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Growth factor regulation of proliferation and survival of multipotential stromal cells

Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of sufficient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to influence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be beneficial that the growth factor does not induce differentiation at an early stage since the number of early-differentiating progenitors would be very low. The present review looks at the effect of and downstream signaling of various growth factors on proliferation and survival in MSCs.

Introduction
Th ere is a growing need for new ways to regenerate and repair injuries in organs. Most organs have limited inherent regenerative capacity, with scarring preventing full organ functioning. For instance, myocardial infarc tion is often followed by the myocardium being replaced with noncontractile scar tissue, which can further result in congestive heart failure [1,2]. In the case of bone, metabolic disorders such as osteoporosis cause abnormal bone loss and traumatic injuries lead to large lesions, which are incapable of self-regeneration. Th e search has therefore turned to novel ways to stimulate the original organogenic process and regenerate normal tissue.
Use of multipotential stromal cells (MSCs) or mesenchymal stem cells to reconstruct tissue looks extremely promising due to their trans-diff erentiation potential. MSCs have the ability to form cells of the connective tissue, muscle, heart, blood vessels and nerves [3][4][5][6]. Th ese cells are easy to isolate from almost all individuals; these cells are relatively safe as they rarely form teratomas [7]. In addition, these stromal cells off er several advantages over conventional therapy. MSCs respond to their environment by diff erentiating into the needed lineages. Th ese cells will therefore grow, remodel and adapt to changes in tissue functions over time. As MSCs derive from bone marrow, these can be isolated from most adults with the potential of autologous transplantation, not requiring immunosuppressive agents. Th is procedure is in contrast to traditional methods of transplantation that lead to infection, immune rejection or simply not enough material for large-scale grafts.
Preclinical animal studies have shown promise of using MSCs for tissue regeneration. Application of these cells has led to the formation of bone, the regain of ventricular function, and the restoration of renal tubular function in rodents [8][9][10]. Mice rendered paraplegic by spinal cord injury have recovered on MSC treatment [11,12]. Th e use of MSCs is limited, however, by their scarceness in the bone marrow, as they constitute only 0.001 to 0.01% of the bone marrow population. Since regeneration of large tissues requires around 10 7 to 10 8 MSCs [13], there exists a need for MSCs to be expanded prior to tissue regeneration. In culture conditions, however, proliferation of these cells is highly inconsistent -which subsequently impacts diff erentiation.
Even if the desired cell numbers are obtained, there is another hurdle to be crossed before diff erentiation of Abstract Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of suffi cient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to infl uence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be benefi cial that the growth factor does not induce diff erentiation at an early stage since the number of early-diff erentiating progenitors would be very low. The present review looks at the eff ect of and downstream signaling of various growth factors on proliferation and survival in MSCs.
MSCs begins: the incorporation of MSCs into regenerating tissue. MSCs applied to regenerate porcine hearts following an infarct display only 5% survival in a 14-day period [14][15][16]. Similar results are seen on implantation of MSCs into mouse hearts with infarcts. While MSCs injected into nonischemic hearts survive better initially, one cannot fi nd viable cells after 4 weeks [17][18][19]. In rat brains with cerebral artery occlusion, allogenic and human MSC transplants also show very low survival [20]. Th e failure of these cells to regenerate tissue may thus simply be that they do not survive to contribute to the new tissue.
Th e reasoning behind low incorporation of MSCs may be attributed to poor viability of cells caused by ischemia, anoikis, loss of trophic factors or infl ammation at the graft site [21]. To test whether nonspecifi c infl ammation induced death of MSCs, Griffi th and colleagues subjected human MSCs to various nonspecifi c infl ammatory cytokines in vitro. MSCs were extremely susceptible to FasL-induced cell death and also died in the presence of TRAIL [22]. Not only does the infl ammatory response challenge transplanted MSCs -these cells are also considered for regeneration of tissues with harsh microenvironments. For example, when used to regenerate cartilage, MSCs need to adapt to an avascular, low oxygen concentration and a low pH microenvironment characteristic of chondrocytes [23]. Taken together, the microenvironment in which MSCs are delivered, the presence of infl am mation, or the loss of trophic factors may play a role in maintaining a proliferating MSC population at the graft site ( Figure 1).
On the contrary, there are reports of improved healing on MSC delivery. In a rat cerebral occlusion model there is signifi cant recovery in motor neuron function after MSC transplantation. Despite the low survival rate of MSCs in ischemic hearts, there is decreased scarring and increased neo-angiogenesis after MSC transplantation [24,25]. MSC injection has also helped to improve pulmo nary emphysema [26]. In all these cases, secretion of various growth factors and cytokines by MSCs is Figure 1. Various threats multipotential stromal cells face at the delivery site leading to loss of cells. Leukocytes and macrophages that are brought into the multipotential stromal cell (MSC) implantation site as part of the nonspecifi c infl ammatory response can secrete proinfl ammatory cytokines and chemokines, as well as increase cellular stress and generate reactive oxygen species (ROS) that might activate apoptotic cascades. Wound and ischemic sites where MSC therapy is usually required are low in vascularity and are hypoxic regions, which might add to MSC cell stress. Also, lack of attachment of MSC to the extracellular matrix (ECM) may cause MSC to detach, undergo anoikis and ultimately lead to cell death.
thought to bring about paracrine signaling and revival of endogenous tissue cells or suppression of harmful infl ammation [27]. Th e lack of demonstrated persistence of the transplanted MSCs has resulted in these aff ects being attributed not to MSC integration and regeneration of tissue, but to trophic eff ects brought about by these unique cells [28].
Th e role of growth factors in increasing proliferation and survival in MSCs has been widely studied over the past few years. Most growth factors are pleiotrophic, causing multiple biological eff ects. Th ey bring about changes in motility, proliferation, morphogenesis and survival. Th e search for the ideal growth factor for use with MSCs is still ongoing. While some groups aim at fi nding a growth factor not aff ecting diff erentiation, other groups opt for a growth factor that has diff erentiation preference towards a specifi c lineage. All groups, however, attempt to fi nd a factor that improves ex vivo expansion and heightens survival on implantation. Th e present review explores the eff ects of various growth factors on MSC expansion and survival and the signaling mechanisms behind these eff ects.

Transforming growth factor beta family of growth factors
Th e choice of growth factors to be used on MSCs was initially determined based on previously existing knowledge about the eff ect of a particular growth factor on cell morphogenesis. Th is was done with the dual pursuit of expanding MSCs and causing them to diff erentiate into the lineage that it was known to favor. Transforming growth factor beta (TGFβ), for example, is known to infl uence cells from the chondrogenic lineage in vivo, promoting initial stages of mesenchymal condensation, prechondrocyte proliferation, production of extracellular matrix and cartilage-specifi c molecule deposition, while inhibiting terminal diff erentiation [29][30][31]. When applied to MSCs in vitro to study chondrocyte regeneration, cells show increased proliferation and a bias towards the chondrogenic lineage [30,32]. TGFβ exists as three isoforms: TGFβ 1 , TGFβ 2 and TGFβ 3 . While all three isoforms induce proliferation of MSCs and chondrocyte formation, TGFβ 3 has been found to have the most pronounced eff ect on chondrogenesis and consistently increases proliferation of MSCs [33,34], making it a prime factor for induction of chondrogenesis from implanted MSCs.
Similarly, bone morphogenetic protein (BMP)-2 through BMP-7 -factors belonging to the TGFβ superfamily -are known to aff ect bone formation. While BMP-2, BMP-4, BMP-6, and BMP-7 induce MSCs to form osteoblasts, BMP-2 has the greatest impact on diff erentiation [35]. MSCs overexpressing BMP-2 and implanted with the extracellular matrix protein collagen I as a hydrogel system increase proliferation of MSC diff erentiation into bone, and this model has been used to study cranial closures in swine [36,37]. Another member of the same family, BMP-3, increases MSC proliferation threefold [38]. Since these factors all aff ect bone formation at diff erent rates and some have a greater eff ect on proliferation, synergistic pairs of these growth factors can be used at optimal doses and at specifi c points during the bone regeneration process. One such search for synergistic pairs led to combination treatment of TGFβ 3 with BMP-2 on MSCs; chondrogenic diff er entiation was found to be enhanced [39].
TGFβ signaling occurs when TGFβ or factors from the family bind a type II serine-threonine kinase receptor recruiting another such transmembrane protein (receptor I). Receptor I phosphorylates the primary intracellular downstream molecules SMADs, causing their translocation into the nucleus and specifi c gene transcription. Receptor I can be ALK-1, ALK-2, ALK-3, or ALK-6 that signal SMAD 1, SMAD 5, and SMAD 8, or can be ALK-4, ALK-5, or ALK-7 that signal SMAD 2 and SMAD 3. Signaling via SMAD 1, SMAD 5, or SMAD 8 is required for chondrocyte diff erentiation while signaling through SMAD 2 or SMAD 3 blocks chondrocyte diff erentiation [40]. TGFβ and members of this growth factor family can also signal via the mitogen-activated protein kinase (MAPK), Rho GTPase and phosphoinositide-3 kinase (PI3K) pathways [41]. Th e eff ect of BMP-2 on proliferation and osteogenic diff erentiation of MSCs has been shown to occur via sustained signaling of the MAPK Erk [42]. Th e mitogenic eff ects of BMP-3, on the other hand, have been found to be mediated by TGFβ/activin signaling and not by any of the MAPK signaling pathways, with ALK-4 and SMAD 2 and SMAD 3 being the key players involved [38]. Figure 2 shows how signaling via SMAD 2 or SMAD 3 leads to proliferation of MSCs but blocks terminal diff erentiation into chondrocytes, while signaling via SMAD 1, SMAD 5, or SMAD 8 potentially leads to chondrocyte diff erentiation in MSCs. Figure 2 also shows how sustained signaling via Erk leads to osteoblast formation.

Fibroblast growth factors
Fibroblast growth factors (FGFs) are a family of growth factors involved in wound healing and angiogenesis. Among the various members of this family, FGF-2 or basic fi broblast growth factor (b-FGF) has been used in MSC-related studies showing increased rabbit, canine and human MSC proliferation in vitro, with the mitogenic eff ect being more pronounced when MSCs are seeded at lower densities [13,[43][44][45]. b-FGF not only maintains MSC proliferation potential, it also retains osteogenic, adipogenic and chondrogenic diff erentiation to fi broblast growth factor receptor (FGFR), binding of epidermal growth factor (EGF) and heparin-binding (HB)-EGF to epidermal growth factor receptor (EGFR) and binding of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) to platelet-derived growth factor receptor (PDGFR) causes phosphorylation of the respective receptors, causes recruitment of the adaptor protein Grb2 and the nucleotide exchange factor SOS, which causes activation of downstream pathways, primarily phosphoinositide-3 kinase (PI3K)-Akt/protein kinase B (PKB) and the mitogen-activated protein kinase (MAPK) Erk. Phosphorylated Erk either enters the nucleus and activates transcription of cellular proliferation genes like c-myc, or activates downstream receptors like Rsk that then activates proliferation genes. Akt similarly prevents the expression of proteins like Myt1 and Wee1, which are involved in inhibiting proliferation. Bone morphogenetic protein (BMP)-2 activates proliferation via the MAPK Erk pathway, unlike BMP-3 that activates Smad2 and Smad3 via Activin signaling. TGFβ 3 is the most potent transforming growth factor beta (TGFβ) mitogen causing proliferation via activation of Smad2, Smad3 and Smad4. Binding of Wnt3a to the Frizzled receptor causes activation of the protein Dishevelled and inactivation of the Axin-APC-Gsk3 complex, which leads to a nuclear infl ux of β-catenin, activating the cell cycle proteins cyclin D 1 and c-myc. TGFβ also causes an infl ux of β-catenin in a Smad3-dependent manner. Binding of hepatocyte growth factor (HGF) to c-Met under low doses causes activation of Erk and Akt, but under higher doses it inhibits proliferation by activating the p38 MAPK pathway and causing the expression of cell cycle progression inhibitors p21Waf1 and p27Kip. APC, adenomatous polyposis coli protein; Gsk3, glycogen synthase kinase 3; RSK, ribosomal S6 kinase; Smad. Sma and Mad related proteins. potentials through the early mitogenic cycles; eventually, however, all of the MSCs diff erentiate into the chondrogenic line. Th ere is a report that b-FGF can extend the proliferation of MSCs for at least 80 population doublings, which is in excess of the Hayfl ick number [45]. Other reports, however, do not fi nd this extension; rather, b-FGF may just decrease the doubling time [46] with the MSCs observing the Hayfl ick limitation [47]. Th is slowing and senescence of MSCs follows that seen in other cell types; as the cells reach senescence, their growth factor receptors become downregulated and signal attenuation is highly increased to bring about resistance to the growth factor stimuli [48,49].
FGF-4, another member of this growth factor family, also increases MSC proliferation at lower densities. In addition to MSC proliferation increasing fi ve times, the number of colony-forming units -indicative of progenitor cell populations -increases by one-half [50]. Th is observation suggests not only that growth factors can drive prolifera tion; they could contribute to stem cell expansion and a greater number of cells undergoing diff erentiation. FGF signals proliferation through the MAPK cascade in various cell types. From microarray analysis, Tanavde and colleagues determined that MAPK-Erk signaling might be involved in increased growth induction by b-FGF [51]. Th e schematic mechanism is presented in Figure 2.

Vascular endothelial growth factor
While investigating ways to better vascularize the MSC transplant site, it was noted that vascular endothelial growth factor (VEGF) increased MSC proliferation on its own [52]. Both endogenous and exogenously secreted VEGF has been found in porcine MSCs [53] but the amounts are too low for autocrine signaling. For in vivo transplantation studies, therefore, MSCs have been either adenovirally transduced with the VEGF gene or injected with a VEGF peptide to bring about increased cell counts [54]. Some signaling studies imply that MSCs do not express the VEGF receptor. Th is could imply that VEGF stimulates MSC proliferation by activation and downstream signaling of the platelet-derived growth factor (PDGF) receptors [55].

Platelet-derived growth factor
PDGFs are potent mitogens of MSCs [56] and these stromal cells express all forms of the growth factor: PDGF-A and PDGF-C at higher levels and PDGF-B and PDGF-D at lower levels. Both receptors PDGFRα and PDGFRβ are also expressed [57]. Th e two receptors homo dimerize or heterodimerize to generate overlapping but distinct cellular signals: PDGFRαα binds PDGF-AA, PDGF-BB, PDGF-CC and PDGF-AB; PDGFRββ binds PDGF-BB and PDGF-DD; and PDGFRαβ binds PDGF-BB, PDGF-CC and PDGF-AB. Several groups have found PDGF-BB to induce both proliferation and migration in MSCs [58][59][60]. While PDGFRβ inhibits osteogenesis, however, PDGFRα has been observed to induce osteo genesis [57]. Akt signaling has been proposed to mediate both the suppression and induction of osteogenesis by PDGFR signaling [58]. As the two receptor isoforms present quanti tatively diff erent preferences for pathway activa tion, due to distinct phosphotyrosine motifs, defi nition of critical signaling elements will await a system's approach to parse the delicate balance of competing impetuses.
Early studies with PDGF showed Erk to be responsible for MSC proliferation [60]. Recently, however, it was shown that while Erk gets phosphorylated in the presence of PDGF, addition of a PDGFR inhibitor does not change phosphorylation levels of Erk [61] -which might imply that Erk activation occurs not by direct PDGFR signaling but via a secondary pathway. Th e same group showed that increase of MSC proliferation occurs in a dosedependent manner due to Akt phosphorylation. Not only was there an increase in proliferation on Akt activation, there was also secretion of VEGF [61]. Further, VEGF was found to act as a ligand to PDGFR in MSCs [61]. Th e mitogenic pathways operative downstream of PDGFR activation are thus still uncertain.

Hepatocyte growth factor
Hepatocyte growth factor (HGF) and its receptor c-Met are expressed at low levels in mouse MSCs [62]. While the low levels of HGF found in culture media are insuffi cient to activate the receptor, exogenous addition of HGF to MSCs triggers the activation of receptor, aff ecting proliferation, migration and diff erentiation. Interestingly, short-term exposure to HGF in MSCs activates Ras-ERK and PI3K-Akt; these are the main pathways activated by HGF in other cell types [63]. Despite activation of these path ways, long-term exposure to the growth factor inhibits mitogenesis. In addition, exposure brings about cytoskeletal rearragement, cell migration and expression of cardiac markers. Th e inhibition of proliferation probably occurs by activation of p38 MAPK and blockade of G 0 -G 1 phase transition. Th is signaling also induces the universal cell cycle progression inhibitor p21waf1 and p27kip proteins [64]. HGF therefore does not seem to be an ideal factor for use with MSCs. Table 1 summarizes the eff ects of the various growth factors on MSCs.

Epidermal growth factor and heparin-binding epidermal growth factor
Th e growth factors described above facilitate MSC prolifer ation but bias diff erentiation into a particular lineage. Th is is helpful in generating specifi cally diff er en tiated cells in culture. When injected into the body or when implanted at the site of requirement for a diff erent or multiple lineage, however, this bias can be counterproductive. Adding to this, if the growth factors initiate diff erentiation, this competes with expansion and thus there may be insuffi cient cell numbers to completely regenerate the desired tissue. Th e search for a growth factor that does not cause MSC diff erentiation led to investigation of growth factors from the near-ubiquitous prototypal growth factor receptor family of ErbB1/ epidermal growth factor receptor (EGFR). EGFR signaling induces proliferation, motility and survival of MSCs. Two of the receptor's ligands, epidermal growth factor (EGF) and heparin-binding EGF, promote ex vivo expansion of MSCs without triggering diff erentiation into any specifi c lineage [60,65]. In addition to its mitogenic eff ect on MSCs, EGF also increases the number of colonyforming units by 25% [15]. Th is observation indicates that treat ment with EGF would also be benefi cial for the maintenance of early progenitor cells.
Classical growth factors, upon binding to their cognate receptors with intrinsic tyrosine kinase activity, activate several downstream pathways that lead to proliferation: Ras GTPase through Raf and MEK to the ERK MAPKs, PI3K activation of Akt/PKB, and the STAT pathways. Tamama and colleagues showed that EGF does not activate STAT3 for proliferation in MSCs, but rather triggers ERK strongly [60]. Heparin-binding EGF, the other EGFR ligand implicated in MSC proliferation, shows activation of ERK1/2 as well as phosphorylation of Akt, but the activa tion of Akt is signifi cantly lower than that by EGF. With activation of EGFR signaling, therefore, the overall population of both MSCs and their early progenitors will be high, leading to enough cells for tissue formation.

Wnt family
Th ere have been several confl icting fi ndings concerning Wnt signaling proliferation in MSCs. One set of studies suggests that canonical Wnt signaling maintains stem cells in an undiff erentiated but self-renewing state. Addition of Wnt3a by activating the canonical Wnt pathway increases both proliferation and survival while preventing diff erentiation into the osteoblastic lineage in MSCs [66]. Frizzled 1 and Frizzled 4 are present on undiff erentiated MSCs and are responsible for canonical Wnt transduction via Wnt3a. In addition, Wnt3a also increases the survival rate of MSCs. Wnt5a, a noncanonical Wnt, competes for Wnt3a binding to the Frizzled receptor and negates the positive eff ect of Wnt3a on MSC proliferation [67]. Th e cell cycle progression factors cyclin D 1 and c-myc have been implied in both these signaling mechanisms [68]. Studies with Wnt4, another noncanonical Wnt, show no change in MSC proliferation [69]. Th e other set of fi ndings connotes that canonical signaling initiated by Wnt3a inhibits human Table 1
MSC proliferation [70]. A third set of studies, however, proposes that canonical Wnt signaling at low levels promotes proliferation while at higher levels inhibits MSC proliferation [71]. Part of the controversy surrounding Wnt signaling is the extensive crosstalk between Wnts and other signaling pathways that aff ect the fate of MSCs. TGFβ 1 , for example, causes rapid nuclear translocation of β-catenin in a Smad3-dependent manner, causing enhanced prolifera tion and suppression of osteogenesis.

MSC survival and role of growth factors
A second cell behavior critical for the successful use of MSCs in regenerative repair is the survival of transplanted cells. Various growth factors -trophic factors as they are called in other cell types -have been queried for promoting this survival. VEGF is one factor that has been extensively used in MSC survival studies. MSCs treated with VEGF in vitro and MSCs carrying the VEGF gene in vivo have been shown to increase survival in these stromal cells. Rodent hearts that have undergone myocardial infarction and have been injected with MSCs along with the VEGF peptide show a higher number of MSCs at the site of injection [52]. Th e surge in survival is attributed to an increase in Akt signaling causing a reduction in infarct size, lesser fi brosis, increased vascularity and thicker ventricular walls [53,72]. Akt signaling in other cell types causes increased expression of prosurvival proteins XIAP, Bcl2 and Bcl-xl, and decreased levels of caspases and apoptotic proteins Bad, Bax and Bim. Akt signaling is also known to inhibit the transcription factors FOX01, FOX02 and FOX03 involved in causing cell cycle arrest and apoptosis [73].
MSCs pretreated with transforming growth factor alpha and implanted at the ischemic site after a myocardial infarction show increased survival. Th is improvement is also attributed to VEGF signaling, although direct signaling through the EGFR receptor cannot be discounted. Transforming growth factor alpha increases VEGF production via the p38 MAPK pathway and enhances recovery [74]. For the ischemic cardiac tissue, MSCs supplanted with VEGF have so far been the best choice for increased survival, leading to improved vascularity in ischemic cardiac tissue and isolated islets. Th e current issue of debate, however, is whether VEGF causes greater incorporation of MSCs and succeeding survival, or whether it brings about paracrine eff ects on surrounding endothelial cells, increasing angiogenesis and formation of more vessels.
Several other growth factors have proven to increase MSC survival. MSC transplantation with brain-derived neurotrophic factor and nerve growth factor into rodents after traumatic brain injury has shown a signifi cantly higher number of engrafted cells compared with MSCs transplanted without any growth factor [75]. PDGF-BB has been found to reduce the 46% loss of cells by apoptosis seen between days 5 and week 3 in rats following acute myocardial infarction [76]. Contrary to its limiting eff ect on MSC expansion, HGF causes a slight increase in MSC survival. PI3K signaling is implicated in this increase [64].
Another major growth factor studied for its eff ects on MSC survival is EGF. Since initial studies showed that EGF in the soluble state did not cause diff erentiation of MSCs but enhanced expansion, it was hypothesized that soluble EGF would similarly enhance survival of MSCs subjected to prodeath cytokines such as FasL and TRAIL in vitro. Contrary to what was expected, soluble EGF did not protect MSCs, but increased cell death in the presence of FasL [22]. Fan and colleagues then presented EGF to MSCs tethered to a biomaterial substratum (tEGF). Th is mode of presen tation of the same growth factor enhanced survival of MSCs in the presence of the proinfl ammatory cytokines. In addition to limiting cell death, tEGF also increased cell attachment and spreading, which might limit cell death by anoikis [22]. Th e survival brought about by tEGF was found to be mainly due to sustained levels of Erk activation, as opposed to transient Erk activation with soluble EGF. Furthermore, tEGF restricts the subcellular localization of activated EGFR. Unlike soluble EGF that causes the internalization and fi nally the degradation of EGFR, tEGF restricts EGFR and EGFR signaling to the plasma membrane, and thereby changes the spatio temporal balance of intracellular signaling pathways [77]. tEGF did not interfere with subse quent diff erentiation into osteoblasts under inducing conditions while increas ing the effi ciency and the number of osteoid colonies [78]. As this was the fi rst study to directly challenge MSCs with proapoptotic infl am matory stimuli, the tech nique holds promise as a quantal advance in protecting MSCs from death in vivo. Th e cross-signaling of survival by various growth factors is represented in Figure 3.

Clinical issues of using growth factors in MSCs
Current limitations to using MSCs for regeneration include providing suffi cient numbers of these stromal cells in a timely manner in the challenging in vivo milieu. To bring about MSC expansion, fetal bovine serum (FBS) is currently employed since human serum does not fully support growth of MSCs in vitro. Complications arise in use of FBS for MSC transplants in vivo, however, since FBS contains undefi ned elements that can vary in inducing proliferation. More importantly, contaminants in FBS can cause infections, and, being of nonhuman origin, the components can trigger host immune reactions [79]. On the commercial front, companies have developed serum-free and animal supplement-free MSC Figure 3. Growth factor signaling pathways mediating survival in multipotential stromal cells. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) bind PDGFR, and hepatocyte growth factor (HGF) binds c-Met, which causes phosphoinositide-3 kinase (PI3K) to be activated, converting PIP2 to PIP3 and activating Akt/protein kinase B (PKB). This leads to the inhibition of the Fork head family of transcription factors Foxo1, Foxo3 and Foxo4, and also causes inhibition of pro-death proteins Bim, Bad and Caspase9. At the same time there is activation of pro-survival proteins XIAP, Bcl2 and Bcl-xl. In addition, Akt activation causes activation of eNOS and HSP90, causing nitric oxide synthesis and angiogenesis that promotes survival. Binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGFR), in addition to activating Akt, brings together the guanine nucleotide exchange factor SOS and the small adapter protein Grb2, which activates the mitogen-activated protein kinase (MAPK) pathway: Ras-Raf-Mek1/2-Erk1/2. Activation of Erk leads to the expression of pro-survival proteins like NF-kB, Bcl2 and Bcl-xL. EGF binding to EGFR also causes PLCg to cleave PIP2 to IP3 and DAG, which activates protein kinase C (PKC). PKC can activate Raf and further cause downstream Erk activation. All these activated receptors, however, are quickly internalized by clathrin machinery or by alternate internalization mechanisms into the endosome where they continue to signal. The fi gure shows internalization of the EGF-EGFR complex continuing to signal in the cytosol, but once inside the lysosome, the receptor along with the ligand completely degrades and the survival signal is lost. Both the Akt and Erk signals generated therefore are acute and transient. Tethering of growth factors near the membrane, as in the case of EGF (tEGF), however, causes a more sustained signaling of Erk and Akt since the receptor-ligand complex signals for longer from the cell membrane, leading to multipotential stromal cell (MSC) survival for a more prolonged time period. Bcl2, B-cell lymphoma 2; DAG, diacyl glycerol; Erk, extracellular signal related kinase; eNOS, endothelial nitric oxide synthase; HSP90, heat shock protein 90, IP3, inositol triphosphate; NF, nuclear factor; PDGFR, platelet-derived growth factor receptor; PLCg, phospholipase C gamma; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphotidylinositol-3,4,5-trisphosphate; XIAP, X-linked inhibitor of apoptosis protein.
media. Th ese media products are comprised of synthetic supplements that are meant to replace serum, thereby reducing variability. Th e companies claim that MSCs grow as well in the media as in media supplanted with FBS. Th e proprietary composition of these products, however, goes against them for clinical use. Details of these products are summarized in Table 2. Th e use of growth factors as culture supplements instead of FBS therefore off ers the most promising alternative [80]. Search for a serum-free media to expand MSCs has led to combination treatments with PDGF-BB, FGF-2 and TGFβ 1 showing the most encouraging results. Th is treatment has not only brought about a synergistic eff ect on MSC proliferation, but has also retained the phenotype, diff erentiation and colony-forming potential of these cells [81].
In addition to using combined treatment of growth factors to improve proliferation, MSCs have been pretreated with a blend of growth factors to boost survival. Pretreatment of these stromal cells with FGF-2, BMP-2 and insulin-like growth factor-1 before delivery into the ischemic heart has shown enhanced rates of survival [82].
One of the reasons MSCs are preferred for regenerative use is their genetic stability. MSCs are shown to maintain their diploid karyotype without aneuploidy, polyploidy or chromosomal structural abnormalities [83]. Th ere has also been a report, however, of MSCs displaying localized genetic alterations in the presence of FBS or autologous serum. Th e same report states that platelet lysate expands cytogenetically normal MSC colonies and that this eff ect may be due to the presence of growth factors such as EGF, PDGF and FGF in the platelet lysate [84]. In short, growth factors should be chosen not only based on expansion potential but also on not altering the MSC genome.
Th ere still remains the scare that while growth factors increase proliferation, this proliferation and the added protection off ered might let MSCs and surrounding cells escape control and lead to tumor growth. It is therefore important to have proper modes of growth factor delivery, which is localized, controlled and of a timelimited nature. Controlled release of growth factors or presentation of the growth factor in bioengineered forms, such as tEGF, are some of the ways in which this can be achieved [39,78,85,86], discussion of which lies beyond the scope of the present review.

Conclusions and future directions
Growth factors are a promising adjuvant to MSCs to circumvent problems of MSC proliferation and expansion, and survival in vivo. Th e choice of growth factor(s), however, depends on three major criteria. First, the growth factor needs to prolong proliferation for several population doublings, to generate a considerable number of MSCs before these cells are to be diff erentiated into the desired tissue. Second, the growth factor should be able to completely replace the use of animal serum for proliferation purposes, to eliminate the use of xenographic substances and reduce variability. Finally, there need to be modes of localized and controlled delivery, which will help present the mitogenic and protective signals in sustained forms without letting MSCs escape into uncontrolled proliferation. While individual growth factors like b-FGF have advantages in steering MSCs down a select lineage after several population doublings, combination treatments of growth factors currently seem to be drawing a lot of attention due to their synergistic eff ect on MSCs. Composite treatment with PDGF, b-FGF and TGFβ 1 appears to be a good alternative for proliferation in vitro to replace serum. More studies need to be performed, however, to look into whether such a combination would accentuate survival and encourage grafting of cells in the wound microenvironment. Th ere also need to be ways by which such combinations can be delivered at the wound region. and clinical purposes Cells are smaller in size [88] 2 Mesencult Stem Cell Serum free, xeno-free. Causes rapid expansion MSCs fail to maintain a similar growth rate beyond the Technologies of cells in the fi rst passage, higher than any fi rst passage and stop growing altogether after the other media [89] sixth passage, while MSCs grown in DMEM-KO and DMEM F12 supplanted with 10% FBS proliferate for up to 25 passages [89] 3 Mesengro StemRD Chemically defi ned, serum free and No published data using this media as yet xeno-free. The company claims that the growth rate of MSCs in this media is the same as that of MSCs supplanted with 10% FBS for up to nine passages in vitro DMEM, Dulbecco's modifi ed Eagle's medium; FBS, fetal bovine serum; MSC, multipotential stromal cell.
While there have been several groups looking into the proliferation eff ects of growth factors and their eff ects on morphogenesis, much less attention has been paid to growth factor signaling for survival. Th is might partly be because MSCs were for a very long time considered to be cells with the advantage of survival. When MSCs were not observed in the body on delivery, the absence was attributed more to cells migrating away rather than to cells dying at the site. Only recently have there been studies showing that MSCs are susceptible to death by proinfl ammatory cytokines and reactive oxygen species, which might be the major reason for their loss at the site of delivery.
Th e advantage with the EGFR family of ligands is that work has been carried out on almost all aspects of MSC biology: eff ects on survival, proliferation, diff erentiation, migration and modes of delivery have been studied. Th is family of ligands appears to be generalized expanders and survival adjuvants while not aff ecting MSC diff er entiation. More over, presenting EGF in a tethered form has been studied with respect to sustained signal ing, making it one of the factors of foremost importance. Taken together, the right choice of growth factors with proper modes of their delivery will help bridge the gap in MSC regenerative therapy and exploit the full potential of MSCs to regenerate tissue in the near future.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Engineering

text:
PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

Background In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. Results In the present study we show that overexpression of PGM2 under control of the HXT7'promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. PGM2 overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When PGM2 was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying PGM2 on the integrative plasmid. Conclusion PGM2 overexpression in S. cerevisiae from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity.

Background
Sustainable bioconversion of renewable lignocellulose raw material requires that all sugar components are efficiently utilized [1]. These raw materials are composed of a number of different monosaccharides one of which is galactose. It may constitute up to 18% of the total released monomeric sugar of pretreated softwood [2]. Actually the amount of galactose is higher than that of arabinose in certain raw materials such as willow [3] and spruce [4]. Galactose also makes up a substantial amount of other industrial media raw materials as beet molasses [5] and cheese whey [6].
Baker's yeast, which is the preferred organism for industrial ethanolic fermentation [7], catabolizes galactose via the Leloir pathway [8]. Galactose catabolism to the glycolytic intermediate glucose 6-phosphate is a complex sequence of reactions including phosphorylation and group transfer. Galactose is consumed and metabolized at a significantly lower rate than glucose since it delays induction of glycolytic genes [9]. In addition the Leloir pathway is controlled by glucose repression even in the presence of galactose [10].
When the structural genes for the galactose metabolizing enzymes were overexpressed, growth on galactose was impaired [11]. However increased galactose utilization and ethanol production was observed when genes encoding the galactose regulatory network were engineered [12]. A transcriptome analysis of two of these engineered S. cerevisiae strains, SO7 (pGAL4, 2 μ) and SO16 (Δgal6 Δgal80 Δmig1), later identified a slight upregulation of PGM2 (GAL5) to be a common denominator [13]. It was also verified that overexpression of PGM2 in S. cerevisiae improved galactose uptake under aerobic conditions while anaerobic conditions were not investigated [13].
The objective of the current study was to investigate the influence of overexpressing PGM2 in S. cerevisiae under anaerobic conditions. PGM2 was expressed both from a multicopy plasmid and from an integrative plasmid. The PGM activity increased 5 to 6-fold in galactose-grown cells when the gene was expressed from the integrative plasmid. PGM2 overexpression reduced the lag phase of glucose pre-grown cells and increased the maximum specific growth rate on galactose. The additional increase of PGM activity in strains expressing PGM2 from a multicopy plasmid did not further increase growth on galactose.

Effect of PGM2 gene dosage
Two strains with different copy numbers of the gene PGM2 were constructed in the same genetic background (Table 1). In both strains, PGM2 was expressed under the control of the constitutive promoter HXT7' [14]. One strain expressed at least one additional integrated copy of PGM2 and was named PGM2 i. Its corresponding control strain was Control i. The other constructed strain overexpressed PGM2 from a multicopy plasmid and was named PGM2 m. Its control strain Control m carried the same plasmid without the structural gene.
The effect of the PGM2 copy number on the specific PGM activity was assessed in crude extracts of cells grown on galactose (Table 2). Strains Control m and Control i displayed the same specific PGM activity, 0.33 and 0.34 U mg -1 protein, respectively, whereas the specific activity in the PGM2 m and PGM2 i strains increased to 10.40 and 1.81 U mg -1 protein, respectively. Whereas PGM activity increased almost 32 fold in the multicopy strain, only a 17 fold increase was observed in the previously reported overexpression of PGM2 on multicopy vector [13] ( Table 2). The difference may be ascribed to different strain background, different multicopy vector and different promoter.
Next, the PGM2 overexpressing strains and their respective control strains were compared with respect to aerobic growth in galactose medium (Table 2, Figure 1). In aerobiosis on galactose, growth, galactose consumption and ethanol production were similar for the two control strains, Control m and Control i (Figure 1). Also the strains overexpressing PGM2, from a multicopy plasmid, PGM2 m, and from an integrated plasmid, PGM2 i, respectively, displayed similar results ( Figure 1). The maximum specific growth rates (μ max ) were 0.22 ± 0.01 and 0.24 ± 0.02 h -1 for strains Control m and Control i respectively, whereas μ max for strains PGM2 m and PGM2 i increased to 0.30 ± 0.02 and 0.32 ± 0.01 h -1 , respectively. Despite the fact that strain PGM2 m had 5 to 6 times higher PGM activity than strain PGM2 i when grown in galactose, μ max did not increase significantly. It was therefore decided to further investigate the influence of increased PGM activity only for strain PGM2 i and its control strain Control i. Additionally the fact that the extra-copy of PGM2 was stably integrated in the genome of the PGM2 i strain enabled us to study the effect of PGM2 overexpression independently of variations in plasmid copy number.
When the strains Control i and PGM2 i were grown aerobically in glucose medium μ max was 0.36 ± 0.01 h -1 and 0.37 ± 0.04 h -1 , respectively, which showed that the additional copy of the PGM2 gene did not affect growth on glucose. Assuming that the maximum specific growth rate on glucose (0.37 h -1 ) is also the theoretical maximum specific growth rate on galactose, overexpression of PGM2 results in a significant contribution to obtaining that growth rate.

Anaerobic fermentation of galactose
Anaerobic fermentation of galactose with the strains Control i and PGM2 i, showed that overexpressing PGM2 reduced the time required to deplete galactose with about 24 h ± 1 h (Figure 2A and 2B). The increased PGM activity consistently reduced the lag phase of fermentative galactose-grown cells (pre-cultivated on glucose) ( Figure  2). This was accompanied by an increase of the maximum specific growth rate from 0.098 h -1 ± 0.003 to 0.139 h -1 ± 0.004 (p-value < 0.01) ( Table 3). The ratio between the different products of galactose was much less influenced by the increased PGM activity. The ethanol yield was the same for strains Control i and PGM2 i. However, the reduced lag phase and the increased maximum specific growth rate translated into a shorter fermentation duration from 69.3 ± 0.8 h in the Control i strain to 45.5 ± 0.4 in the PGM2 i strain.

Discussion
The present study demonstrates for the first time that the introduction in the chromosome of an additional copy of the gene PGM2 encoding Pgm2p under the control of a strong and constitutive promoter increased the maximum specific growth rate on galactose in anaerobic fermentation. In addition the lag phase of glucose-pregrown cells in anaerobic galactose medium was significantly reduced. The extra integrated copy of the gene PGM2 increased the PGM activity 5 to 6 times in galactosegrown cells. Further overexpression of PGM2 from a multicopy plasmid increased the PGM activity almost 32 times as compared to the control strain but this did not further increase aerobic growth on galactose compared with overexpression from an extra chromosomal copy.
Pgm2p catalyses the last step in the Leloir pathway [8] where glucose 1-phosphate (G1P) is reversibly converted to the glycolytic intermediate glucose 6-phosphate (G6P). Pgm2p is the major isoform of the PGM enzyme in S. cerevisiae accounting for 80 to 90% of the PGM activity [15,16]. Pgm2p was originally believed to be constitu-tively present in high levels [10,15]. However, it was later found that the exposure to galactose gives lower induction of Pgm2p activity compared to other structural GAL genes that are at least 1000-fold induced [10,16]. The fact that overexpression of PGM2 under a strong constitutive promoter enhanced growth on galactose indicates that PGM is a controlling enzyme in the Leloir pathway.
Overexpression of PGM2 has previously been shown to increase the galactose uptake rate and the specific growth rate under aerobic conditions [13]. In the current study we confirmed these results in another strain background. When compared with the results of Bro et al, higher specific growth rates on galactose were obtained for both the control strains and the PGM2 overexpressing strains under aerobiosis. However higher concentrations of galactose were used in the present study. Nevertheless, in both studies the aerobic specific growth rates on galactose increased by 33-36% when the overexpressing PGM2 gene was overexpressed. We also found that, in our strain background, the increased PGM activity brought the aerobic specific growth rate in 50 g l -1 galactose medium, 0.32 h -1 , close to that in 20 g l -1 glucose medium, 0.37 h -1 . Absence of anaerobic growth on galactose has been frequently observed for S. cerevisiae [17][18][19] and it has recently been ascribed to low cellular energy charge [18]. In the present study, an increased PGM activity resulted in improved anaerobic galactose utilization, which indicated that the energy charge deficiency may not be the only limitation of the Leloir pathway. For instance PGM upregulation has been shown to increase the intracellular levels of glucose 6-phosphate [11,13,20] that is known to activate glycolysis [9,21,22].

Conclusions
Chromosomal integration of an additional copy of the PGM2 gene was sufficient to increase the anaerobic growth rate with 42% and reduce the fermentation time by 34%. Chromosomal integration allows for the construction of stable industrial strains that are expected to decrease the ethanol production time. These results have practical implications for industrial ethanolic fermentation of raw materials containing galactose, such as raffinose from the sugar industry [5], whey from the dairy industry [6] and lignocellulose for fuel ethanol production [2][3][4].

Strains
Escherichia coli DH5α and JM101 (Life Technologies, Rockville, MD, USA) were used for cloning. Plasmids and Saccharomyces cerevisiae strains (Table 1) were stored at -80°C in 15% glycerol. Freshly streaked plates from frozen stocks were used to inoculate pre-cultures.

Molecular biology techniques
Standard molecular biology techniques were used for cloning [23]. Restriction enzymes, T4-DNA ligase and Shrimp Alkaline Phosphatase (SAP) were obtained from Fermentas (Vilnius, Lithuania). Yeast genomic DNA was purified with Easy-DNA Kit (Invitrogen, Groningen, The Netherlands). Escherichia coli DH5α competent cells were prepared as described before [24] and transformed using the calcium chloride method [25]. Yeast was transformed with the lithium acetate method [26]. Plasmids were extracted from bacteria either with GeneJET TM (Fermentas, Vilnius, Lithuania) or with QIAGEN Mini Plasmid Purification kit (Qiagen GmbH, Hilden, Germany). The QIAquick ® kit was used for Gel Extraction of DNA fragments from agarose gel as well as for PCR purification of amplified DNA products. Taq-polymerase (Fermentas, Vilnius, Lithuania) was used for analytical PCR, while Pfu-polymerase (Fermentas, Vilnius, Lithuania) and Pwo-polymerase (Roche Diagnostics GmbH, Mannheim, Germany) were used for High-fidelity PCR. Abi-Prism Big-Dye cycle sequencing kit (Applied Biosystems, Weiterstadt, Germany) was used for DNA sequencing that was performed by BM labbet AB (Furulund, Sweden).

Construction of strains expressing multiple copies of the PGM2 gene
The YEplacHXT vector [27] that enables strong constitutive expression under the yeast promoter HXT7' [14] was

Genomic integration of PGM2
The HIS3 amplicon was first transformed into S. cerevisiae CEN.PK 113-11C and transformants were selected on defined mineral medium supplemented with uracil. The resulting strain TMB 3134 (Table 1) was used to construct the strains Control i, TMB 3135 and PGM2 i, TMB 3136 (Table 1) as follows. The purified plasmid YEplacHXT-PGM2 (Table 1) from strain PGM2 m was used as template to PCR amplify the amplicon HXT7'p-PGM2-PGKt using Pwo-polymerase and the following primers: PGKSalI (5' ATCTGTCGACGACATA-GAAATATCGAATGG 3') with sequence homologous to PGKt (in bold) and HXTSalI (5' ATCTGTCGACAG-GAACAATTTCGGGCC 3') with sequence homologous to HXT7'p (in bold). The PCR product HXT7'p-PGM2-PGKt and the integrative vector YIplac211 [30] were cleaved with restriction enzyme SalI and ligated. The ligation mixture was transformed into E. coli DH5α competent cells and transformants were selected on LB plates with 100 mg l -1 ampicillin. PCR and restriction analyses were used to confirm the proper size of the resulting plasmid YIplac211 HXT-PGM2.
Purified plasmids YIplac211 and YIplac211 HXT-PGM2 (Table 1) from E. coli were cleaved with EcoRV to target the URA3 locus in strain TMB 3134. The prototrophic strain Control i and PGM2 i were generated by integration of cleaved YIplac211 and YIplac211 HXT-PGM2 (Table 1), respectively. Genomic integration of HXT7'p-PGM2-PGKt was verified by analytical PCR of genomic DNA.

Aerobic cultivation conditions
E. coli was grown and selected on Luria-Bertani medium (LB) [23] with 100 mg l -1 ampicillin. For yeast strains 6.7 g l -1 Yeast Nitrogen Base medium (YNB, Difco Laboratories-Becton, Dickinson and Co., Sparks, MD, USA) was supplemented either with 50 g l -1 galactose or 20 g l -1 glucose as sole carbon source to assess growth. YNB liquid medium was buffered with potassium hydrogen phthalate (10.21 g l -1 phthalate, 2.1 g l -1 KOH, pH 5.5) [31]. For growth on plates 20 g l -1 glucose and 20 g agar l -1 was used. The concentration of YNB was doubled when the sugar concentration was above 20 g l -1 to avoid nutrient limitation. Pre-cultures grown in YNB with 20 g l -1 glucose until mid-late exponential phase overnight in 50 ml tubes were used to inoculate batch cultures at OD 620 nm 0.1-0.2 in cotton-stoppered baffled 500 ml flasks. To ensure initial aerobic conditions, the growth media was only 10% of the flask volume. YNB medium was supplemented with 40 mg l -1 histidine and 20 mg l -1 uracil for auxotrophic strains. Cultivation was performed at 30°C and 180-200 rpm agitation (Gallenkamp INR-200, Leicester, UK) at least in duplicate.

Anaerobic fermentation
For anaerobic fermentation and corresponding pre-cultures defined mineral medium [32] was used. The medium for anaerobic fermentation was supplemented with 0.4 g l -1 Tween 80 and 0.01 g l -1 ergosterol and 20 g l -1 galactose. The pre-culture medium contained 20 g l -1 glucose and was buffered with phthalate buffer (10.21 g l -1 phthalate, 2.1 g l -1 KOH, pH 5.5) [31] whereas the bioreactor pH was controlled at 5.5 with automatic addition of 3 M KOH.
A first pre-culture was grown until late exponential phase in 50 ml tubes. The culture was used to inoculate a second aerobic pre-culture in 1000 ml cotton-stoppered baffled shake flasks. Both pre-cultures were grown in media, constituting 10% of vessel volume to assure maximum aeration. Cultures were grown at 30°C (Gallenkamp INR-200, Leicester, UK) and 180-200 rpm. Cells from the second pre-culture were grown until late exponential phase, washed twice with sterile water, centrifuged at 5000 rpm for 10 min, and used to inoculate anaerobic batch cultures at OD 620 nm of 0.1-0.2.
Anaerobic batch fermentation was performed in 3 l Applikon ® Bio Reactors (Applikon, Schiedam, The Netherlands) with a working volume of 1.5 l, at 30°C and 200 rpm stirring. Prior to inoculation the bioreactor was flushed with nitrogen gas containing less than 5 ppm O 2 (AGA Gas, Sundbyberg, Sweden) from the bottom of the bioreactor at a flow rate of 0.2 l min -1 controlled by a gas mass flow-meter (Bronkhorst, HI-TECH, Ruurlo, The Netherlands). Outlet carbon dioxide and oxygen were monitored by a Carbon Dioxide and Oxygen Monitor type 1308 (Brüel & Kjaer, Copenhagen, Denmark). Offgas condensers were cooled to 4°C to minimize ethanol  evaporation and a dissolved oxygen probe monitored 0% oxygen tension. Anaerobic fermentation experiments were performed in at least biological duplicates and metabolite measurements varied with less than 10%. Cultivation was performed both with and without nitrogen gas flushing. Flushing resulted in an increased lag phase but did not affect the outcome of the experiment. Physiological characterization of recombinant strains was only performed with the prototrophic strains.

Enzymatic activity measurements
PGM activity was determined in crude extracts of cells grown on YNB medium containing 20 g l -1 galactose. For every strain, at least three independent biological replicates were assayed with at least two independent enzymatic measurements. Cells were harvested in exponential phase, centrifuged at 5000 rpm for 5 min, washed twice with cold sterile water and permeabilized with Y-PER (Pierce, Rockford, IL, USA). The protein concentration was determined with Coomassie Protein Assay Reagent (Pierce, Rockford, IL, USA), using bovine serum albumin as standard. Phosphoglucomutase activity was determined at 30°C by monitoring NAPDH production at 340 nm [13] with a Ultrospec 2100 pro spectrophotometer (Amersham Biosciences, Uppsala, Sweden). The chemicals used to determine enzyme activity were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Determination of metabolites and maximum specific growth rate
Concentrations of galactose, glycerol, acetic acid and ethanol were determined by high-pressure liquid chromatrography (HPLC) (Waters, Milford, MA, USA or Beckman Instruments Fullerton, CA, USA) with an Aminex HPX-87H ion exchange column (Bio-Rad, Hercules, USA) at 45°C. The mobile phase was 5 mM H 2 SO 4 at a flow rate of 0.6 ml min -1 . Cell dry weight was determined in at least duplicate at different time points of the fermentation experiment by filtering 5 ml culture through a pre-weighed and microwaved dried hydrophilic polyethersulfone 0.45 μM filter (PALL, Life Sciences, Michigan, USA). The filters were dried at 350 W for 8 min in a microwave oven and cooled prior to weighing. The maximum specific growth rate was calculated by linear regression on a logarithmic plot of the phase of constant and maximum values.


CONTRASTED ECOLOGICAL REPARTITION OF THE NORTHERN FOWL MITE ORNITHONYSSUS SYLVIARUM (MESOSTIGMATA: MACRONYSSIDAE) AND THE CHICKEN RED MITE DERMANYSSUS GALLINAE (MESOSTIGMATA: DERMANYSSIDAE)

The two hematophagous mite species, the Northern Fowl Mite Ornithonyssus sylviarum (Macronyssidae) and the Chicken Red Mite Dermanyssus gallinae (Dermanyssidae), are considered serious pests in fowl farms in the USA and in Europe respectively. However, neither of them seems to be restricted to either of these two continents according to sparse records in the literature. The aim here was to explore the respective ecological repartitions of these two species in France and to compare them with data from the USA. We thus analyzed hematophagous mesostigmatid mites collected in France in natura, in orchards agroecosystems, in pet bird and fowl farms and compared DNA sequences from some North American and French mites in both species. It is remarkable that O. sylviarum has been recurrently encountered in bird nests from France and yet been absent from French layer farms, whereas it is a serious pest in layer farms in the USA. On the other hand, O. sylviarum has been isolated from ornamental bird farms in France (canary, pheasant). This suggests either a strong impact of different farming practices between continents or a colonization in process in Europe. It remains to be explored whether the opposite applies to D. gallinae in the USA. Lastly, mites belonging to the special lineage D. gallinae L1 have been isolated from North American pigeons providing mt and nDNA sequences very close to French L1 isolates. This confirms the specific status of this cryptic entity.

INTRODUCTION
The two hematophagous mite species, the Northern Fowl Mite Ornithonyssus sylviarum (Canestrini and Fanzago, 1877) (Mesostigmata: Dermanyssoidea: Macronyssidae) and the Chicken Red Mite Dermanyssus gallinae (De Geer, 1778) (Mesostigmata:Dermanyssoidea:Dermanyssidae) are obligatory ectoparasites primarily parasitizing birds. They are serious pests in fowl farms in the USA and in Europe respectively. Neither of them seems to be restricted to either of these two continents according to sparse records in the literature.
Ornithonyssus sylviarum has been recorded in nests of various birds in several European countries (Ambros et al., 1992;Kristofík et al., 2007) as well as in some French layer farms (Bruneau et al., 2002), but is not considered a problem in layer farms in France. In contrast, it is is a well-known pest in permanent parasites (Moss, 1978). Likely due to this difference between the two economic pest species, although both are able to be starved over long periods, Kirkwood (1963) has shown that marked difference in the duration of their starvation ability separated them, since in identical conditions and in the absence of any host, D. gallinae lived for as long as 34 weeks, compared to only 3 weeks for O. sylviarum. Chen and Mullens (2008) have shown that the on-host survival duration in O. sylviarum may increase with respect to ambient temperature and humidity as well as to the mite's development stage, but nonetheless remaining far more inferior to the range noted in D. gallinae.
Most of the repartition data of these species are based upon morphological identifications. These appeared to be partly irrelevant concerning D. gallinae according to Moss et al. (1970). These authors have shown that non gallinae species of Dermanyssus had likely been previously misidentified and confused with D. gallinae in some North American records. And yet, Roy et al. (2009a, b), by using both morphological and molecular data, have shown that previous European records might have been incorrect as well, since non gallinae species revealed to be much more host specific than previously believed. This is also probably the key to the amazing and parentally transmitted behaviour differences noted by Nakamae et al. (1997) between two assumed D. gallinae populations.
As a result, one might wonder whether recorded distribution of populations in both species and for both continents are relevant or not. Second, for each species are mites parasitizing wild and domestic birds in each species are truly conspecific respectively?
Our aim is to establish the respective ecological segregation of these two species in France and to compare these findings with some data from the USA, at the strictly specific level and regardless of intraspecific population differentiation. Especially, we test whether O. sylviarum is present in the French wild avifauna and in French layer farms, as the presence of D. gallinae in French wild and domestic birds has already been shown by Roy et al. (2009b).
To this end, an inventory of hematophagous 208 mesostigmatid mites in birds at the specific level has been carried out in France and based on both morphological and DNA data. The obtained DNA sequences for each species are compared with sequences obtained from other countries, including the USA.

Biological material
A detailed list of the analyzed French samples is available in Appendix 1. Additionally, a few samples collected in other countries, including the USA, are detailed in Appendix 2. Mites were sampled between 2005 and 2009. Two types of samples are considered here:   1) Material sampled in the bird's immediate environment (nest or litter). This mainly concerns wild avifauna.
2) Some mites have been directly sampled as such: mite aggregates or individuals found in the host environment (crevices, under dried droppings. . . ) or on host. This mainly concerns birds which are reared or kept by humans.
In order to get an overview of the different bird ecologies, samples have been collected in a diversity of environments, both in wild avifauna and in domestic birds. Within each of these two categories, several different types may be distinguished. The wild avifauna has been sampled in crop agroecosystems or not, and some on urban systems. Domestic birds' samples come from industrial layers, industrial broilers, amateur fowl, pet bird facilities, and even one isolate collected in game bird breeding facilities. Finally, a diversity of geographical origins within France has been sampled, in order to avoid artifacts due to endemism.
Additionally, mites have been sampled in several other countries throughout Europe, as well as in the USA, Brazil, Australia (see Appendix 2).

Nests/litter analysis
Mites were isolated from nests following the method described in de Lillo (2001), slightly modified as described in (Roy et al., 2009b). This method is based on the immersion of the nest or litter sample in a fixed quantity of water and subsequent filtration using stacked sieves. Next, the obtained filtrate is reimmersed into water and observed using a dissecting microscope. Compared to the Berlese funnel method used in Burtt et al. (1991), de Lillo's method allows detection of both living and dead mites. All detected hematophagous Mesostigmata were isolated, since their heterogenous coloration due to blood ingestion and digestion makes them highly recognizable using stereoscopic magnifying glasses. They then were digested by proteinase K, then mounted into Hoyer's medium and identified using a photonic miscroscope. In some individuals, DNA contained into the digestion product was purified and used in molecular analyses (see below).
Mites are not counted in the present study. A simple occurrence assessment is used per sample (presence/absence of each species).
A Chi-square test was used to assess the association between species (D. gallinae, O. sylviarum) and their ecological distribution assuming as the null hypothesis that ecological distribution of hematophagous Mesostigmata species is homogenous. The distribution between the observed occurence of D. gallinae and O. sylviarum in Mesostigmata containing nest/litter and their theoretical distribution was also tested under the same null hypothesis.

DNA analyses
DNA sequencing of some gene portions (16S rRNA, COI coding gene, Tropomyosin) was performed in some isolates following Roy et al. (2009a, b) and Roy (2009) in order to check the specific identity and/or assess conspecificity. One to five individuals were sequenced per isolate. Phylogenetic analyses of obtained partial sequences of the mitochondrial coding gene for cytochrome oxidase I were run using both maximum-likelihood and maximumparsimony methods. Maximum-likelihood (ML) analysis has been performed using the online software PHYML (Guindon et al., 2005) with the substitution model GTR. Branch supports were estimated by approximate Likelihood Ratio Tests (aLRT) following Anisimova and Gascuel (2006). Maximumparsimony (MP) analysis has been performed using PAUP* 4.0b10 (Swofford, 2001): a heuristic analysis has been run with TBR branch swapping and 10,000 random additions saving all most parsimonious trees. Heuristic searches in TNT 1.1 (Goloboff et al., 2008, program available with the sponsorship of the Willi Hennig Society from http://www.cladistics.org/tnt.html) were used to obtain relative Bremer (Goloboff and Farris, 2001) and bootstrap support values. TNT searches recovered the same topology and tree length as PAUP, but calculation of support values is much more efficient in TNT.

French samples
Overall, 729 French samples were analyzed, of which 361 contained at least one hematophagous Mesostigmata individual (see Table 1 Table 1). Within D. gallinae, the special lineage L1 (Roy et al., 2009a) has been shown strongly isolated on a reproductive point of view (Roy, 2009). Therefore, it is noted D. gallinae L1 all along present paper, and non L1 gallinae mites are labelled D. gallinae s. str. Concerning the specific identification of Ornithonyssus, only two species have been isolated in present samples. O. sylviarum individuals provided rather clear morphological characteristics sensu Micherdziński (1980), similar to the North American isolate OSBM's. Ornithonyssus sp. 1 was very similar to, but slightly differed from O. bacoti (Hirst, 1913), according to Micherdziński (1980) and compared with individuals of O. bacoti from a lab strain cultured in the French Museum of Natural History (O. Bain). It did not key to another of the Ornithonyssus species sensu Micherdziński (1980).

Concerning the specific identification within
Dermanyssus, all mites from domestic birds have been successfully identified at the specific level. As for samples from wild avifauna, some mites were too damaged for morphological identification, and/or DNA characterization was not yet fully de-  veloped at the moment of sample exploration (before or at the beginning of the study of Roy et al. 2009a), thus 51 samples contained Dermanyssus mites not identified at the specific level.
Only 12 samples (ie 3.3% hematophagous Mesostigmata positive samples) allowed for detection of two different species in a single nest, eight of which grouped together species of different families (Macronyssidae + Dermanyssidae or Macronyssidae + Laleapidae). The four remaining cases (1.1% hematophagous Mesostigmata positive samples) grouped together D. gallinae either with D. apodis (3 cases) or with D. carpathicus (1 case) (see Appendix 1).
As in Roy et al. (2009b), D. gallinae s. str. appeared to be the most generalist species in France, not only from the host point of view but also with regard to the environment (see Figure 2). Note that it was present in free-range as well as in caged layer farms, and in layer as well as in broiler farms (see Appendix 1). The special lineage D. gallinae L1 (Roy et al., 2009a, b) was present not only in wild but also in farm environments, almost exclusively on pigeons and never on hens. Other Dermanyssus species have been found only in wild avifauna. O. sylviarum has been found in almost all situations, but never in poultry farms in France (see Appendix 1), which is significant given the number of farms sampled (n=132). Moreover, only D. gallinae s. str. has been isolated from other European farm samples under test (see Appendix 2), but the number of samples (n=8) is insufficient to estimate the pres-ence/absence of O. sylviarum.
Lastly, the percentage of samples containing hematophagous Mesostigmata is presented in Table  2. The presence of mites in direct samples (collection of mites directly in the bird's environment or on the host) was not significant as such with regards to a prevalence estimate. Most farms had been visited as a result of farmers' complaints. As for the wild avifauna, the exact number of wild birds examined is not known. Consequently, only nests or litters are considered here. At the generic level, Dermanyssus appeared present more often than Ornithonyssus. But most of the Dermanyssus positive nest samples contained non gallinae species. On the contrary, when considering solely D. gallinae and O. sylviarum, the latter appeared slightly more often than the former (P < 0.001). It is important to keep in mind that these data are mainly composed of wild bird nests. Overall, both D. gallinae and O. sylviarum have been regularly encountered in the wild avifauna, and in distant points in France (see Figure 1).

Other samples
In addition to the French samples, 24 different mite samples of non French origin are listed in Appendix 2. Hematophagous mesostigmatid mites from layer farms collected in other European countries, as well as from Australia and from Brazil, all belong to D. gallinae s. str. In contrast, none of the three North American gallinae-like isolates belong to D. gallinae s. str. The only isolate from a North American  (Roy et al., 2009a;Roy, 2009) and have shown COI being a powerful factor in species delineation within Dermanyssus while not as powerful for interspecific relationships' resolution. Moreover, on the morphological basis, mites in this isolate key to D. gallinae, except that setae j1 and s1 are off the dorsal shield, sensu Moss (1978). These characters make it closer to D. gallinoides, however, leg chaetotaxy is typical of D. gallinae. As a result, the single-molecule analysis is considered here to be a sufficient in order to estimate that isolate JOW is strongly isolated from D. gallinae s. str. as well as L1 on the reproductive point of view. From this point, here will be referred to this entity as D. sp. JOW.
Two other gallinae-like isolates have been sampled in some pigeons in the USA (JOW_N33 and JOW_123). Both revealed to belong to the special lineage L1 of D. gallinae (Roy et al., 2009a, b). This special lineage has been recorded almost exclusively on pigeons at several locations in France and proves here to be also present on North American pigeons. COI (accession numbers FN646512-21) and Tropomyosin (accession numbers FN646507-8) sequences obtained from these two isolates are extremely close to the L1 French isolates haplotypes (1-2% divergence in COI, 0-1% in Tropomyosin intron n between American and French isolates, as well as between French isolates).
Finally, the DNA sequencing of some mitochondrial regions showed that isolates of O. sylviarum from French bird nests under test in the present study were conspecific to the North American poultry farm isolate OSBM. Based on an rRNA 16S region isolated following Roy et al. (2009a), isolates sampled in France (EMBL accession numbers: FN599080, FN646506) and isolate OSBM (accession number: FN599081) are diverging by 2-3% from each other, and by 1-2% from an isolate sampled from African Gold Breasted Starlings (Cosmopsarus regius) (accession number AY185362) (Schrenzel et al., 2003). Based on the COI coding region, OSBM (accession number FN599077) and isolates sampled in France (accession numbers FM179677, FN432541, FN432544, FN432545, FN432547, FN432548, FN599074) were diverging by 2-3% from each other. Within these French isolates, individuals sampled in tit nests located in the South of France from orchards were diverging by 1% from the two isolates sampled in nests of Montagu's Harrier, Circus pygargus (Linnaeus, 1758) in the Western North of France (FS5 and FS6, accession numbers FN432544, FN432545, FN432547, FN432548) and from individuals sampled in a canary cage in the east-central France (Rh1, accession number FN646526). FS5, FS6 and Rh1 provided exactly the same haplotype. In contrast, 14-15% divergence separates all these North American and Branch length are much shorter (more than 3 times shorter) between each other than between their common clade and their common ancestor with O. bacoti (Figure 3). All that strongly suggests that variations between included O. sylviarum samples do be intraspecific variations, not interspecific ones.

DISCUSSION
Among the eight species of hematophagous Mesostigmata recorded in the present study, the Dermanyssus species have been encountered more frequently than the Ornithonyssus species. Nevertheless, O. sylviarum is the only species of Ornithonyssus which has been repeatedly isolated, whereas each of the five species of Dermanyssus have been isolated rather frequently. The laelapid Myonyssus is a genus known to parasitize shrews and its presence is likely to be fortuitous (a single individual in a single nest).  It is obvious that O. sylviarum and D. gallinae are both present in the wild avifauna in France, and in some non-industrial bird breeding facilities (pet birds, game birds), but the former is absent to date from industrial fowl farms in France, especially from layer farms.
This contradicts the report of Bruneau et al. (2002). And yet, arguments advanced by these authors for mite identification are not discriminating at the specific level, nor even at the generic level, but are characters allowing a family-level discrimination.
Phylogenetic topologies based on mtDNA sequences do not highlight important distances between populations from layer farms (American isolate OSBM) and from the wild avifauna (French and African samples). Present data do not show that populations from farms and from the wild avi-fauna are different. However, this does not either mean that these populations are closely related and what's the more that transfers are at all possible. A close relationship between poultry and wild populations of D. gallinae is also established in Roy et al. (2009b), but no common COI haplotype has been isolated between wild and domestic birds, although mites collected in some very distant farms share the same haplotypes. In both cases, much larger sample batches along with analyses using population genetic tools are needed in order to assess the real closeness of populations parasitizing wild and domestic birds.
In any case, it is worth noting that O. sylviarum is present in environments where synthetic molecules targeting arthropoda are largely applied such as conventional orchards and wheat fields (see Appendix 1). O. sylviarum has shown being able to develop resistance to inhibitors of acetylcholinesterase as well as to pyrethinoids in the USA (Mullens et al., 2004). Populations in this species are regularly exposed to organophosphates (Ravap EC ® ) or carbamates (sevin-80s ® ) in North American poultry farms, not only by spraying, but also in some cases by digging (Mullens et al., 2009;Rubinoff I., pers. comm.), due to the typical parasitic habits of O. sylviarum described above. And yet, it is also exposed to various pesticides in French pet birds breeding facilities, including organophosphates and carbamates (M. Serre, Association Ornithologique Rhodanienne, pers. comm.), which may have resulted in a selection of resistant populations in France too. This might explain its presence in conventionally controlled crop farm agroecosystems. European layer farms are regularly treated with organophosphates and carbamates too, as well as other synthetic pesticides (such as amitraze, for instance). In France, in particular, an organophosphate-based pesticide (phoxim, Bye-Mite ® /Bye-Pou ® ) is allowed for usage not only between, but also during flocks since 2007. And many products composed of various molecules, including organophosphates (e.g. dichlorvos-based products) or carbamates (e.g. Carbyl ® ), have long been used illegally during flocks, as a consequence of the lack of allowed products for this period. All these prod-ucts are sprayed in the farm buildings, which does not differ so much from North American use. As a result, it is very unlikely that pesticide use might be the cause of the disbalance in the red mites' distribution between North American and European layer farms.
It is not impossible that O. sylviarum is currently in the process of colonizing European farms, since a few cases of infestations in layer farms have been recently reported in Northern Europe (J. Chirico, pers. comm.). And some pet bird as well as pheasant breeders provided accounts suggesting that O. sylviarum is in the process of invading their facilities (red mites staying on host since a few years). Some population genetics would be needed in order to determine whether populations found in farms in Europe come from wild birds or correspond to different populations, which might have potentially been transferred by human activities.
In the USA, no mite belonging to D. gallinae s. str. has been isolated in this study. The two samples from North American wild avifauna containing mites belonging to D. gallinae clearly belong to the special lineage L1. This confirms the specific status of this cryptic entity as already suggested in Roy et al. (2009a) and Roy (2009). Indeed, cryptic species are species which do not possess any morphological divergence with their closest sister(s) and are usually the result of a recent speciation event. Establishing the specific status of cryptic entities is not so easy. Overall, reproductive isolation corroborations need the congruence of more than one line of evidence, for instance morphology and DNA sequences. In the case of cryptic species, morphological characters do not help. Consequently, it was necessary to explore some other characters (DeSalle et al., 2005), such as life histories or ecological associations. In the case of lineage L1, molecular assessment has been checked (Roy, 2009) by comparing topologies based on mt-and nDNA and including isolates of several different origins in France. Isolates under test in this study were almost solely pigeons. In the present study, the close association of L1 with pigeons is confirmed. This characteristic, along with specific COI (mtDNA) and Tropomyosin (nDNA) haplotypes is maintained throughout Eu-rope and America. This is a considerable insight in favour of a complete reproductive isolation between this lineage and other D. gallinae entities, which appears to be more a cryptic species complex.
As for mite samples from layer farms collected in the USA, no D. gallinae s. str. has been isolated, but the number of samples is far too small to establish the presence/absence of this species in North American layer farms. J. Owen and B. Mullens, although having performed recurrent experiments in layer farms (Mullens et al., 2001(Mullens et al., , 2009Mullens, 2004, Owen et al., 2009, . . . ), only sampled mites belonging to O. sylviarum with one exception (J.O. and B.M. pers. comm.), which provided mites belonging to a non gallinae species (D. sp. JOW). Their studies did not target D. gallinae, which should be searched for by different means, due to its nidicolous habits. As a result, it is difficult to establish the presence/absence of D. gallinae in North American layer farms, due to the lack of sampling data in such environments. Nevertheless, the isolation of a non gallinae species in a North American layer farm is very interesting. Roy et al. (2009a, b) have shown that previous European records based on morphological identification alone were at least partly erroneous in the morphologically plastic species composing the gallinae group sensu Moss (1968Moss ( , 1978. And yet, Moss et al. (1970) have highlighted the likely frequent misidentifications which might have led to consider D. gallinae more often present than it was in the USA. These authors have shown that non gallinae species in the genus Dermanyssus have likely been repeatedly confused with D. gallinae. Consequently, given that no study involving morphological along with molecular analyses in the North American Dermanyssus acarofauna have been performed, the ecological distribution of D. gallinae in this continent is to be considered unkown. D. gallinae s. str. is the only species of Dermanyssus recorded in layer farms in other parts of the world, including South America according to present data. Moreover, in France, non gallinae species of Dermanyssus appear strictly restricted to the wild avifauna. Is D. sp. JOW a second Dermanyssus species adapted to layer farm environments? Or was its presence in a small free range hen house a fortuitous one? It is worth noting that D. gallinae s. str. is a relatively recent problem in Brazilian layers, since O. bursa was the predominant ectoparasite around 1939 and D. gallinae was established being more common in 1997 (Tucci et al., 2008). The causes of this colonization in process in Brazilian farms remain unclear. It is not known whether D. gallinae s. str. was present in the wild avifauna in Brazil before.
Many questions remain unsolved here. It would be especially very useful to further explore the "dermanyssofauna" in North American layer farms as well as in the wild avifauna, to check whether D. sp. JOW is recurrently present or not, and whether D. gallinae s. str. is also present or not.

CONCLUSION
It is remarkable that O. sylviarum is so commonly encountered in French bird nests under test, yet absent from French layer farms, whereas it is a serious pest in layer farms in the USA. This suggests that different farming practices between both continents might explain the paradox of the omnipresence of both species in wild avifauna and a selective presence in French layer farms. Nevertheless, this difference does not seem to be attributable to pesticides commonly used in these farms. In any case, it remains to be checked whether the opposite applies to D. gallinae s. str. in the USA. It will be also interesting to look for additional samples of D. sp. JOW and characterize it using some other DNA sequences, in order to check it specific status and establish whether it is recurrently present in North American layers.
In any case, it is not excluded that populations under test in either O. sylviarum (France and USA) or D. gallinae (Europe, Brazil, Australia), although respectively conspecific, are genetically different and do not possess the same ability to parasitize hens in farms. Some population genetics in both species are needed in order to assess it. And more generally further surveys of ectoparasites in both wild nests and domestic fowl farms are lacking in the USA and are desperately needed in order to answer such important questions.