Erythropoietin Delivery by Genetically Engineered Bone Marrow Stromal Cells for Correction of Anemia in Mice with Chronic Renal Failure
Nicoletta Eliopoulos*,
Raymonde F. Gagnon,
Moira Francois* and
Jacques Galipeau*,
* Lady Davis Institute for Medical Research, Division of Nephrology, McGill University Health Center, and Division of Hematology/Oncology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada
Address correspondence to: Dr. Jacques Galipeau, Department of Medicine and Oncology, Sir Mortimer B. Davis Jewish General Hospital & Lady Davis Institute for Medical Research, McGill University, 3755 Cote Sainte-Catherine Road, Montreal, Quebec, Canada H3T 1E2. Phone: 514-340-8214; Fax: 514-340-8281; E-mail: jgalipea{at}lab.jgh.mcgill.ca
Received for publication October 6, 2005.
Accepted for publication March 26, 2006.
The goal of this research was to develop a strategy to couplestem cell and gene therapy for in vivo delivery of erythropoietin(Epo) for treatment of anemia of ESRD. It was shown previouslythat autologous bone marrow stromal cells (MSCs) can be geneticallyengineered to secrete pharmacologic amounts of Epo in normalmice. Therefore, whether anemia in mice with mild to moderatechronic renal failure (CRF) can be improved with Epo gene-modifiedMSCs (Epo+MSCs) within a subcutaneous implant was examined.A cohort of C57BL/6 mice were rendered anemic by right kidneyelectrocoagulation and left nephrectomy. In these CRF mice,the hematocrit (Hct) dropped from a prenephrectomy baselineof approximately 55% to 40% after induction of renal failure.MSCs from C57BL/6 donor mice were genetically engineered tosecrete murine Epo at a rate of 3 to 4 units of Epo/106 cellsper 24 h, embedded in a collagen-based matrix, and implantedsubcutaneously in anemic CRF mice. It was observed that Hctincreased after administration of Epo+MSCs, according to celldose. Implants of 3 million Epo+MSCs per mouse had no effecton Hct, whereas 10 million led to a supraphysiologic effect.The Hct of CRF mice that received 4.5 or 7.5 million Epo+MSCsrose to a peak 54 ± 4.0 or 63 ± 5.5%, respectively,at 3 wk after implantation and remained above 48 or 54% for>19 wk. Moreover, mice that had CRF and received Epo+MSCsshowed significantly greater swimming exercise capacity. Inconclusion, these results demonstrate that subcutaneous implantationof Epo-secreting genetically engineered MSCs can correct anemiathat occurs in a murine model of CRF.
Anemia, a hallmark of ESRD, is due in part to the reduced productionof erythropoietin (Epo) by the kidney. Recombinant human Epo(rhEpo) utilization has considerably improved the managementof anemia of patients with ESRD and markedly reduced the requirednumber of blood transfusions (1–3). With rhEpo treatment,patients also benefit from secondary advantages, such as improvedcardiac function, enhanced exercise capacity, and better qualityof life (3). However, rhEpo administration also includes undesirableaspects: the intermittent high-dose nature of rhEpo treatmentunlike the normal continuous endogenous Epo production; thepossibility of antibody formation against rhEpo and its cross-reactingwith endogenous Epo, potentially leading to pure red cell aplasia;the possible noncompliance of patients to self-administer rhEpoinjections or to follow recommended instructions; and the highcost and limited financial resources that confine rhEpo useto patients with severe anemia. In view of the aging populationand consequently predicted rise in renal disease, rhEpo-relatedexpenditures are anticipated to escalate, as will the need todevelop cost-effective alternatives. One such alternative treatmentthat we propose herein is a strategy to couple cell and genetherapy, whereby patients with anemia would receive subcutaneousimplantation of a neo-organoid that encloses autologous cellsthat have been genetically engineered to secrete Epo.
One autologous cell type, bone marrow stromal cells (MSCs),which are important in hematopoiesis and in the bone marrowmicroenvironment, also has valuable characteristics that renderit a desired autologous cellular vehicle for the systemic deliveryof therapeutic proteins (4–8). MSCs are plentiful in humansof all ages; can be obtained with minimal discomfort; can beexpanded, genetically engineered, and clonally selected in vitroeasily; and can be reimplanted in the donor without a conditioningregimen (9–12). We therefore hypothesized that MSCs thatare harvested from a patient can be gene-modified in vitro toexpress Epo and subsequently implanted subcutaneously in thesame patient as a neo-organoid that allows sustained, clinicallyrelevant production of Epo to correct anemia, reducing morbidityand improving health status.
We previously published proof-of-concept studies with murineMSCs that were genetically engineered to secrete murine Epo(mEpo). In brief, we reported significant mEpo production frommEpo gene-modified MSCs (Epo+MSCs) that were implanted in nonmyeloablated,immunocompetent mice via the intraperitoneal or subcutaneousroute of delivery (13). We showed that embedding these Epo+MSCswithin a matrix before their subcutaneous administration ledto a more significant and prolonged pharmaceutical effect, asascertained by resulting hematocrit (Hct). We observed thatthis enhanced outcome that was achieved when Epo+MSCs were embeddedwithin the mouse-compatible material Matrigel (13) also occurredwhen the human-compatible, FDA approved, bovine collagen-basedmatrix Contigen was used (14). Furthermore, we demonstrateda retrievability safety feature of our strategy, confirmed byour observation that upon retrieval of implants of Contigen-embeddedEpo+MSCs, the effect on the Hct was reversed (14). The sum ofthese observations supported the notion that a cell and genetherapy approach with genetically engineered MSCs would be feasibleand advantageous for the treatment of anemia in ESRD.
In this investigation, we tested our strategy in a mouse modelof anemia secondary to experimental renal failure, a well-toleratedprocedure, and consequently noted that subcutaneous implantationof Contigen-embedded Epo+MSCs in these anemic mice led to acell dose–dependent increase in plasma Epo and in Hctlevels, as well as significantly enhanced exercise capacity.
Generation of Retrovirus-Producing Cells and Gene-Modified Primary Murine MSCs
We generated a retroviral plasmid construct (pEpo; Figure 1)that contained the cDNA for mEpo and used it to prepare retrovirus-producingcells GP+E86-Epo, as reported previously (14). Control GP+E86-EmptyVectorvirus producers were similarly produced (14).
Figure 1. Retroviral plasmid DNA construct (pEpo). pEpo was generated as described in Materials and Methods. The murine Epo (mEpo) cDNA was inserted into the multiple cloning site (MCS) of pEmptyVector that contained an ampicillin resistance gene (AmpR), allowing for selection of successful clones. The cytomegalovirus (CMV) promoter drives transgene expression in cells that are transfected with the retroviral plasmid, whereas the long terminal repeat (LTR) promoter controls expression in cells that are transduced with the retroviral particles that are derived from transfected retrovirus-producing cells. pEpo, as all retroviral vectors, contains a packaging signal to enable the RNA transcribed to be packaged within retroviral particles.
A female 15- to 20-g C57BL/6 mouse (Charles River, LaprairieCo., St. Constant, QC, Canada) was killed, and whole bone marrowwas collected by flushing femurs and tibias with complete media(DMEM supplemented with 10% FBS and 50 U/ml Pen/Strep); after5 d at 37°C with 5% CO2, the nonadherent hematopoietic cellswere removed, and the adherent MSCs were maintained for approximately15 passages. MSCs were genetically engineered by transductionwith filtered retroviral supernatant from GP+E86-Epo, givingrise to the polyclonal population of mEpo gene-modified MSCs,as described previously (14). The monoclonal subpopulation ofmEpo gene-modified MSC that was used in this study (Epo+MSCs)was generated after the polyclonal population was plated atlimiting dilutions and one clone was isolated and expanded.A supernatant sample from Epo+MSCs was used in an ELISA thatwas specific for hEpo (Roche Diagnostics, Indianapolis, IN).Control EmptyVector, which lacks the mEpo cDNA, MSCs likewisewere prepared and referred to as control MSCs (14). Animalswere handled under the guidelines promulgated by the CanadianCouncil on Animal Care.
Differentiation of MSCs
To verify the in vitro differentiation capabilities, we exposedEpo+MSCs to specific media. For induction of osteogenic differentiation,Epo+MSCs were cultured for 4 wk in complete medium that wassupplemented with -glycerol phosphate (10 mM), ascorbic acid2-phosphate (5 µg/ml), and dexamethasone (10–8 M;Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) (15,16), andsubsequently stained with Alizarin Red S (Sigma-Aldrich Canada;pH 4.1 using ammonium hydroxide) (16). For induction of adipogenicdifferentiation, Epo+MSCs were cultured for 7 d in completemedium that was supplemented with insulin (10 µg/ml),dexamethasone (1 µM), 3-isobutyl-methylxanthine (0.5 mM),and indomethacin (46 µM; Sigma-Aldrich Canada) (17), andthen stained with Oil Red O (Sigma-Aldrich Canada) (15).
Generation of Murine Model of Anemia from Renal Failure
Renal failure was produced in female C57BL/6 mice (15 to 20g) by electrocoagulation of the right kidney surface, leavinga small portion of the hilum intact, and 2 wk later surgicalablation of the contralateral left kidney, as previously published(18–21). Blood samples were collected from the saphenousvein, using heparinized micro-Hct capillary tubes (Fisher Scientific,Pittsburgh, PA), before and after induction of anemia, and usedfor determination of Hct by standard micro-Hct method, of plasmaurea nitrogen concentration by colorimetric method (Urea NitrogenReagent, Colorimetric Method; Teco Diagnostics, Anaheim, CA),and of plasma Epo concentration by ELISA (Roche Diagnostics).These test mice developed, within approximately 3 wk of leftnephrectomy, mild to moderate anemia with Hct of approximately40%. The Hct of normal C57BL/6 mice was 56 ± 0.4%. Moreover,this model of CRF with anemia has been described to induce inmice the development of a constellation of other abnormalitiesalso noted in humans with CRF (18–20). In this well-characterizedmurine model, the anemia was reported to respond to rhEpo ina dose-dependent manner (18,19).
Implantation of Gene-Modified MSCs and Analysis on Recipient Mice
Once the Hct remained steady at its lowered levels at approximately1 mo after left nephrectomy, i.e., induction of CRF, gene-modifiedMSCs were implanted subcutaneously within a matrix scaffolding,similar to our previous work (14). In this study, taking intoaccount our published findings in normal mice, various doses(3 to 10 million cells per mouse) of Epo+MSCs were implantedin cohorts of anemic C57BL/6 mice (referred to as CRF mice;n = 6 to 8 per group) to allow for effect comparison. NormalC57BL/6 mice likewise received implants. More specific, Epo+MSCswere trypsinized; concentrated by centrifugation; and 3 x 106,4.5 x 106, 7.5 x 106, or 107 cells were resuspended in 50 µlof RPMI medium (Wisent, Rocklin, CA) and then approximately500 µl of Contigen (C.R. Bard Inc., Covington, GA). Contigen-embeddedcells were injected subcutaneously in the right flank of CRFand of normal mice. In addition, control CRF mice were generatedthrough implantation with Contigen-embedded control MSCs. Wholeperipheral blood was collected from the saphenous vein beforeimplantation and once per week or more after implantation andused for measurements of Hct, plasma urea nitrogen, and plasmamEpo levels. Furthermore, mice that received implants of either4.5 x 106 or 7.5 x 106 cells were assessed for exercise capacityby recording of the length of time they were able to swim continuouslywhen placed in a container with 32°C water.
Statistical Analyses
The t test was conducted to compare the various groups, andP < 0.05 was deemed significant.
Characterization of Gene-Modified MSCs
Murine MSCs were genetically engineered by transduction withretroviral particles from virus-producing cells that were generatedby transfection with retroviral plasmid pEpo (Figure 1). A subpopulationof resulting Epo+MSCs, which were shown to secrete in vitro3 to 4 units of Epo/106 cells per 24 h, was used in this study.Control MSCs, engineered with a control retroviral vector thatlacks Epo, were shown by ELISA to secrete <0.02 units ofEpo/106 cells per 24 h. No difference was noted in the in vitrogrowth of Epo+MSCs and of control MSCs that were maintainedand expanded in culture plates.
The cell surface marker phenotype of Epo+MSCs was analyzed previouslyby flow cytometry and shown to be CD31–, CD45–,Mac1–, CD34+, CD44+, CD73+, CD90+, and CD105+ (22). Thesephenotypic markers confirm that MSCs are not hematopoietic cells(by the absence of CD45) and do not express the endothelialmarker CD31 (also known as platelet-endothelial cell adhesionmolecule-1). In fact, MSCs often are referred to as mesenchymalstem cells because of their ability to differentiate into mesenchymalcell types (23–25), and this functional cellular featuredefines and distinguishes MSCs from endothelial cells and otherend-differentiated somatic and progenitor cells that residein marrow. For us to verify the mesenchymal differentiationcapability of our cells, Epo+MSCs were exposed in vitro to specificagents that induce osteogenic or adipogenic differentiation.As shown in Figure 2, when our undifferentiated cells (Figure 2A)were exposed to -glycerol phosphate, ascorbic acid 2-phosphate,and dexamethasone, they developed an osteogenic phenotype (Figure 2B),whereas when exposed to insulin, dexamethasone, 3-isobutyl-methyl-xanthine,and indomethacin, they acquired an adipogenic phenotype (Figure 2C).
Figure 2. Differentiation ability of marrow stromal cells (MSCs). As described in Materials and Methods, Epo gene-modified MSCs (Epo+MSCs) undifferentiated (A), when 70 to 80 or 50 to 60% confluent, were cultured in conditions that were inductive of osteogenic or adipogenic differentiation, respectively. After osteogenic differentiation, calcium in the mineralized extracellular matrix was shown by Alizarin Red S staining (B). After adipogenic differentiation, lipid droplets were evident by light microscopy (C) as well as by their staining with Oil Red O (C, insert). Epo+MSCs, before and after differentiation, were photographed under bright-field light microscopy using an Axiovert25 Zeiss microscope attached to a Contax167MT camera. Magnifications: x100 in A, x50 in B, x400 in C.
Hct of Mice Implanted with Gene-Modified MSCs
Our experimental procedure to evaluate the effect of mEpo-secretingMSCs on the anemia of mice with experimental renal failure isillustrated in Figure 3 and is described in Materials and Methods.A cell-dose-finding series of experiments were performed todetermine the optimal amount of Epo+MSCs in neo-organoids forphysiologic correction of anemia in CRF mice. We tested implantsthat comprised 3 x 106, 4.5 x 106, 7.5 x 106, and 10 x 106 cells.As seen in Figure 4, a dose-response was observed between thenumber of Epo+MSCs and the resulting effect on the Hct. A correlationwas noted between the number of Epo+MSCs that were implantedin CRF mice and the percentage increase in Hct (R2 = 0.911 atday 80 after electrocoagulation of right kidney). With the lowestdose of 3 x 106 Epo+MSCs per mouse, no correction of anemiaoccurred, as no Hct change was noted in anemic mice, althoughan increase in Hct arose in normal mice, up to a maximum valueof 71 ± 1.8% (mean ± SEM) at approximately 4 wkafter implantation (Figure 4A). The dissimilarity in the responseto the 3 x 106 Epo+MSCs between normal and CRF mice indicatedthat although CRF mice had a deficiency in Epo, they, unlikenormal mice, are resistant to its action. CRF mice that received3 x 106 Epo+MSCs or 3 x 106 control MSCs showed similar Hctover time. Epo resistance, as we observed here in CRF mice withthis lowest tested amount of Epo+MSCs, resembles observationsmade in humans with renal impairment and is attributed to theeffect of the uremic environment on the bone marrow (26–28).Erythropoiesis may be influenced not only by Epo deficiencybut also by other factors, such as intramedullary hemolysisand reduced red cell lifespan (29–31).
Figure 3. Overview of implantation procedure. As detailed in Materials and Methods, we first harvested the femurs and tibias of a normal C57BL/6 mouse, harvested the whole marrow and placed it in culture, recovered the MSCs, retrovirally engineered them to secrete mEpo, embedded resulting Epo+MSCs within the matrix Contigen, and implanted them subcutaneously in syngeneic mice that previously were rendered anemic by right kidney thermal injury and subsequent left nephrectomy. Blood from recipient mice was collected over time and analyzed for the determination of hematocrit (Hct) and of plasma Epo and urea nitrogen levels. As negative controls, anemic mice similarly received implants of control MSCs. Moreover, as positive controls, normal mice received implants of Epo+MSCs.
Figure 4. Hct of mice that received matrix-embedded MSCs. As described in Materials and Methods, mice with chronic renal failure (CRF) received implants of various amounts of Epo+MSCs or of control MSCs, and their peripheral blood was collected and Hct was determined over time. More specific, electrocoagulation of the right kidney was performed at day 0, the left kidney was removed at the day indicated by vertical line, and MSCs were implanted at the day indicated by the arrow. CRF mice received Epo+MSCs () at 3 x 106 (A), 4.5 x 106 (B), 7.5 x 106 (C), or 10 x 106 (D) cells/mouse or control MSCs () at 3 x 106 (A), 7.5 x 106 (B and C), or 10 x 106 (D) cells/per mouse. Normal mice also received implants of Epo+MSCs (, dotted curve) at 3 x 106 (A), 4.5 x 106 (B), 7.5 x 106 (C), or 10 x 106 (D) cells/mouse.
In contrast, with the highest dose of 10 x 106 Epo+MSCs permouse, Hct response was supraphysiologic both in normal andin CRF mice (Figure 4D). The Hct in CRF mice, as in normal mice,rose to levels >80%, more precise, to 85 ± 1.1%, byapproximately 4 wk after Epo+MSCs implantation, whereas theHct was not significantly altered in CRF mice that receivedthe control MSCs (Figure 4D).
The intermediate doses of 4.5 x 106 and 7.5 x 106 Epo+MSCs ledto more physiologic Hct levels in CRF mice, as shown in Figure 4,B and C, respectively. More specific, in CRF mice that received4.5 x 106 Epo+MSCs, the Hct that had fallen from basal 57 ±0.6 to 40 ± 1.1% after induction of mild to moderaterenal failure increased to 54 ± 4.0% at approximately3 wk after Epo+MSCs implantation and remained above 48% for>16 wk more (Figure 4B). In CRF mice that received 7.5 x106 Epo+MSCs, the Hct that had declined from basal 55 ±0.7 to 40 ± 0.8% after renal failure induction rose to63 ± 5.5% at 3 wk after Epo+MSCs implantation and persistedat >54% for more than another 16 wk (Figure 4C). In contrast,in CRF mice that each received 7.5 x 106 control MSCs, the Hctremained low, below normal, at values of 39 to 44% (Figure 4,B and C). A significant statistical difference was noted betweenthe Hct data of CRF mice that received Epo+MSCs at the celldoses of 4.5 x 106, 7.5 x 106, and 10 x 106 cells/mouse andthat of CRF mice that received control MSCs (P < 0.005, Ttest; Figure 4, B through D). Moreover, in normal mice thatreceived 4.5 x 106 or 7.5 x 106 Epo+MSCs, the Hct rose to levelsthat were higher than the increased levels that were achievedin CRF mice, as revealed in Figure 4, B and C, pointing to thepresence of a relative Epo resistance in CRF mice.
Plasma Urea Nitrogen and Epo Concentrations in Relation to Hct and Exercise Capacity
To ascertain whether our groups of experimentally induced CRFmice had comparable kidney dysfunction, we assessed blood plasmafor urea nitrogen levels. As shown in Figure 5A, plasma ureanitrogen concentrations in CRF mice at 2 wk after implantationof Epo+MSCs or of control MSCs were elevated similarly, approximatelythree-fold higher than normal, with no significant differencedetected between CRF groups. More precise, CRF mice that received4.5 x 106 Epo+MSCs, 7.5 x 106 Epo+MSCs, or 7.5 x 106 controlMSCs had plasma urea nitrogen levels of 78 ± 5.8 (mean± SEM), 76 ± 8.1, or 86 ± 4.7 mg/dl, respectively,in contrast to 25 ± 2.0 mg/dl in normal mice that didnot receive implants (Figure 5A). In addition, the concentrationof urea nitrogen in plasma samples of anemic mice that received3 x 106 or 10 x 106 Epo+MSCs was 69 ± 2.9 or 79 ±5.3 mg/dl, respectively, at approximately 2 wk after implantation,similar to that of anemic mice that received 3 x 106 or 10 x106 control MSCs (79 ± 8.0 or 72 ± 3.8 mg/dl,respectively; n = 6 to 9).
Figure 5. Levels of plasma urea nitrogen and Epo, Hct, and exercise capacity of mice. As indicated in Materials and Methods, CRF mice that received Epo+MSCs or control MSCs at 4.5 x 106 or 7.5 x 106 cells/mouse were evaluated at 2 wk after implantation for plasma levels of urea nitrogen (A) and Epo (B), for Hct (C), as well as for exercise capacity, as determined by swimming duration (D). In addition, normal mice that did not receive implants were evaluated likewise (right).
To detect any changes in plasma Epo levels in CRF mice thatreceived Epo+MSCs, we evaluated the plasma concentration ofmEpo by ELISA for hEpo, as similarly used in other reportedstudies (13,32,33). The ELISA’s specificity for hEpo rendersa poorer sensitivity for mEpo (34), indicating that the plasmamEpo concentrations that are detected are likely to be underestimationsbut nevertheless valuable for comparison purposes. As shownin Figure 5B, plasma Epo concentrations in CRF mice 2 wk afterEpo+MSCs implantation were significantly greater than in CRFmice that received control MSCs (P < 0.05, t test). In brief,levels were 35 ± 8.9 or 83 ± 32 mU/ml in CRF micethat received 4.5 x 106 or 7.5 x 106 Epo+MSCs, respectively,in contrast to 17 ± 2.4 mU/ml in CRF mice that receivedcontrol MSCs (Figure 5B).
We show that Hct response correlates with cell dose and plasmaEpo levels in treated animals. In CRF mice that received 4.5x 106 or 7.5 x 106 Epo+MSCs, the Hct was 47 ± 2.7 or57 ± 3.9%, respectively, 2 wk later, both significantlysuperior to the Hct of CRF mice that received control MSCs (P 0.05, t test; Figure 5C). Moreover, the concentration of Epoin plasma of anemic mice that received 3 x 106 Epo+MSCs embeddedin Contigen was 6.8 ± 0.9 mU/ml at approximately 2 wkafter implantation, similar to that of anemic mice that received3 x 106 control MSCs (6.0 ± 1.7 mU/ml), whereas the plasmaEpo concentration of anemic mice approximately 2 wk after implantationwith 10 x 106 Epo+MSCs was considerably increased at 160 ±40 mU/ml, in contrast to that of anemic mice that received 10x 106 control MSCs (11 ± 2.1 mU/ml).
It is recognized that anemia alters exercise ability in humansand that treatment with rhEpo of patients with renal failureprovides improved energy and physical activity (35,36). Therefore,to demonstrate a physiologic correlate to increased Hct thatwas induced by mEpo-secreting MSCs, we subjected mice to a swimmingendurance test. As displayed in Figure 5D, CRF mice that receivedEpo+MSCs, as compared with those that received control MSCs,showed significantly greater swimming endurance at approximately2 mo after implantation (P < 0.05, t test). CRF mice thateach received 4.5 x 106 or 7.5 x 106 Epo+MSCs swam for 155 ±24 or 173 ± 19 s, respectively, whereas CRF mice thatreceived control MSCs swam for 95.6 ± 13 s before stopping.
Recipient mice were weighed over time, and no significant differencewas noted among the various groups of mice. In addition, nosignificant difference was observed in the survival of treatedmice.
Studies on the treatment of renal anemia, other than with rhEpoadministration, have included gene therapy approaches, wherebythe Epo gene is transferred directly to the host either throughviral or nonviral means, as well as cell and gene therapy strategies,such as ours, whereby the Epo gene is transferred to cells invitro and the cells subsequently are implanted in vivo to serveas Epo-releasing vehicles. Indeed, several studies, includingour previous published work, have reported the use of autologousMSCs for the delivery of plasma-soluble therapeutic proteinsin vivo in immunocompetent hosts and via various routes of administration(9,11,37–40). The main purpose of our study was to adaptthis biopharmaceutical approach for the delivery of Epo in vivofor the treatment of anemia consequent to ESRD. The potentialbenefits of this biopharmaceutical strategy can be contextualizedto past published experience in cell and gene therapy approachesto Epo-deficient anemia.
Gene therapy studies for anemia correction have included theutilization of plasmid DNA and of adenoviral and adeno-associatedviral vectors that express Epo. With the use of plasmid DNAencoding for rat Epo, studies reported significantly increasedHct long term after plasmid electroporation into skeletal musclesin rats that were rendered anemic by 5/6 or subtotal nephrectomy.The Hct response was proportional to the amount of plasmid DNAdelivered (41,42). It must be noted that the Epo gene in theEpo+MSCs retroviral construct is not under physiologic hypoxia-induciblefactor-1 control, and, as such, constant, high-level productionof Epo may lead to polycythemia in otherwise normal experimentalmice (13,14), as well as in CRF mice (Figure 4D). One remedyto decrease the incidence of polycythemia is to control thetherapeutic effect by the number of implanted Epo-secretingMSCs. As shown in Figure 4, we determined that the magnitudeof the increase in blood Hct that was caused by Epo releasein vivo depended on the amount of implanted Epo+MSCs. We controlledthe rise in Hct and obtained a more physiologic effect in CRFmice (Figure 4, B and C) by adjusting the number of Epo+MSCs.Furthermore, the effect can be regulated by the number of neo-organoidsimplanted, and it also may be adjusted after implantation throughthe removal of an implant. As we have published previously,our neo-organoid approach is reversible, as removal of the implantsof Contigen-embedded Epo+MSCs from recipient mice led to a dropand return of Hct levels to preimplantation basal values within14 d (14). The complete reversal of the endocrine effect afterthe removal of the collagen-based organoids indicates that thebulk of engineered MSCs that are sequestered within the implantdo not migrate out of the organoid (14). In a further study,in which normal mice received two implants each, we showed thatremoval of one of two implants led to a lowering of the Hctlevel (data not shown). Hence, adjusting cell dose can avoidthe pitfalls of polycythemia and skirts the need for hypoxia-sensinggenetic devices that may operate aberrantly or ineffectivelyoutside the kidney parenchyma and microvasculature. Moreover,MSCs can be genetically engineered to allow regulatable transgeneexpression, as we have published previously using a glucocorticoid-induciblesystem (43).
With the use of viral vectors, reports revealed increased Hctlevels through intraperitoneal administration of a replication-defectiveadenoviral vector that expresses hEpo in mice with CRF frompolycystic kidney disease (44), as well as through intravenousinjection of a fully deleted helper-dependent adenoviral vectorthat expresses mEpo in subtotally nephrectomized rats (45).Amelioration of the anemia occurred through Epo but not of theuremia (45), as we also noted in our study. In a further investigation,the intramuscular injection of an adeno-associated virus vectorconstruct with a hypoxia responsive promoter brought about physiologicallyregulated expression of mEpo and resulting long-term normalizedHct in anemic Epo-deficient Epo-TAgh transgenic mice (46). However,the use of adenoviral and adeno-associated viral vectors isassociated with well-documented potential risks, including thethreat of serious, possibly lethal, host immune response (47–50).Moreover, regardless of whether the gene delivery vehicle isviral or nonviral, efficiency of gene transfer in vivo is unpredictable.Unforeseen negative events in fact have arisen in gene therapyof anemia studies that used virus vectors directly in vivo.In one report, severe anemia occurred through an acquired autoimmuneresponse to Epo after intramuscularly delivered adeno-associatedvirus vector that expressed Epo (51). We have not observed redcell aplasia or acquired anemia in any mouse—kidney failureor not—that received Epo-secreting autologous MSCs. Alikely explanation is that MSCs are genetically engineered invitro with nonimmunogenic replication-defective retroviral vectors.This gene expression system does not lead to expression of viralxenoproteins, and care was taken to avoid the co-expressionof any reporter/selection xenoprotein (e.g., drug-resistancegenes) that may lead to an immune response to gene-enhancedcells. Furthermore, because cells are gene-modified in vitrounder highly controlled conditions, there is absolutely no riskfor in vivo viral dissemination or transfer, and transgene expressionlevel is measured before implantation.
Combined cell and gene therapy strategies for treatment of anemiahave been reported using an array of cell types. Mesothelialcells have been investigated, specifically human and murinemesothelial cells that were transfected or transduced to produceEpo and subsequently implanted into 5/6 nephrectomized Balb/cor nude mice by intraperitoneal or intramuscular administration(52). Epo-secreting human mesothelial cells in nude mice, whenaffixed onto the peritoneum with a collagen matrix, led to detectablesystemic levels of Epo (52). Epo gene-modified murine mesothelialcells corrected the anemia of uremic mice and led to a greaterHct rise in nonuremic mice, although transiently (52). The higherHct enhancement in normal versus anemic mice, as we also noted,likewise seems to indicate an Epo resistance. In contrast toprevious work in the field of gene-enhanced cell delivery, ourplatform technology led to a long-term pharmaceutical effectthat persisted for >19 wk. Such a long-term beneficial outcomeis desired when looking for a clinically applicable substituteto rhEpo injections for overcoming anemia. As we previouslyreported (14), we did not note any vasculogenic differentiationof MSCs when embedded in the human-compatible collagen-basedmatrix Contigen, in contrast to our earlier observations withthe use of Matrigel (13). However, this does not seem to interferewith the desirable biopharmaceutical characteristics of theContigen organoid base in terms of its ability to provide astable extracellular matrix environment for genetically engineeredMSCs and to permit long-term, sustained delivery of the plasma-solublegene product in vivo. The effect, however, does subside slowlyover time and this may be associated with the physical decreasein size of the implant, likely as a result of gradual resorptionof the Contigen material as we have noted previously (14). Therefore,our cell and gene therapy strategy as used here in this formdoes have its limitations as a long-term approach to anemiatherapy. A loss of effect over time or, conversely a supraphysiologicresponse, may occur after a first treatment with our matrix-embeddedEpo-secreting cells and consequently may necessitate furthersubcutaneous injections or a resection of the neo-organoid,respectively. Therefore, clinical relevance would be enhancedthrough the use of an improved matrix and/or via prolongationof in vivo cell survival.
Another cell type that is used in cell and gene therapy investigationsis myoblasts. The C2 myoblast cell line that was transfectedwith hEpo and injected intramuscularly into nude mice with renalfailure caused significant Hct augmentation (53). In anotherstudy, C2C12 myoblasts that were transfected with mEpo and implantedsubcutaneously within semipermeable polyethersulfone hollowfibers restored hemopoiesis for up to 1 yr in anemic Epo-TAghmice after their immunosuppression (54). With our subcutaneousimplants consisting of collagen-embedded mEpo gene-modifiedprimary murine MSCs, a long-lasting effect occurred in nonmyeloablated,immunocompetent mice. Therefore, our alternative to rhEpo utilizationdoes not require any toxic conditioning or immunosuppressionpreparatory regimen of the host. We recently revealed that long-termuse of MSCs for cell and gene therapy applications, at leastin mice, is restricted to autologous settings, because a cellularand humoral immune response against allogeneic MSCs as wellas against Epo arose in allogeneic hosts (22). Therefore, Epogene-modified autologous MSCs, not allogeneic MSCs, would beused for Epo delivery for anemia treatment.
A clinical trial involving cell and gene therapy was performedin patients with anemia of CRF (55). Autologous dermal sampleswere genetically engineered with an adenoviral vector that expresseshEpo and returned to patients by subcutaneous implantation,subsequently revealing minimal morbidity, transient rise inEpo serum concentration, and increased reticulocyte numbers(55). The transient nature of the increased Epo levels, up toapproximately 10 d, was attributed to the occurrence of a cellularimmune response to the adenovector gene-modified dermal cores,likely against viral proteins that were expressed by the adenovirus(55). Besides adenoviruses, use of autologous terminally differentiatedsomatic cells, such as dermal fibroblasts, has its shortcomings.Skin fibroblasts may inactivate introduced vector sequencesafter transplantation (56,57) and may have an expansion abilityrestricted by the donor’s age (58), leading possibly tosuboptimal amounts for clinical utilization. In contrast, MSCspossess a strong expansion ability in vitro (5), also allowingfor high-efficiency retroviral engineering, and the selection,proliferation in culture, and utilization in vivo of selectedpopulations of gene-modified cells.
CRF mice that received Epo+MSCs acquired improved swimming durationconsequent to the persistent mEpo delivery (Figure 5). The beneficialeffect that was observed on exercise capacity, as determinedby swimming duration of CRF mice that received Epo+MSCs, pointsto advantages of such continuous Epo delivery that may be linkednot only to the correction of anemia. Epo is a multifunctionaltrophic factor with different sites of endogenous expression,a tissue-specific regulation, and several mechanisms of action(59–62). Functional Epo receptors not only have been identifiedon erythroid progenitor cells but also have been detected onnonerythroid blood cells, such as megakaryocytes, lymphocytes,and myeloid cells, and on various nonhematopoietic cells, suchas endothelial cells, myocardial cells, smooth muscle fibers,mesangial cells, cells of neural origin, and renal cells (62–64).The expression of Epo receptors on renal cells possibly mayallow Epo to exert a reparative effect on damaged kidney (64).Therefore, although the effect on Hct of Epo delivered by Epo+MSCsmay be similar to that of intermittent pulsed rhEpo, there maybe additional clinical benefit on organ systems that expressthe Epo receptor, such as on myocardium. Epo’s many effectsinclude cardioprotection and improved myocardial function (62,65–67).Therefore, the greater swimming time in CRF mice that receivedEpo-secreting MSCs may have arisen not only from the resultingincrease in Hct but also from a direct biologic effect on myocardiumthat would be observed in a setting of continuous Epo delivery,such as provided by our neo-organoid system.
Gene-enhanced autologous MSCs can serve as an effective customEpo production organoid to avoid pitfalls such as autoimmunecomplications that are associated with immunogenic vector systemsand allogeneic cell source. The high ex vivo proliferative capacityof MSCs makes it a more desirable cellular source than senescence-susceptibleautologous somatic cells. The subcutaneous retrievable implantstrategy allows for cell dosing and avoids the complexitiesof replicating physiologically responsive hypoxia sensors. Last,continuous low-dose Epo delivery provided by the cell-basedplatform may lead to unheralded desirable effects on organ systems,such as myocardium, which directly use Epo as a means to preserveand enhance function in addition to the Epo-dependent erythroideffects.
Acknowledgments
N.E. is a fellow of the US Army Medical Research and MaterialCommand Breast Cancer Research program (award DAMD17-02-1-0447).J.G. is a Canadian Institutes of Health Research Clinician-Scientist,and this project is supported by the Fonds de la Recherche enSanté au Québec Programme Hémovigilanceand by the Anemia Institute for Research and Education of Canada.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
The content of the information in this article does not necessarilyreflect the position or the policy of the US government.
Winearls CG, Oliver DO, Pippard MJ, Reid C, Downing MR, Cotes PM: Effect of human erythropoietin derived from recombinant DNA on the anaemia of patients maintained by chronic haemodialysis. Lancet 2
: 1175–1178, 1986[CrossRef][Medline]
Eschbach JW, Egrie JC, Downing MR, Browne JK, Adamson JW: Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial. N Engl J Med 316
: 73–78, 1987[Abstract]
Macdougall IC: Present and future strategies in the treatment of renal anaemia. Nephrol Dial Transplant 16
: 50–55, 2001[Abstract/Free Full Text]
Clark BR, Keating A: Biology of bone marrow stroma. Ann N Y Acad Sci 770
: 70–78, 1995[Medline]
Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276
: 71–74, 1997[Abstract/Free Full Text]
Gerson SL: Mesenchymal stem cells: No longer second class marrow citizens. Nat Med 5
: 262–264, 1999[CrossRef][Medline]
Caplan AI, Bruder SP: Mesenchymal stem cells: Building blocks for molecular medicine in the 21st century. Trends Mol Med 7
: 259–264, 2001[CrossRef][Medline]
Hurwitz DR, Kirchgesser M, Merrill W, Galanopoulos T, McGrath CA, Emami S, Hansen M, Cherington V, Appel JM, Bizinkauskas CB, Brackmann HH, Levine PH, Greenberger JS: Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells. Hum Gene Ther 8
: 137–156, 1997[Medline]
Horwitz EM: Bone marrow transplantation: It’s not just about blood anymore! Pediatr Transplant 7
: 56–58, 2003
Prockop DJ: Marrow stromal cells as stem cells for continual renewal of nonhematopoietic tissues and as potential vectors for gene therapy. J Cell Biochem 284–285, 1998
Pochampally RR, Horwitz EM, DiGirolamo CD, Stokes DS, Prockop DJ: Correction of a mineralization defect by overexpression of a wild-type cDNA for COL1A1 in marrow stromal cells (MSCs) from a patient with osteogenesis imperfecta: A strategy for rescuing mutations that produce dominant-negative protein defects. Gene Ther 12
: 1119–1125, 2005[CrossRef][Medline]
Eliopoulos N, Al-Khaldi A, Crosato M, Lachapelle K, Galipeau J: A neovascularized organoid derived from retrovirally engineered bone marrow stroma leads to prolonged in vivo systemic delivery of erythropoietin in nonmyeloablated, immunocompetent mice. Gene Ther 10
: 478–489, 2003[CrossRef][Medline]
Eliopoulos N, Lejeune L, Martineau D, Galipeau J: Human-compatible collagen matrix for prolonged and reversible systemic delivery of erythropoietin in mice from gene-modified marrow stromal cells. Mol Ther 10
: 741–748, 2004[CrossRef][Medline]
Phinney DG, Kopen G, Isaacson RL, Prockop DJ: Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation. J Cell Biochem 72
: 570–585, 1999[CrossRef][Medline]
Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O: Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57
: 11–20, 2003[CrossRef][Medline]
Zhang F, Laneuville P, Gagnon RF, Morin B, Brox AG: Effect of chronic renal failure on the expression of erythropoietin message in a murine model. Exp Hematol 24
: 1469–1474, 1996[Medline]
Brox AG, Zhang F, Guyda H, Gagnon RF: Subtherapeutic erythropoietin and insulin-like growth factor-1 correct the anemia of chronic renal failure in the mouse. Kidney Int 50
: 937–943, 1996[Medline]
Gagnon RF, Gallimore B: Characterization of a mouse model of chronic uremia. Urol Res 16
: 119–126, 1988[CrossRef][Medline]
Gagnon RF, Duguid WP: A reproducible model for chronic renal failure in the mouse. Urol Res 11
: 11–14, 1983[CrossRef][Medline]
Eliopoulos N, Stagg J, Lejeune L, Pommey S, Galipeau J: Allogeneic marrow stromal cells are immune rejected by MHC class I and II mismatched recipient mice. Blood 106
: 4057–4065, 2005[Abstract/Free Full Text]
Short B, Brouard N, Occhiodoro-Scott T, Ramakrishnan A, Simmons PJ: Mesenchymal stem cells. Arch Med Res 34
: 565–571, 2003[CrossRef][Medline]
Gregory CA, Prockop DJ, Spees JL: Non-hematopoietic bone marrow stem cells: Molecular control of expansion and differentiation. Exp Cell Res 306
: 330–335, 2005[CrossRef][Medline]
Prockop DJ, Gregory CA, Spees JL: One strategy for cell and gene therapy: Harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci U S A 100
: 11917–11923, 2003[Abstract/Free Full Text]
Rao DS, Shih MS, Mohini R: Effect of serum parathyroid hormone and bone marrow fibrosis on the response to erythropoietin in uremia. N Engl J Med 328
: 171–175, 1993[Abstract/Free Full Text]
Koury MJ: Investigating erythropoietin resistance. N Engl J Med 328
: 205–206, 1993[Free Full Text]
Scharte M, Fink MP: Red blood cell physiology in critical illness. Crit Care Med 31[Suppl]
: S651–S657, 2003[CrossRef][Medline]
Macdougall IC: Poor response to erythropoietin. BMJ 310
: 1424–1425, 1995[Free Full Text]
Macdougall IC: Hyporesponsiveness to anemia therapy: What are we doing wrong? Perit Dial Int 21
: S225–S230, 2001[Abstract/Free Full Text]
Svensson EC, Black HB, Dugger DL, Tripathy SK, Goldwasser E, Hao ZP, Chu LE, Leiden JM: Long-term erythropoietin expression in rodents and non-human primates following intramuscular injection of a replication-defective adenoviral vector. Hum Gene Ther 8
: 1797–1806, 1997[Medline]
Regulier E, Schneider BL, Deglon N, Beuzard Y, Aebischer P: Continuous delivery of human and mouse erythropoietin in mice by genetically engineered polymer encapsulated myoblasts. Gene Ther 5
: 1014–1022, 1998[CrossRef][Medline]
Coleman T, Brines M: Science review: Recombinant human erythropoietin in critical illness: A role beyond anemia? Crit Care 8
: 337–341, 2004[CrossRef][Medline]
Lewis NP, Macdougall IC, Willis N, Coles GA, Williams JD, Henderson AH: Effects of the correction of renal anaemia by erythropoietin on physiological changes during exercise. Eur J Clin Invest 23
: 423–427, 1993[Medline]
Oyama M, Tatlock A, Fukuta S, Kavalkovich K, Nishimura K, Johnstone B, Robbins PD, Evans CH, Niyibizi C: Retrovirally transduced bone marrow stromal cells isolated from a mouse model of human osteogenesis imperfecta (oim) persist in bone and retain the ability to form cartilage and bone after extended passaging. Gene Ther 6
: 321–329, 1999[CrossRef][Medline]
Stagg J, Lejeune L, Paquin A, Galipeau J: Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy. Hum Gene Ther 15
: 597–608, 2004[CrossRef][Medline]
Zhang XS, Linkhart TA, Chen ST, Peng HR, Wergedal JE, Guttierez GG, Sheng MHC, Lau KHW, Baylink DJ: Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice. J Gene Med 6
: 4–15, 2004[CrossRef][Medline]
Sato H, Kuwashima N, Sakaida T, Hatano M, Dusak JE, Fellows-Mayle WK, Papworth GD, Watkins SC, Gambotto A, Pollack IF, Okada H: Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther 12
: 757–768, 2005[CrossRef][Medline]
Maruyama H, Ataka K, Gejyo F, Higuchi N, Ito Y, Hirahara H, Imazeki I, Hirata M, Ichikawa F, Neichi T, Kikuchi H, Sugawa M, Miyazaki J: Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats. Gene Ther 8
: 461–468, 2001[CrossRef][Medline]
Rizzuto G, Cappelletti M, Mennuni C, Wiznerowicz M, DeMartis A, Maione D, Ciliberto G, La MN, Fattori E: Gene electrotransfer results in a high-level transduction of rat skeletal muscle and corrects anemia of renal failure. Hum Gene Ther 11
: 1891–1900, 2000[CrossRef][Medline]
Jaalouk DE, Eliopoulos N, Couture C, Mader S, Galipeau J: Glucocorticoid-inducible retrovector for regulated transgene expression in genetically engineered bone marrow stromal cells. Hum Gene Ther 11
: 1837–1849, 2000[CrossRef][Medline]
Osada S, Ebihara I, Setoguchi Y, Takahashi H, Tomino Y, Koide H: Gene therapy for renal anemia in mice with polycystic kidney using an adenovirus vector encoding the human erythropoietin gene. Kidney Int 55
: 1234–1240, 1999[CrossRef][Medline]
Maione D, Wiznerowicz M, Delmastro P, Cortese R, Ciliberto G, La MN, Savino R: Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector. Hum Gene Ther 11
: 859–868, 2000[CrossRef][Medline]
Binley K, Askham Z, Iqball S, Spearman H, Martin L, de Alwis M, Thrasher AJ, Ali RR, Maxwell PH, Kingsman S, Naylor S: Long-term reversal of chronic anemia using a hypoxia-regulated erythropoietin gene therapy. Blood 100
: 2406–2413, 2002[Abstract/Free Full Text]
Lochmuller H, Petrof BJ, Allen C, Prescott S, Massie B, Karpati G: Immunosuppression by FK506 markedly prolongs expression of adenovirus-delivered transgene in skeletal muscles of adult dystrophic [MDX] mice. Biochem Biophys Res Commun 213
: 569–574, 1995[CrossRef][Medline]
Miller HI: Gene therapy on trial. Science 287
: 591–592, 2000[CrossRef][Medline]
Wadman M: NIH under fire over gene-therapy trials. Nature 403
: 237, 2000[Medline]
Jenks S: Gene therapy death: 'Everyone has to share in the guilt.’ J Natl Cancer Inst 92
: 98–100, 2000[Free Full Text]
Gao GP, Lebherz C, Weiner DJ, Grant R, Calcedo R, McCullough B, Bagg A, Zhang Y, Wilson JM: Erythropoietin gene therapy leads to autoimmune anemia in macaques. Blood 103
: 3300–3302, 2004[Abstract/Free Full Text]
Einbinder T, Sufaro Y, Yusim I, Byk G, Passlick-Deetjen J, Chaimovitz C, Douvdevani A: Correction of anemia in uremic mice by genetically modified peritoneal mesothelial cells. Kidney Int 63
: 2103–2112, 2003[CrossRef][Medline]
Hamamori Y, Samal B, Tian J, Kedes L: Myoblast transfer of human erythropoietin gene in a mouse model of renal failure. J Clin Invest 95
: 1808–1813, 1995[Medline]
Rinsch C, Dupraz P, Schneider BL, Deglon N, Maxwell PH, Ratcliffe PJ, Aebischer P: Delivery of erythropoietin by encapsulated myoblasts in a genetic model of severe anemia. Kidney Int 62
: 1395–1401, 2002[CrossRef][Medline]
Lippin Y, Dranitzki-Elhalel M, Brill-Almon E, Mei-Zahav C, Mizrachi S, Liberman Y, Iaina A, Kaplan E, Podjarny E, Zeira E, Harati M, Casadevall N, Shani N, Galun E: Human erythropoietin gene therapy for patients with chronic renal failure. Blood 106
: 2280–2286, 2005[Abstract/Free Full Text]
Ramesh N, Lau S, Palmer TD, Storb R, Osborne WR: High-level human adenosine deaminase expression in dog skin fibroblasts is not sustained following transplantation. Hum Gene Ther 4
: 3–7, 1993[Medline]
Palmer TD, Rosman GJ, Osborne WR, Miller AD: Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci U S A 88
: 1330–1334, 1991[Abstract/Free Full Text]
Cristofalo VJ, Volker C, Francis MK, Tresini M: Age-dependent modifications of gene expression in human fibroblasts. Crit Rev Eukaryot Gene Expr 8
: 43–80, 1998[Medline]
Ebert BL, Bunn HF: Regulation of the erythropoietin gene. Blood 94
: 1864–1877, 1999[Free Full Text]
Lappin TR, Maxwell AP, Johnston PG: EPO’s alter ego: Erythropoietin has multiple actions. Stem Cells 20
: 485–492, 2002[Abstract/Free Full Text]
Jelkmann W, Wagner K: Beneficial and ominous aspects of the pleiotropic action of erythropoietin. Ann Hematol 83
: 673–686, 2004[CrossRef][Medline]
Maiese K, Li F, Chong ZZ: New avenues of exploration for erythropoietin. JAMA 293
: 90–95, 2005[Abstract/Free Full Text]
Weiss MJ: New insights into erythropoietin and epoetin alfa: Mechanisms of action, target tissues, and clinical applications. Oncologist 8[Suppl 3]
: 18–29, 2003
Westenfelder C, Biddle DL, Baranowski RL: Human, rat, and mouse kidney cells express functional erythropoietin receptors. Kidney Int 55
: 808–820, 1999[CrossRef][Medline]
Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, Salio M, Cerami A, Brines M: Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling. Proc Natl Acad Sci U S A 100
: 4802–4806, 2003[Abstract/Free Full Text]
Mancini DM, Kunavarapu C: Effect of erythropoietin on exercise capacity in anemic patients with advanced heart failure. Kidney Int Suppl S48
–S52, 2003
Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB, Thompson RB, Petrofski JA, Annex BH, Stamler JS, Koch WJ: A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest 112
: 999–1007, 2003[CrossRef][Medline]
This article has been cited by other articles:
T. Kucic, I. B. Copland, J. Cuerquis, D. L. Coutu, L. E. Chalifour, R. F. Gagnon, and J. Galipeau Mesenchymal stromal cells genetically engineered to overexpress IGF-I enhance cell-based gene therapy of renal failure-induced anemia
Am J Physiol Renal Physiol,
August 1, 2008;
295(2):
F488 - F496.
[Abstract][Full Text][PDF]
N. Eliopoulos, M. Francois, M.-N. Boivin, D. Martineau, and J. Galipeau Neo-Organoid of Marrow Mesenchymal Stromal Cells Secreting Interleukin-12 for Breast Cancer Therapy
Cancer Res.,
June 15, 2008;
68(12):
4810 - 4818.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
-glycerol phosphate (10 mM), ascorbic acid 2-phosphate (5 µg/ml), and dexamethasone (10–8 M; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) (15,16), and subsequently stained with Alizarin Red S (Sigma-Aldrich Canada; pH 4.1 using ammonium hydroxide) (16). For induction of adipogenic differentiation, Epo+MSCs were cultured for 7 d in complete medium that was supplemented with insulin (10 µg/ml), dexamethasone (1 µM), 3-isobutyl-methylxanthine (0.5 mM), and indomethacin (46 µM; Sigma-Aldrich Canada) (17), and then stained with Oil Red O (Sigma-Aldrich Canada) (15). 
0.05, t test; Figure 5C). Moreover, the concentration of Epo in plasma of anemic mice that received 3 x 106 Epo+MSCs embedded in Contigen was 6.8 ± 0.9 mU/ml at approximately 2 wk after implantation, similar to that of anemic mice that received 3 x 106 control MSCs (6.0 ± 1.7 mU/ml), whereas the plasma Epo concentration of anemic mice approximately 2 wk after implantation with 10 x 106 Epo+MSCs was considerably increased at 160 ± 40 mU/ml, in contrast to that of anemic mice that received 10 x 106 control MSCs (11 ± 2.1 mU/ml). 
packaging signal to enable the RNA transcribed to be packaged within retroviral particles.
) at 3 x 106 (A), 4.5 x 106 (B), 7.5 x 106 (C), or 10 x 106 (D) cells/mouse or control MSCs (
) at 3 x 106 (A), 7.5 x 106 (B and C), or 10 x 106 (D) cells/per mouse. Normal mice also received implants of Epo+MSCs (
, dotted curve) at 3 x 106 (A), 4.5 x 106 (B), 7.5 x 106 (C), or 10 x 106 (D) cells/mouse.