-functionality


Simultaneous

expression

of

and

genes

of

three

germ

layers

in

single

cell-derived

multipotent

adult

progenitor

cells

Future

application

of

adult

stem

cells

in

clinical

therapies

largely

depends

on

the

successful

isolation

of

homogeneous

stem

cells

with

high

plasticity

Multipotent

adult

progenitor

cells

-LRB-

MAPCs

-RRB-

are

thought

to

be

a

more

primitive

stem

cell

population

capable

of

extensive

in

vitro

proliferation

with

no

senescence

or

loss

of

differentiation

capability

The

present

study

was

aimed

to

find

a

less

complicated

and

more

economical

protocol

for

obtaining

single

cell-derived

MAPCs

and

understand

the

molecule

mechanism

of

multi-lineage

differentiation

of

MAPCs

We

successfully

obtained

a

comparatively

homogeneous

population

of

MAPCs

and

confirmed

that

single

cell-derived

MAPCs

were

able

to

transcribe

and

genes

of

three

germ

layers

simultaneously

and

differentiate

into

multiple

lineages

Our

observations

suggest

that

single

cell-derived

MAPCs

under

appropriate

circumstances

could

maintain

not

only

characteristics

of

stem

cells

but

multi-lineage

differentiation

potential

through

quantitative

modulation

of

corresponding

regulating

gene

expression

rather

than

switching

on

expression

of

specific

genes

Introduction

Bone

marrow

mesenchymal

stem

cells

-LRB-

MSCs

-RRB-

are

the

most-studied

adult

stem

cells

and

have

been

widely

used

owing

to

their

advantages

in

autografting

and

multi-lineage

differentiation

potential

which

is

also

referred

to

as

the

plasticity

of

stem

cells

-LSB-

-RSB-

However

some

studies

in

recent

years

indicated

that

MSCs

might

have

no

plasticity

or

only

have

a

limited

plasticity

-LSB-

-RSB-

and

that

the

in

vivo

multi-lineage

differentiation

of

MSCs

might

be

a

result

of

their

fusion

with

the

host

cells

-LSB-

-RSB-

That

is

why

recently

people

have

been

suspicious

about

the

plasticity

of

MSCs

Some

researchers

think

that

the

previously

separated

MSCs

are

actually

a

mixed

cell

population

containing

several

kinds

of

stem

cells

with

different

differentiation

potentials

-LSB-

-RSB-

Therefore

how

to

isolate

homogeneous

MSCs

with

high

plasticity

becomes

a

ready

task

of

researchers

Isolation

of

MAPCs

from

adult

bone

marrows

was

firstly

reported

by

Verfaillie

's

team

-LSB-

-RSB-

Compared

with

MSCs

these

cells

were

thought

to

be

a

more

primitive

cell

population

with

partially

overlapping

ontogeny

Moreover

MAPCs

have

been

proven

to

be

capable

of

extensive

in

vitro

proliferation

with

no

senescence

or

loss

of

differentiation

capability

A

few

researchers

held

that

when

embryonic

stem

cells

differentiate

into

different

somatic

germ

layers

and

tissue

cells

some

pluripotent

cells

remain

in

specific

tissues

for

future

use

-LSB-

-RSB-

judging

from

their

differentiation

property

MAPCs

should

belong

to

this

type

of

cell

Although

the

above

hypothesis

needs

to

be

further

verified

this

subpopulation

of

pluripotent

stem

cells

provides

a

new

thought

and

may

serve

as

a

new

cell

source

for

tissue

engineering

Based

on

the

above

understanding

we

started

our

experiment

with

isolation

of

MAPCs

from

bone

marrow

of

adult

rats

and

then

fully

studied

their

differentiation

potentials

MAPCs

were

isolated

using

a

modified

protocol

of

Verfaillie

team

-LSB-

-RSB-

By

screening

for

cells

highly

expressing

from

clone-like

clusters

we

successfully

obtained

a

comparatively

homogeneous

population

of

MAPCs

with

multi-lineage

differentiation

potential

The

present

study

was

aimed

to

find

a

less

complicated

and

more

economical

protocol

for

obtaining

single

cell-derived

MAPCs

and

understand

the

molecule

mechanism

of

multi-lineage

differentiation

of

MAPCs

Our

observations

confirmed

that

single

cell-derived

MAPCs

were

able

to

transcribe

and

genes

of

three

germ

layers

simultaneously

and

differentiate

into

multiple

lineages

Materials

and

methods

Culture

and

clone

expansion

of

MAPCs

Bone

marrow

was

obtained

from

male

Sprague

Dawley

-LRB-

SD

-RRB-

rats

With

fat

and

red

blood

cells

removed

the

remaining

cells

were

seeded

at

a

density

of

in

mm

dishes

coated

with

ng/ml

fibronectin

-LRB-

FN

Sigma-Aldrich

USA

-RRB-

and

incubated

at

°C

with

in

a

fully

humidified

atmosphere

The

expansion

medium

of

MAPCs

consisted

of

DMEM-LG

-LRB-

Gibco-Invitrogen

USA

-RRB-

and

-LRB-

Sigma

-RRB-

with

fetal

bovine

serum

-LRB-

FBS

Gibco-Invitrogen

-RRB-

insulin/transferring/selenium

-LRB-

ITS

-RRB-

linoleic

acid

bovine

serum

albumin

-LRB-

LA-BSA

-RRB-

dexamethasone

-LRB-

Dex

-RRB-

ascorbic

acid

-LRB-

all

from

Sigma-Aldrich

-RRB-

U/ml

penicillin

U/ml

streptomycin

-LRB-

Gibco-Invitrogen

-RRB-

ng/ml

EGF

-LRB-

Sigma-Aldrich

-RRB-

ng/ml

PDGF-BB

-LRB-

R&D

Systems

-RRB-

and

ng/ml

LIF

-LRB-

Chemicon

International

USA

-RRB-

The

nonadherent

cells

were

removed

by

medium

change

at

h

and

every

days

thereafter

until

confluence

was

reached

Cells

were

passaged

once

with

trypsin-ethylenediaminetetraacetic

acid

-LRB-

EDTA

Gibco-Invitrogen

-RRB-

and

the

passage

-LRB-

-RRB-

cells

were

replated

at

a

dilution

under

the

same

culture

condition

After

being

passaged

for

at

least

four

generations

-LRB-

-RRB-

cells

were

seeded

on

a

plate

coated

with

ng/ml

FN

at

a

density

of

cell/well

by

limited-dilution

method

in

expansion

medium

The

wells

containing

more

than

one

cell

under

a

phase-contrast

microscope

were

discarded

When

cells

grew

to

confluence

they

were

pipetted

from

one

well

seeded

to

one

of

the

wells

and

then

serially

reseeded

to

a

six-well

plate

mm

dish

and

mm

dish

in

the

presence

of

trypsin-EDTA

When

cells

reached

a

density

of

in

mm

dish

they

were

replated

at

a

dilution

and

the

fastest-growing

clone

was

selected

after

being

passaged

for

times

Total

RNA

isolation

and

RT-PCR

analysis

Total

RNA

was

extracted

from

the

clonal

cells

using

RNA

Mini

kit

-LRB-

Huashun

China

-RRB-

First-strand

cDNA

was

synthesized

with

total

RNA

using

a

mixture

of

oligo

-LRB-

dT

-RRB-

and

random

hexamers

-LRB-

Roche

Diagnostics

Germany

-RRB-

primers

with

AMV

-LRB-

Promega

USA

-RRB-

and

Superscript

II

RNase

H

Reverse

Transcriptase

-LRB-

Invitrogen

Germany

-RRB-

Polymerase

chain

reaction

-LRB-

PCR

-RRB-

was

conducted

with

approximately

ng

cDNA

for

amplification

of

the

following

marker

genes

pluripotent

marker

endodermal

marker

-LRB-

CYP

-RRB-

mesodermal

marker

and

and

ectodermal

marker

N-methyl-d-aspartic

acid

-LRB-

NMDA

-RRB-

glutamate

binding

protein

The

housekeeping

gene

encoding

was

used

as

an

internal

control

The

PCR

primers

are

listed

in

Table

After

initial

denaturation

at

°C

for

min

PCR

amplification

was

performed

with

Sawady

Taq-DNAPolymerase

-LRB-

Takara

Bio

Japan

-RRB-

at

°C

for

s

followed

by

cycles

with

each

cycle

consisting

of

annealing

at

°C

for

s

and

a

final

extension

at

°C

for

s.

PCR

products

were

subjected

to

agarose

gel

electrophoresis

and

the

bands

were

visualized

by

ethidium

bromide

and

photographed

with

Chemi

Doc

XRS

-LRB-

Bio-Rad

Laboratories

Fudan

China

-RRB-

Each

experiment

was

performed

in

triplicate

Table

marker

genes

of

three

germ

layers

-LSB-

-RSB-

GenesPrimers

-LRB-

-RRB-

Product

size

-LRB-

bp

-RRB-

CGCCAGAAGGGGAAAAGA

Reverse

CYP

ATCCTGACCGCTACCTACA

Reverse

GGCTTCCCTGAAATCCTA461LiverEndodermSM22αForward

GTCCACAAACGACCAAGC

Reverse

TGAGCCCACCAGAAAAGG

Reverse

TCCCCACAAGGCTATCCA

Reverse

CAAGAACGAAAGTCGGAGGTTCG

Reverse

Immunocytofluorescence

and

fluorescence-activated

cell

sorter

analysis

Clonal

cultured

MAPCs

were

fixed

with

paraformaldehyde

for

min

at

room

temperature

After

being

blocked

with

phosphate-buffered

saline

-LRB-

PBS

-RRB-

containing

BSA

cells

were

permeabilized

with

for

min

Slides

were

incubated

sequentially

overnight

at

°C

with

the

following

primary

antibodies

-LRB-

Santa

Cruz

USA

-RRB-

Vimentin

-LRB-

DAKO

USA

-RRB-

α-SMA

-LRB-

Boster

Biotechnology

China

-RRB-

and

-LRB-

Santa

Cruz

USA

-RRB-

then

cells

were

incubated

for

min

at

room

temperature

with

fluorescein

isothiocyanate

-LRB-

FITC

-RRB-

or

IgG

secondary

antibody

-LRB-

Jackson

-RRB-

stained

with

DAPI

-LRB-

Sigma

Aldrich

-RRB-

and

observed

under

a

fluorescence

microscope

-LRB-

Olympus

Japan

-RRB-

For

fluorescence-activated

cell

sorter

-LRB-

FACS

-RRB-

analysis

cells

pelleted

were

incubated

on

ice

for

min

with

FITC

or

PE-conjugated

monoclonal

antibodies

against

-LRB-

Becton

Dickinson

CA

-RRB-

and

MHC-I

-LRB-

Abcam

UK

-RRB-

After

two

washes

with

cold

PBS

the

labeled

cells

were

analyzed

by

a

FACStar

flow

cytometer

-LRB-

Becton

Dickinson

CA

USA

-RRB-

In

vitro

differentiation

of

MAPCs

To

induce

differentiation

clonal

cultured

MAPCs

were

cultured

in

the

media

containing

no

FCS

EGF

PDGF-BB

or

LIF

unless

otherwise

indicated

All

the

experiments

were

repeated

at

least

three

times

Adipogenic

differentiation

of

MAPCs

MAPCs

were

seeded

at

a

density

of

in

six-well

plates

allowed

to

grow

confluence

and

cultured

for

another

days

Subsequently

cells

were

allowed

to

differentiate

for

days

in

the

expansion

medium

of

MAPCs

containing

mM

-LRB-

Serva

Electrophoresis

Germany

-RRB-

nM

Dex

μM

indomethacin

and

μg/ml

insulin

At

the

end

of

the

culture

cells

were

fixed

with

formaldehyde

-LRB-

Shenggong

China

-RRB-

for

min

and

were

stained

with

fresh

Oil

Red

O

-LRB-

Sigma-Aldrich

-RRB-

for

h.

Osteogenic

differentiation

of

MAPCs

MAPCs

at

a

density

of

were

induced

with

osteogenic

medium

containing

Dex

M

ascorbic

acid

and

M

glycerolphosphate

for

weeks

NBT/BCIP

staining

was

used

to

analyze

alkaline

phosphatase

-LRB-

AKP

all

from

Sigma-Aldrich

-RRB-

expression

during

MAPCs

differentiation

Meanwhile

cells

were

immunocytochemically

stained

with

osteopontin

to

detect

osteogenic

differentiation

of

MAPCs

Neuroectodermal

differentiation

of

MAPCs

MAPCs

were

plated

at

a

density

of

on

FN

in

a

basal

medium

containing

ng/ml

basic

fibroblast

growth

factor

-LRB-

bFGF

R&D

Systems

-RRB-

The

cells

were

allowed

to

differentiate

for

days

and

then

immunofluorescently

stained

for

GFAP

-LRB-

-RRB-

and

TAU

-LRB-

both

from

Boster

Biotechnology

China

-RRB-

Hepatocyte

differentiation

of

MAPCs

MAPCs

were

plated

at

on

plastic

chamber

slides

coated

with

Matrigel

in

a

basal

medium

supplemented

with

ng/ml

and

ng/ml

HGF

-LRB-

both

from

R&D

Systems

-RRB-

After

days

immunofluorescent

staining

for

albumin

-LRB-

DAKO

USA

-RRB-

was

performed

as

described

above

Results

Stable

in

vitro

proliferation

and

expansion

of

clonal

cultured

MAPCs

On

the

third

day

of

primary

culture

very

few

triangle-like

cells

adhered

to

the

culture

surface

under

a

phase

contrast

microscope

coexisting

with

plenty

of

hematopoietic

cells

which

were

round

in

shape

and

small

in

size

shining

under

the

microscope

-LRB-

Fig.

-RRB-

During

the

fourth

to

seventh

day

of

culture

the

triangle-like

cells

gradually

increased

and

had

a

typical

colony

growth

with

an

obvious

three-dimensional

appearance

and

growth

circle

The

colony

cells

grew

confluent

and

became

morphologically

homogenous

after

days

-LRB-

Fig.

-RRB-

After

treatment

with

trypsin

the

cultured

cells

no

longer

grew

in

clusters

but

in

a

homogeneous

manner

-LRB-

Fig.

-RRB-

Fig.

of

isolated

MAPCs

a

Phase-contrast

micrographs

of

MAPCs

at

day

-LRB-

-RRB-

day

-LRB-

-RRB-

of

primary

culture

-LRB-

-RRB-

and

at

day

-LRB-

-RRB-

after

passages

-LRB-

original

magnification

-RRB-

b

MAPCs

cloned

by

limiting

dilution

A

single

cell

adherent

to

the

plate

in

one

well

proliferated

in

the

primary

culture

for

days

-LRB-

-RRB-

days

-LRB-

-RRB-

and

days

-LRB-

original

magnification

-RRB-

Single-cell

clonal

culture

was

performed

by

infinite

dilution

technique

Thirty

percent

of

single

cell-derived

MAPCs

formed

larger

clones

during

culture

-LRB-

Fig.

-RRB-

and

six

cell

clones

-LRB-

and

-RRB-

were

obtained

in

the

following

expansion

When

being

cultured

at

an

extremely

low

density

-LRB-

-RRB-

this

six-cell

population

still

demonstrated

strong

capability

of

proliferation

with

the

doubling

time

being

about

h.

cells

still

possessed

the

typical

properties

of

MAPCs

polygonal-shaped

with

larger

nuclei

vacuoles

around

the

nuclei

and

sparse

cytoplasm

-LRB-

Fig.

-RRB-

Expression

of

and

genes

of

three

germ

layers

in

single

cell-derived

MAPCs

The

expression

of

self-renewal

gene

was

detected

in

the

six

cell

clones

after

being

passaged

for

more

than

times

All

the

six

cell

clones

were

found

to

have

different

levels

of

expression

-LRB-

Fig.

-RRB-

Specific

markers

of

the

three

germ

layers

were

detected

in

the

three

cell

clones

-LRB-

and

-RRB-

which

strongly

expressed

The

specific

markers

included

-LRB-

present

in

the

liver

of

adults

-RRB-

for

endoderm

and

-LRB-

present

in

the

skeleton

muscle

the

heart

of

adults

-RRB-

for

mesoderm

and

NMDA

-LRB-

present

in

the

brain

of

adults

-RRB-

for

neuroectoderm

As

for

the

markers

for

the

three

germ

layers

clone

had

a

very

weak

expression

of

and

all

the

rest

clones

had

high

expression

of

their

corresponding

markers

-LRB-

Fig.

-RRB-

especially

for

single

cell-derived

Therefore

we

subjected

single

cell-derived

to

further

experiments

Fig.

analysis

of

expression

of

and

specific

markers

for

three

somatic

germ

layers

a

was

highly

expressed

in

six

cell

clones

b

CYP

and

NMDA

were

positive

in

three

cell

clones

NEG

Negative

controls

POS

positive

controls

for

CYP

-LRB-

fetal

liver

-RRB-

-LRB-

fetal

smooth

muscles

-RRB-

-LRB-

fetal

heart

-RRB-

and

NMDA

and

-LRB-

fetal

brain

tissues

n

-RRB-

Identification

of

molecular

markers

in

single

cell-derived

Single

cell-derived

was

chosen

for

immunofluorescence

assay

It

was

found

that

it

expressed

-LRB-

a

surface

antigen

of

stem

cells

-RRB-

Vimentin

-LRB-

a

stromal

cell

skeleton

protein

-RRB-

α-SMA

-LRB-

a

marker

of

early

smooth

muscle

cells

-RRB-

and

-LRB-

a

marker

for

more

primitive

stem

cells

Fig.

-RRB-

suggesting

that

it

still

harbored

the

surface

antigen

of

stem

cells

the

stromal

cell

skeleton

protein

and

the

marker

of

early

smooth

muscle

cells

Fig.

Expression

of

cell

markers

in

the

cloned

MAPCs

Immunofluorescence

analyses

of

single

cell-derived

were

positive

for

Vimentin

-LRB-

with

nuclear

counterstained

by

-RRB-

α-SMA

and

-LRB-

with

nuclear

counterstained

by

DAPI

all

original

magnification

-RRB-

b

FACS

analysis

MAPCs

were

negative

for

and

MHC-I

antigen

The

results

of

flow

cytometry

showed

that

clone

did

not

express

or

-LRB-

markers

for

hematopoietic

stem

cells

-RRB-

For

the

expression

of

the

stromal

cell

marker

and

MHC

I

the

major

histocompatibility

complex

class

I

antigens

MAPCs

were

all

negative

-LRB-

Fig.

-RRB-

Differentiation

potential

of

single

cell-derived

into

three

somatic

germ

layers

consisting

of

osteoblasts

adipocytes

neurons

and

hepatocytes

Single

cell-derived

MAPCs

were

seen

to

have

strong

refractive

oil

drops

around

the

nuclei

on

as

early

as

the

eighth

day

under

the

adipogenic

condition

and

the

oil

drops

gradually

increased

and

fused

into

larger

ones

with

the

prolongation

of

the

induction

time

-LRB-

Fig.

-RRB-

Twenty-one

days

after

induction

with

osteoblastic

medium

AKP

staining

of

MAPCs

showed

purple-bluish

deposits

in

the

cytoplasm

-LRB-

Fig.

-RRB-

and

immunocytochemistry

staining

was

positive

for

osteopontin

-LRB-

Fig.

-RRB-

Fourteen

days

after

MAPCs

were

directed

to

differentiate

into

neuronal

cells

in

vitro

cell

morphology

underwent

obvious

changes

the

cytoplasm

was

shrinking

and

bipolar

or

multipolar

processes

were

seen

growing

out

from

the

spindle-shaped

cells

-LRB-

Fig.

-RRB-

Immunofluorescence

staining

showed

the

presence

of

Tau

protein

the

specific

marker

of

neurons

-LRB-

Fig.

-RRB-

and

GFAP

the

specific

marker

of

glia

cells

-LRB-

Fig.

-RRB-

in

the

cells

after

neurogenic

induction

Fourteen

days

after

MAPCs

were

programmed

to

differentiate

into

hepatocytes

the

spindle-shaped

cells

-LRB-

Fig.

-RRB-

turned

into

round

or

oval

shape

and

became

wider

and

larger

with

narrowed

intercellular

junctions

and

appearance

of

cell

patches

-LRB-

Fig.

-RRB-

Immunofluorescence

analysis

showed

that

the

induced

cells

expressed

albumin

the

marker

of

hepatocytes

-LRB-

Fig.

-RRB-

The

above

findings

suggested

that

clonal

cultured

MAPCs

were

able

to

differentiate

into

osteoblasts

adipocytes

neuronal

cells

and

hepatocytes

under

proper

culture

conditions

Fig.

of

cloned

MAPCs

into

three

somatic

germ

layers

a

Differentiation

into

adipogenic-like

cells

and

osteogenic-like

cells

differentiated

MAPCs

were

positive

in

oil

red

O

staining

counterstained

with

hematoxylin

and

differentiated

MAPCs

were

positive

for

alkaline

phosphatases

-LRB-

AKP

staining

-RRB-

and

osteopontin

-LRB-

immunocytochemical

staining

-RRB-

b

Differentiation

into

neuronal-like

cells

Under

phase

microscope

apparent

branches

were

observed

to

grow

out

from

the

spindle-shaped

cells

and

Immunofluorescence

staining

showed

the

presence

of

GFAP

and

Tau

protein

c

Differentiation

into

hepatocytes-like

cells

DAPI

counterstaining

showed

that

the

MAPCs

untreated

with

hepatogenic

medium

retained

the

spindle

shape

MAPCs

turned

from

spindle

into

round

or

oval

after

hepatogenic

induction

Immunofluorescence

analysis

showed

expression

of

albumin

in

differentiated

MAPCs

-LRB-

all

original

magnification

-RRB-

Discussion

There

is

increasing

evidence

that

isolation

strategies

of

stem

cells

differ

for

different

source

tissues

and

different

stem

cell

types

even

though

the

tissue

is

from

the

same

source

different

isolating

protocols

may

obtain

different

stem

cell

types

-LSB-

-RSB-

For

example

stromal

stem

cells

screened

out

with

different

molecular

markers

demonstrated

certain

varieties

in

growth

and

plasticity

and

were

therefore

given

different

names

including

MSCs

-LSB-

-RSB-

MAPCs

-LSB-

-RSB-

MPCs

-LRB-

mesodermal

progenitor

cells

-RRB-

-LSB-

-RSB-

and

marrow-isolated

adult

multi-lineage

inducible

cells

-LRB-

MIAMI

-RRB-

-LSB-

-RSB-

In

the

present

study

we

used

culture

conditions

similar

to

those

of

Verfaillie

's

team

and

successfully

obtained

a

comparatively

homogeneous

population

of

MAPCs

through

the

following

protocol

isolating

mononuclear

cells

from

rat

total

bone

marrow

by

direct

adherence

eliminating

non-adherent

cells

after

three

to

four

passages

subjecting

the

remaining

cells

to

single

cell

clonal

culture

by

limiting

dilution

technique

plating

the

cells

at

a

low

density

of

for

further

passage

and

finally

screening

for

clone-like

cells

We

found

that

single

cell-derived

MAPCs

did

not

express

and

MHC-I

which

was

consistent

with

the

results

of

Verfaillie

's

team

who

found

that

MAPCs

were

able

to

reserve

their

pluripotency

when

plated

at

a

low

density

and

unable

to

do

so

when

plated

at

a

much

higher

density

With

pluripotency

decreasing

and

MHC

I

turned

from

negative

to

strongly

positive

in

MAPCs

and

therefore

could

serve

as

a

marker

for

the

loss

of

pluripotency

It

should

be

notified

that

our

protocol

for

MAPC

isolation

which

involved

direct

adherence

passage

selection

single

cell

clonal

culture

and

low

density

passage

is

less

complicated

and

more

economical

than

that

of

Verfaillie

's

team

who

employed

the

technique

of

immunomagnetic

microbeads

The

reliability

of

our

protocol

is

verified

by

its

results

Single

cell-derived

MAPCs

retained

their

multi-potential

differentiation

capability

even

after

being

passaged

for

generations

and

could

not

only

give

rise

to

adipocytes

and

osteoblasts

-LRB-

mesoderm

-RRB-

but

to

neurons

glia

cells

-LRB-

ectoderm

-RRB-

and

hepatocytes

-LRB-

endoderm

-RRB-

It

has

been

demonstrated

that

the

isolation

method

and

cultivation

condition

are

vital

to

the

isolation

of

bone

marrow

stem

cells

and

the

maintenance

of

stem

cell

property

-LSB-

-RSB-

The

latest

research

showed

that

in

vitro

culture

of

human

MSCs

with

autologous

serum

is

superior

to

FBS

for

the

amplification

and

for

maintaining

the

pluripotency

of

MSCs

-LSB-

-RSB-

and

the

original

culture

strategy

might

lead

to

the

loss

of

differentiation

potential

of

MSCs

Moreover

some

researchers

found

that

serum-free

culture

could

upgrade

the

differentiation

potential

of

MSCs

-LSB-

-RSB-

So

recently

researchers

have

made

persistent

efforts

to

optimize

the

isolation

method

of

MSCs

For

example

people

have

isolated

another

population

of

pluripotent

cells

called

marrow-isolated

adult

multilineage

inducible

-LRB-

MIAMI

-RRB-

cells

from

human

bone

marrow

by

plating

whole

bone

marrow

cells

initially

in

media

containing

FBS

and

subsequently

maintaining

them

in

the

medium

containing

FBS

in

fibronectin-coated

dishes

These

cultures

were

then

maintained

in

hypoxic

conditions

at

-LSB-

-RSB-

MIAMI

cells

still

expressed

telomerase

and

even

after

being

expanded

for

more

than

population

doublings

In

the

present

study

our

medium

contained

a

low

level

of

serum

but

was

supplemented

with

growth

factor

and

LIF

which

promoted

the

proliferation

of

MAPCs

to

some

extent

and

retained

their

dedifferentiated

state

Single

cell

clonal

culture

and

low

density

passage

may

help

to

isolate

a

more

homogenous

cell

population

with

strong

proliferation

capability

As

the

marker

for

pluripotent

stem

cells

is

closely

linked

with

the

pluripotency

of

MAPCs

-LSB-

-RSB-

Our

results

showed

that

single

cell-derived

MAPCs

not

only

highly

expressed

but

expressed

the

early

markers

of

endoderm

mesoderm

and

ectoderm

-LSB-

-RSB-

-LSB-

-RSB-

and

NMDA

-LSB-

-RSB-

which

is

the

molecular

basis

for

their

differentiation

into

three

germ

layers

Woodbury

's

team

-LSB-

-RSB-

demonstrated

that

MSCs

expressed

germline

ectodermal

endodermal

and

mesodermal

genes

before

neurogenesis

The

present

research

extended

their

observations

Our

observations

confirm

that

single

cell-derived

MAPCs

transcribe

and

genes

of

the

three

germ

layers

simultaneously

and

could

differentiate

into

multiple

lineages

It

may

be

concluded

that

under

appropriate

circumstances

single

cell-derived

MAPCs

can

maintain

not

only

characteristics

of

stem

cells

but

multi-lineage

differentiation

potential

which

has

expressed

corresponding

regulating

genes

and

single

cell-derived

MAPCs

could

differentiate

into

multiple

lineages

through

quantitative

modulation

of

regulating

gene

expression

rather

than

switching

on

expression

of

specific

genes

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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