A Humanized Mouse Model of Idiopathic Nephrotic Syndrome Suggests a Pathogenic Role for Immature Cells
Anne-Laure Sellier-Leclerc*,
Arnaud Duval,
Stéphanie Riveron*,
Marie-Alice Macher,
Georges Deschenes,
Chantal Loirat,
Marie-Christine Verpont,
Michel Peuchmaur||,
Pierre Ronco,
Renato C. Monteiro* and
Elie Haddad
* INSERM U699, Faculté de Médecine Denis Diderot, Université Paris 7; Service de Néphrologie and || Service de Pathologie, EA 3102, Hôpital Robert Debré, AP-HP, and Université Paris 7, and INSERM UMR S 702, Université Pierre et Marie Curie-Paris 6, Hôpital Tenon, Paris, France; and Centre de Recherche du CHU Ste-Justine, Département de Pédiatrie, Université de Montréal, Montréal, Québec, Canada
Correspondence: Dr. Elie Haddad, Centre de Recherche du CHU Suite-Justine, Département de Pédiatrie, Université de Montréal, 3175 Cote Sainte-Catherine, Montréal, QC H3T1C5, Canada. Phone: 514-345-4713; Fax: 514-345-4897; E-mail: elie.haddad{at}umontreal.ca
Received for publication December 12, 2006.
Accepted for publication June 26, 2007.
Idiopathic nephrotic syndrome is characterized by glomerularproteinuria in the absence of infiltrating cells or immunoglobulindeposits. Although it is suspected that T cells secrete a circulatingfactor that leads to proteinuria by altering the permeabilityof the glomerular filtration barrier, the precise etiology ofthis syndrome is unknown. Because an animal model that mimicshuman idiopathic nephrotic syndrome does not exist, we developeda humanized mouse model of the disease by injecting CD34+ stemcells or CD34– peripheral blood mononuclear cells fromafflicted patients into immunocompromised mice. Even thoughboth CD34+ and CD34– cells induced the engraftment ofhuman CD45+ leukocytes in mice, only the injection of CD34+stem cells induced albuminuria. Ultrastructural analysis ofglomeruli from the resulting proteinuric mice revealed effacementof podocyte foot processes, similar to the pathology observedin the human disease. Therefore, our data suggest that the cellsresponsible for the pathogenesis of idiopathic nephrotic syndromeare more likely to be immature differentiating cells ratherthan mature peripheral T cells.
Idiopathic nephrotic syndrome (INS) is a disease of unknowncause that can be associated with two different histologic varieties:Minimal-change nephrotic syndrome (MCNS) with minimal histologicchanges in the glomeruli and FSGS, characterized by glomerularscarring.1 MCNS is usually steroid sensitive, whereas FSGS isoften steroid resistant and progresses to end-stage renal failurein approximately 20% of patients. After transplantation, upto 40% of patients rapidly relapse to their initial disease.2,3Although neither immune cell infiltration nor immune complexdeposits can be identified, accumulating data suggest that steroid-sensitiveMCNS and a subset of FSGS (particularly those recurring aftertransplantation) have an immunologic basis. Indeed, many findings(e.g. sensitivity to immunosuppressive drugs; relapse initiatedby immune challenge with infectious or allergic stimuli; theoccurrence of MCNS and FSGS in patients with Hodgkin's disease,non-Hodgkin's lymphoma, or thymoma) have led Shalhoub4 to proposethat MCNS and FSGS are caused by an immunologic disorder. Animmunologic basis for MCNS and FSGS is a widely accepted hypothesis.1,5The most likely pathologic process is that T cells promote theproduction of a circulating factor that alters the glomerularpermeability of the renal filtration barrier.5,6 This conceptcame from the observation that proteinuria recurred in the veryfirst samples of urine from patients who received a transplant7and was supported by studies demonstrating increased proteinexcretion in rats after the injection of serum from patientswho relapsed after transplantation for FSGS.8 Nevertheless,both the nature of pathogenic circulating factor and the mechanismsby which the immune system is involved in the pathologic processof FSGS and MCNS remain unknown. Several experimental modelsof glomerular lesions resembling human FSGS or MCNS have beendescribed in rodents,9–12 but these models are of limitedvalue in the characterization of the circulating factor andthe underlying immunologic abnormalities. It was shown thatthe Buffalo/Mna rat model was similar to human FSGS13; however,it has not aided in determining the mechanisms of FSGS. In thework presented here, we developed a new animal model of FSGSand MCNS by using the technology of humanizing NOD/SCID mice.The concept relies on the fact that very immunocompromised micecan be engrafted with human cells, either peripheral blood mononuclearcells (PBMC)14–18 or stem cells (CD34+ cells).19–22When PBMC are injected, mature cells, mostly mature activatedhuman T cells, proliferate in mice, whereas when CD34+ stemcells are injected, immature stem cells proliferate and maydifferentiate into immature T cell progenitors that migrateto thymus and differentiate into more mature T cells.20,22 Wehypothesized that if such mice were engrafted with either PBMCor CD34+ stem cells from patients with FSGS or MCNS, they couldreproduce the disease, providing an animal model in which theunderlying mechanisms are similar to those observed in humans.
To engraft human cells into mice, we used two strategies: injectionof PBMC and injection of CD34+ stem cells. Twelve blood samplesfrom six patients (Table 1) and 12 samples from 12 control subjectswere separated into CD34+ cells and CD34– PBMC. We obtained1.28 ± 0.21 x 104 CD34+ cells and 22 ± 4.8 x 106CD34– PBMC from patients’ blood samples and 1.5± 0.25 x 104 CD34+ cells and 20 ± 4 x 106 CD34–PBMC from control blood samples.
Assessment of Engraftment
Intraperitoneal injection of CD34– PBMC from control subjectsor patients into sublethally irradiated NOD/SCID mice that weretreated with anti-NK antibody (12 mice in each group) inducedthe development of human CD45+ cells in the peripheral bloodcompartment in all cases. The percentage of human CD45+ cellsincreased to approximately 13% and then decreased to <0.1%without any significant differences between groups (Figure 1A).We showed that human CD45+ cells were CD3+ and CD19– (Figure 2A).These data demonstrate that intraperitoneal injection of PBMCinduces a transient peripheral engraftment of human peripheralmature T lymphocytes (CD3+ cells) into mice in a reproduciblemanner.
Figure 1. Kinetics of engraftment of human cells in NOD/SCID mice. Engraftment of human cells was determined by the percentage of human CD45+ cells in blood of mice that were administered an injection of CD34– PBMC (A) of patients () or control subjects () and with CD34+ cells (B) of patients () and control subjects (). Percentages of human CD45+ cells were calculated as % human CD45+ = human CD45+ cells/(human CD45+ cells + mouse CD45+ cells) x 100. Data are means ± SEM of 12 mice except for those that were administered an injection of CD34+ cells from patients in which only the 11 mice with engraftment have been considered. CD45 is a marker of all hematopoietic and lymphoid cells except erythrocytes and platelets.
Figure 2. Phenotype of engrafted CD45+ human cells in NOD/SCID mice. Representative analysis by flow cytometry of peripheral blood from a mouse that received an intraperitoneal injection of CD34– PBMC from patient 3 (A) and from a mouse injected by IO route with CD34+ cells from patient 2 (B). 7AAD– (living) human CD45+ cells were gated (left) and analyzed for CD3 (marker of T lymphocytes) and CD19 (marker of B lymphocytes) expression (right).
Because we obtained <2 x 104 CD34+ cells and it has beenshown that intraosseous (IO) injection induces better engraftmentof human CD34+ cells,23 we delivered CD34+ cells by IO injection.We observed an engraftment of human cells into all 12 mice thatwere administered an injection of CD34+ cells from control subjectsand in 11 of 12 mice that were administered an injection ofCD34+ cells from patients. The only mouse in which engraftmentdid not occur was administered an injection of CD34+ cells frompatient 6, from whom it was possible to harvest CD34+ cellsonly once. In mice with engraftment, we detected >0.2% humanCD45+ cells in peripheral blood 3 wk after injection. This percentageincreased to approximately 0.8% when mice were killed (Figure 1B),without any significant differences between groups. None ofthe human CD45+ cells detected in mice were CD3+ or CD19+ (Figure 2B).We tested seven mice that were administered an injection ofCD34+ engraftment and detected in six of them human CD34+ cellsin both the injected femur and the contralateral femur, suggestingan engraftment of human stem cells and a migration of thesehuman CD34+ stem cells to other osseous localizations (Figure 3A).Moreover, 10 mice that were administered an injection of CD34+cells from patients (n = 9) or from control subjects (n = 1)were tested for the presence of human cells in the thymus whenthe mice were killed. In one mouse that was administered aninjection of CD34+ from patient 3, no human CD45+ cells weredetected; however, in the remaining nine mice, 1.61 ±1% CD45+ cells were identified, suggesting that migration ofhuman cells, likely thymic progenitors, had occurred into thethymus. Human cells identified in thymus were CD45RA+, CD45RO+,or CD45RA+RO+ (Figure 3B), but none was CD3+ neither CD4+ norCD8+, suggesting that human cells were in a differentiationprocess in the thymus. Also, no human engrafted cells couldbe detected in the secondary lymph nodes (spleen and lymph nodes),suggesting that until 9 wk after transplantation, no matureT cells had developed and emigrated from thymus and no matureB cells had developed and emigrated from bone marrow. Our resultsdemonstrate that approximately 104 CD34+ cells are sufficientto establish an engraftment of human cells into immunocompromisedmice when injected by the IO route.
Figure 3. Detection of human cells in femur and thymus of NOD/SCID mice that were engrafted with human CD34+ cells. (A) Representative analysis by flow cytometry of bone marrow cells harvested in both femurs from a mouse that was administered an IO injection of CD34+ cells from patient 1 and killed 9 wk after injection. (B) Representative analysis of cells in thymus from one mouse that was administered an IO injection of CD34+ cells from patient 4 and killed 9 wk after injection. CD45RA and CD45RO both are isoforms of CD45, markers of thymocytes at different stages of differentiation.
Assessment of Albuminuria
We first analyzed albumin and creatinine of 20 NOD/SCID mice,15 times for each mouse, and determined a mean ± SD ofalbumin-to-creatinine ratio (ACR) value of 11.88 ± 4.69µg albumin/mg creatinine. We established the limit ofnormal value of ACR as being mean + 2 x SD (i.e., 21.26 µgalbumin/mg creatinine).
In mice that were administered an injection of CD34+ cells orCD34– PBMC from controls, ACR level did not change. Inmice that were administered an injection of cells from patients,we showed that CD34– PBMC engraftment did not change ACRlevels, whereas all 11 mice with CD34+ cell engraftment showeda significant increase in ACR (Table 2). In these 11 mice, ACRincreased significantly during the fourth week after injectionand then remained significantly increased until mice were killed(Figure 4). Therefore, the increase in ACR levels paralleledthe detection of CD45+ human cells. That the one mouse thatdid not display albuminuria, although administered an injectionof CD34+ cells from a patient, was the one in which no engraftmentwas observed suggests that engraftment of patient cells wasneeded to induce albuminuria. To confirm this finding, we administeredan injection of CD34+ cells from patients 1 and 2 to two othermice but without treating mice with anti-NK antibody. No humanCD45+ cells were detected, and no increase of ACR occurred.
Figure 4. Evolution of ACR in mice that were administered an injection of CD34– PBMC from patients () or control subjects () or of CD34+ cells from patients () or control subjects (). Data are means ± SEM of 12 mice in each group except for mice that were administered an injection of CD34+ cells from patients in which only the 11 mice with engraftments have been considered. *Significance at P < 0.001 versus all other mice or each group of mice. Dashed line represents the superior limit of ACR as determined as mean + 2 x SD in 20 control NOD/SCID mice.
In mice that were administered an injection of CD34– PBMCfrom patients, ACR did not increase (Figure 4) despite the engraftmentof human CD3+ cells that increased up to approximately 13%,suggesting that mature T cells from patients are not capableof inducing ACR increase in mice. One could hypothesize thatproliferation of human T cells in mice was inhibited and thatthis inhibition prevented an increase of ACR. This inhibitioncould be due to the presence of mature CD25+ regulatory T cells(Treg) within the injected CD34– PBMC or to the residualimmune system of mice that reject human T cells. To rule outthese hypotheses, we purified CD25– cells from CD34–PBMC from two patients (patients 1 and 2) and two control subjectsand injected them into sublethally irradiated NOD/SCIDc–mice in which the c chain was mutated, thereby preventing NKcell development. These NOD/SCIDc– mice are more immunocompromisedthan NOD/SCID mice that are administered an injection of anti-NKantibody and are known to allow a higher engraftment of humancells.24 Human CD45+ cells, made of 93 ± 5% mature CD3+T cells, raised up to 82.26 ± 9%, and mice died fromxenograft-versus-host disease 4 to 8 wk after injection withoutany ACR increase (monitored every 3 d). Therefore, despite ahuge engraftment of mature activated T cells from patients,albuminuria could not be transferred into mice.
Assessment of Renal Pathology
To test whether renal pathology induced by the injection ofCD34+ cells from patients was similar to that observed in humandisease, we analyzed kidneys of mice with increase of ACR usinglight microscopy and immunofluorescence (n = 3) and electronmicroscopy (n = 6). As shown in Figure 5, light microscopy wasnormal as revealed by nonexpanded mesangium and cells per glomerularcross-section at 36 ± 7 (normal) in mice that were administeredan injection of CD34+ cells from patients and 35 ± 4(normal) in mice that were administered an injection of CD34+cells from control subjects. Also, no Ig or complement componentdeposits were observed with immunofluorescence microscopy (datanot shown). However, electron microscopy revealed a partialwidening and effacement of epithelial cell foot processes, asis also observed in humans with INS, whereas epithelial cellfoot processes of mice that were administered an injection ofcells from control subjects were normally distributed alongthe basal membrane (Figure 6). Glomerular basement membranethickness was 175.8 ± 23.8 nm in mice that were administeredan injection of CD34+ cells from control subjects (n = 3) and198.4 ± 19.7 nm in mice that were administered an injectionof CD34+ cells from patients (n = 6; P = 0.16, NS). Also, noelectron-dense deposits were present. These data suggest thatthe increase in ACR level observed in mice was strong enoughto induce effacement of epithelial cell foot processes and thatthe mechanism of albuminuria in mice is likely similar to theetiology hypothesized in humans; that is, a secretion of a vascularpermeability factor by cells of the immune system.
Figure 5. Normal aspect of glomeruli on light microscopy. Representative light microscopy of glomeruli from control mice (A) and albuminuric mice (B). Formalin-fixed, paraffin-embedded section stained with periodic acid-Schiff. Magnification, x1000.
Figure 6. Epithelial cell foot fusion in mice receiving CD34+ cells. Representative electron microscope images of a glomerulus from albuminuric (A) and control (B) mice. The albuminuric mouse was administered an injection of CD34+ cells from patient 1 and shows partial widening and effacement of epithelial cell foot processes (arrows, A), whereas the mouse that was administered an injection of CD34+ cells from a control subject has normal and regular epithelial cell foot processes (arrow, B).
We show that CD34+ cells that are from patients and injectedinto mice may engraft and induce albuminuria. However, ACR didnot increase in mice that were administered an injection ofCD34– PBMC from patients despite both a higher numberof cells injected and a higher number of human cells detectedin mouse blood. It has been shown that injection of PBMC intoimmunocompromised mice induces the expansion of mature T cells,14–17whereas injection of CD34+ cells induces the development ofa human immune and hematopoietic system,19,21,22 with immatureT cells migrating to the thymus and undergoing differentiation.Therefore, it is likely that, in our model, the cells responsiblefor albuminuria are immature cells undergoing differentiation.Also, ACR increase occurred 3 to 4 wk after injection of CD34+cells and paralleled the detection of human CD45+ cells in peripheralblood, suggesting that the cells that are responsible for albuminuriawere likely to originate from stem cells and their subsequentproliferation and differentiation rather than from the CD34+cells themselves. Data from the literature suggest that cellsthat are responsible for MCNS are from the immune system, morespecifically from the T cell subset. A possible role for T cellsdeveloped from the observation that infusion of supernatantsof cultured PBMC from patients with MCNS relapses induced proteinuriain rats25–28 and that T cell hybridomas obtained froma patient with NS secreted a factor that caused proteinuriain rats.29 Studies of T cell compartments in MCNS demonstratedexpansion of CD4+ and CD8+ T cell populations30,31 and an increasedsynthesis of several cytokines, such as TNF-,31,32 soluble IL-2receptor,32 and IL-13.33 In our model, we showed that peripheralCD3+ cells of patients were not capable of inducing albuminuriain mice, even in cases in which CD3+ cells could proliferatein mice, after depletion of Treg and injection in more immunocompromisedmice. Given all of the data in the literature suggesting theinvolvement of T cells and considering that, in our model, albuminuriaoccurs after engraftment of CD34+ cells but not after engraftmentof CD3+ cells, it is tempting to propose that cells that areresponsible for albuminuria are undifferentiated cells undergoingdifferentiation into T cells. Our results showing that engraftedhuman cells could migrate to the mouse's thymus in mice thatwere administered an injection of CD34+ stem cells and thatno mature CD3+ T cells could be found in mice until they werekilled are consistent with a role for immature rather than matureT cells. However, one cannot rule out the hypothesis that immaturecells of other lineages may be responsible for albuminuria.
Other experimental animal models resembling MCNS or FSGS havebeen described, such as age-associated nephropathy,11 nephronreduction,12 or puromycin or aminonucleoside toxic–inducednephrosis.9,10 However, despite some similarities with humanFSGS, the outcome of proteinuria in these models does not involvethe immune system and does not involve a circulating factor.Therefore, these models are of limited value in terms of understandingthe pathologic process of human primary FSGS. The Buffalo/Mnarat model in which rats spontaneously develop lesions mimickinghuman idiopathic FSGS34 is more interesting. Indeed, proteinuriarelapsed after transplantation of normal kidneys from healthyLEW.1W rats into Buffalo/Mna recipients, suggesting that thismodel resembles human primary FSGS.13 Moreover, Buffalo/Mnarats exhibit renal macrophage activation and TH2 polarizationthat precedes the development of NS.35 It is noteworthy thatBuffalo/Mna rats were first reported because of the spontaneousformation of a thymoma associated with muscular weakness. Alsoin humans, FSGS may be associated with thymoma, and it was recentlysuggested, by T cell transcriptome analysis, that INS was likelya thymic disorder.36 Given this experimental data and our resultssuggesting that immature T cells may be responsible for proteinuria,it is tempting to propose that immune cells passing throughthe thymus to undergo differentiation and selection may be involvedin the pathogenesis of FSGS or MCNS. However, neonatal37 andadult thymectomy35 had no effect on proteinuria in the Buffalo/Mnarat model, making it an unlikely hypothesis for this model.
The outcome of albuminuria and the structural abnormalitiesobserved using the electron microscope strongly suggest thatwe can reproduce the pathologic process underlying albuminuriain FSGS and MCNS. Indeed, these structural abnormalities togetherwith the significant ACR increase observed in all mice thatwere engrafted with CD34+ cells from patients but not in micethat were engrafted with CD34+ from control subjects suggestthat the observed changes in mice are related to a pathologicmechanism similar to one involved in FSGS in human, despitethe low magnitude of ACR increase in comparison with what isobserved in INS in human. Also, it is likely that the modestincrease of ACR is because engraftment of patients’ cellswas very low. One could hypothesize that a higher engraftmentcould induce a higher ACR increase, and this would need to bedemonstrated in models allowing a higher engraftment of humanstem cells. That we did not observe adhesions in glomeruli islikely due to the short period of time during which mice displayedalbuminuria before being killed. Also, that albuminuria wasobserved with both similar kinetics and ranges after injectionof CD34+ cells from four patients with FSGS and from one patientwith MCNS suggests that MCNS and FSGS share the same pathologicprocess and likely represent a different spectrum of the samedisease.
Our data demonstrate that NOD/SCID humanized mice may be a usefultool to study human disease. In the context of FSGS and MCNS,we could reproduce albuminuria in mice together with renal lesions,seen using electron microscopy. Our model suggests that thecells that are responsible for albuminuria are likely to beimmature cells rather than mature CD3+ peripheral T cells. Theseresults can also be seen as a proof of concept for using thetechnology of humanized mice to study human diseases that arelinked to a pathologic process involving the hematopoietic orthe immune system.
For more details, see supplemental Concise Methods online.
Patients
Blood samples (10 ml) were obtained from six patients and 12control subjects. Among control subjects, 10 were normal volunteersand two were adults under chronic hemodialysis for end-stagerenal failure not related to INS. This work was approved bythe local ethics committee. Informed consent was obtained fromthe parents of pediatric patients (and whenever possible fromthe pediatric patients themselves), as well as from adult patientsand normal volunteers.
Purification of Cells
Blood samples were separated into CD34+ and CD34– PBMCusing magnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach,Germany). The CD34+ fraction (purity >85%) and CD34–fraction (purity = 100%) were centrifuged and resuspended inPBS before being injected to mice. In two experiments, the CD25–fraction of CD34– PBMC was also sorted, and its puritywas 100%.
Injection of Human Cells into NOD/SCID Mice
NOD/SCID mice were sublethally irradiated (2.5 Gy) and treatedwith TM-1 (gift from Dr. T. Tanaka, Tokyo Metropolitan Instituteof Medical Science, Tokyo, Japan), an antibody blocking themouse NK cell function, shown to induce better engraftment ofhuman cells.20
Engraftment and Albuminuria
Peripheral blood was aspired weekly from the retro-orbital sinusunder anesthesia, and bone marrow, thymus, and spleen were harvestedwhen mice were killed. The presence of human cells was analyzedby flow cytometry. At the indicated time points, urine samples(minimum of 50 µl) were collected. Urine creatinine andalbumin concentrations were determined in the same samples withan Olympus analyzer AU400 (Olympus Instruments, Japan). Urinealbumin excretion was expressed as the ACR.
Microscopic Examination
Mouse kidney samples were obtained at the time of killing, 9wk after injection. Kidney specimen were analyzed by optic microscopy,direct immunofluorescence, and electronic microscopy. All analyseswere performed in a masked manner. Kidney pathology was assessedaccording to a slightly modified procedure previously described.38
A.L.S.L. received a grant from the "Fonds de roulement pourla recherche de l'AP/HP" and from the "SociétéFrançaise de Néphrologie." A.D. received a grantfrom the CHU Sainte-Justine Research Center (Montreal, Quebec,Canada), and S.R. received a grant from the "Fondation Dassault."
We acknowledge Dr. William Vainchenker for helpful discussionsand for access to animal facilities.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Supplemental information for this article is available onlineat http://www.jasn.org/.
See related editorial, "A New Approach to Idiopathic NephroticSyndrome," on pages 2621–2622.
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Joshua Y. Kausman and A. Richard Kitching
J. Am. Soc. Nephrol. 2007 18: 2621-2622.
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J. Am. Soc. Nephrol. 2007 18: A13.
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J. Y. Kausman and A. R. Kitching A New Approach to Idiopathic Nephrotic Syndrome
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Abstract
Introduction
c– mice in which the 
,31,32 soluble IL-2 receptor,32 and IL-13.33 In our model, we showed that peripheral CD3+ cells of patients were not capable of inducing albuminuria in mice, even in cases in which CD3+ cells could proliferate in mice, after depletion of Treg and injection in more immunocompromised mice. Given all of the data in the literature suggesting the involvement of T cells and considering that, in our model, albuminuria occurs after engraftment of CD34+ cells but not after engraftment of CD3+ cells, it is tempting to propose that cells that are responsible for albuminuria are undifferentiated cells undergoing differentiation into T cells. Our results showing that engrafted human cells could migrate to the mouse's thymus in mice that were administered an injection of CD34+ stem cells and that no mature CD3+ T cells could be found in mice until they were killed are consistent with a role for immature rather than mature T cells. However, one cannot rule out the hypothesis that immature cells of other lineages may be responsible for albuminuria. 
1 (gift from Dr. T. Tanaka, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), an antibody blocking the mouse NK cell function, shown to induce better engraftment of human cells.20 
) or control subjects (
) and with CD34+ cells (B) of patients (
) and control subjects (
). Percentages of human CD45+ cells were calculated as % human CD45+ = human CD45+ cells/(human CD45+ cells + mouse CD45+ cells) x 100. Data are means ± SEM of 12 mice except for those that were administered an injection of CD34+ cells from patients in which only the 11 mice with engraftment have been considered. CD45 is a marker of all hematopoietic and lymphoid cells except erythrocytes and platelets.
