Renal Stem Cells

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Isolation and Characterization of Multipotent ProgenitorCells from the Bowmans Capsule of Adult Human Kidneys

Costanza Sagrinati,* Giuseppe Stefano Netti, Benedetta Mazzinghi,* Elena Lazzeri,*Francesco Liotta,* Francesca Frosali,* Elisa Ronconi,* Claudia Meini,* Mauro Gacci,

Roberta Squecco,

Marco Carini,

Loreto Gesualdo,

Fabio Francini,

Enrico Maggi,*Francesco Annunziato,* Laura Lasagni,* Mario Serio,* Sergio Romagnani,* andPaola Romagnani**Excellence Center for Research, Transfer and High Education DENOthe, Department of Medical and Surgical Critical

Care, and Department of Physiological Sciences, University of Florence, Florence, and Department of Biomedical

Sciences, University of Foggia, Foggia, Italy

Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult

multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal

epithelial cells (PEC) in the Bowmans capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem

cellspecific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24 CD133 PEC

population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloningefficiency. Under appropriate culture conditions, individual clones of CD24 CD133 PEC could be induced to generate

mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and

cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24 CD133 PEC but not of

CD24 CD133 renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of

different portions of the nephron. More important, treatment of acute renal failure with CD24 CD133 PEC significantly

ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the

characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new

avenues for the development of regenerative medicine in patients who have renal diseases.

J Am Soc Nephrol 17: 24432456, 2006. doi: 10.1681/ASN.2006010089

Chronic renal failure is a leading cause of mortality and

morbidity in Western countries (1). The number of

patients with ESRD is growing consistently, and the

cumulative ESRD costs are even greater than the direct treat-

ment costs of cancer (1). Therefore, the potential use of stem

cells (SC) for regenerative medicine to treat kidney diseases

represents a critical clinical goal (2).

The postnatal kidney has a high capacity to regenerate and

repair, as illustrated by its functional recovery after glomerular

or tubular injury (2,3); however, the origin of newly generated

renal cells has not yet been defined. Some cells seem to derive

from the division of fully differentiated cells, and recent reports

suggested that these cells might represent tubular progenitors,

expressing lineage-specific markers (48). Another study re-ported that potential tubular progenitors are present in renal

interstitium (9). Moreover, although glomerular injury is criti-

cal for initiation of irreversible renal failure, the existence of

progenitors within glomerular structures has not yet been de-

scribed (2,49). Previous reports suggested that bone marrow

may be a source of progenitors for tubule turnover and/or

repair (2,10,11). However, recent studies have shown that un-

identified intrarenal, not bone marrowderived, cells mostly

are responsible for regeneration in ischemic acute renal failure

(ARF) (12,13).

The best strategy to identify and amplify multipotent pro-

genitors and/or SC has been their selection on the basis of

functional properties of self-renewal, clonogenicity, multidif-

ferentiation, and/or expression of specific markers (14,15). To

identify multipotent progenitors and/or SC in adult human

kidney, we assessed the presence of both CD24, a surface

molecule that has been used to identify different types of hu-

man SC (16,17) and also is expressed by uninduced metaneph-

ric mesenchyme during renal embryogenesis (18), and CD133,

a marker of adult tissue SC (19,20). The results showed that

both markers were coexpressed by a subset of parietal epithe-

lial cells (PEC) in the Bowmans capsule. Once isolated,

CD24CD133 PEC were found to lack lineage-specific mark-

ers; to express transcription factors that are characteristic of

multipotent SC; and to exhibit self-renewal, high clonogenic

efficiency, and multidifferentiation potential. When injected

intravenously in SCID mice that had ARF, CD24CD133 PEC

Received January 30, 2006. Accepted June 4, 2006.

Published online ahead of print. Publication date available at www.jasn.org.

Address correspondence to: Dr. Paola Romagnani, Interdepartmental Laboratory

of Cellular and Molecular Nephrology, University of Florence, Viale Pieraccini 6,

50139, Firenze, Italy. Phone: 0039-055-4271356; Fax: 0039-055-4271371; E-mail:

[email protected]

Copyright 2006 by the American Society of Nephrology ISSN: 1046-6673/1709-2443


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regenerated tubular structures in different portions of the

nephron and also reduced the morphologic and functional

kidney damage with important implications for the develop-

ment of regenerative medicine in patients who have renal dis-

eases.

Materials and Methods

AntibodiesThe following antibodies (Ab) were used: Anti-CD24 and anti-vimen-

tin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-CD133/1 (clone

AC133) and anti-CD133/2 (clone 293C3; Miltenyi Biotec GmbH, Ber-

gisch Gladbach, Germany); anti-CD105, anti-CD31, anti-CD34, anti-

CD35, anti-CD45, and mouse IgG1 and IgG2b (BD Biosciences, San

Diego, CA); anti-CD29 mAb, rabbit anticholine acetyl-transferase

(ChAT), antineurofilament M (NFM), and antimicrotubule-associ-

ated protein-2 (MAP-2; Chemicon International, Temecula, CA); anti-

CD106 and antiepithelial membrane antigen-1 (EMA-1; Dako,

Glostrup, Denmark); anti-cytokeratin, antihuman HLA-I, anti-

smooth muscle actin (-SMA), and rabbit antineurofilament H

(NF200; Sigma-Aldrich, St. Louis, MO); anti-CD54, PE-conjugated anti-

CD106 and IgG2a mAb, goat anti-mouse IgG1, and rabbit anti-goat IgGAb (Southern Biotech, Birmingham, AL). PE-conjugated anti-CD105

mAb was from Ancell Corp. (Bayport, MN). The anti-LAP (TGF-1)

mAb was from R&D Systems (Minneapolis, MN). The antiTamm-

Horsfall glycoprotein (THG) goat polyclonal Ab was from MP Biomedi-

cals (Verona, Italy). Alexa Fluor 488, 546, 633, and 647labeled goat

anti-mouse IgG1; Alexa Fluor 488labeled goat anti-mouse IgG2a or

goat anti-rabbit IgG; and Alexa Fluor 488 and 546labeled goat anti-

mouse IgG2b or rabbit anti-goat IgG Abs were from Molecular Probes

(Eugene, OR).

TissuesNormal kidney fragments were obtained from 20 patients who had

localized renal tumors and underwent nephrectomy, in accordancewith the recommendations of the Regional Ethical Committee on hu-

man experimentation.

Isolation and Culture of CD24CD133 PECTo obtain CD24CD133 PEC, we minced the cortex and isolated

glomeruli by a standard sieving technique through graded mesh

screens (60, 80, and 150 mesh). The glomerular suspension was col-

lected, washed with endothelial growth mediummicrovascular (EGM-

MV; Cambrex Bio Science, East Rutherford, NJ) without serum, and

plated on fibronectin-coated dishes (10 g/ml; Sigma-Aldrich) at a

density of 200 glomeruli/100-mm plate. To save the Bowmans capsule,

we performed no enzymatic digestion. After 4 to 5 d of culture, isolated

glomeruli adhered to the plate, resulting in cellular outgrowth thatusually was detectable after 5 d of culture. Glomeruli then were de-

tached, and adherent cells were cultured as bulk. Several culture media

were compared. EGM-MV 20% FBS (Hyclone, Logan, UT) yielded the

highest degree of purity and the best amplification efficiency and

therefore was used in subsequent experiments. Bulk cultures were

checked for simultaneous expression of CD133 and CD24 by flow

cytometry and then used for cloning. Generation of clones from

CD24CD133 PEC that were obtained from glomerular outgrowth

was achieved by limiting dilution in fibronectin-coated 96-well plates in

EGM-MV 20% FBS.

CD24CD133 PEC also were maintained in culture as bulk, and

routine cell passaging was performed. Medium was changed twice a

week. The cell counts and cellular dilution factor were recorded at each

passage. This process was repeated for a 4-mo period. The number of

population doublings (PD) was calculated by solving the following

equation: n of PD log2(Ni/No), where Ni is the number of cells

yielded and No is the number of cells plated.

Cell CulturesHuman mesangial cells and human glomerular visceral epithelial

cells were obtained as described (21,22). Human renal proximal tubular

cells, human microvascular endothelial cells, and human aortic smooth

muscle cells were obtained from Cambrex Bio Science, and the HEK-

293 cell line was obtained from ECACC (Sigma-Aldrich).

Immunomagnetic Cell SortingSingle-cell suspensions were obtained from kidney cortical tissue

specimens by mechanical disaggregation using the Medimachine Sys-

tem (BD Biosciences). Anti-CD45 MicroBeads, antiglycophorin A Mi-

croBeads, anti-FITC Multisort Kit, anti-PE Multisort Kit, and CD133

Cell Isolation Kit were obtained from Miltenyi Biotec GmbH.

Isolation of CD24CD133 and CD24CD133 cells was performed

by high-gradient magnetic cell sorting (23). The positive cell fractions

consisted of97% of CD24CD133 cells. Generation of clones from

CD24CD133 and CD24CD133 cells was achieved by limiting di-

lution in fibronectin-coated 96-well plates in EGM-MV 20% FBS.

Confocal MicroscopyConfocal microscopy was performed on 5-m sections of renal fro-

zen tissues or on cells that were cultured on chamber slides as de-

scribed (24) by using an LSM 510 META laser confocal microscope

(Carl Zeiss, Jena, Germany).

Staining with Alexa Fluor 488 Phalloidin (Molecular Probes), FITC-

labeled Dolichos Biflorus Agglutinin (DBA), and FITC-labeled Lotus

Tetragonolobus lectin (LTA; Vector Laboratories, Burlingame, CA)

were performed following the manufacturers instructions.

For quantification of fibrosis in glycerol-injected SCID mice (see

later), four random sections of kidney tissue that stained for -SMA or

TGF-1 were recorded using a 20 objective and scanned from each

tissue of 16 mice (eight mice that were treated with CD24CD133 PEC

and eight mice from the saline-treated group). All random scans of the

kidney tissue for each treatment group were recorded at the same

photo multiplier tube, pinhole aperture, and laser voltage setting and

analyzed using LSM 510 confocal microscopy software 3.0. This anal-

ysis resulted in a data set expressed as fibrotic tissue (-SMA and

TGF-1 positive) area in m2 per image field.

Real-Time Quantitative Reverse TranscriptasePCRTaq-Man reverse transcriptasePCR (RT-PCR) was performed as de-

scribed (25). BmI-1, Tau protein, MAP-2, necdin, neural enolase, nestin,

-tubulin III, Na/H exchanger, aminopeptidase A, Na/glucose co-

transporter (Na/Gluc1), -glutamyltransferase (-GT), aquaporin-1(AQP1), aquaporin-3 (AQP3), Na/Cl transporter, Runx2, and adiponec-

tin quantification was performed using Assay on Demand kits (Applied

Biosystems, Warrington, UK). Oct-4 mRNA expression and quantifica-

tion were performed as described (24).

Flow CytometryFlow Cytometry was performed as described (23).

In Vitro DifferentiationTubulogenic differentiation was obtained by culturing clones of

CD24CD133 PEC in commercially available REBM medium that

contained SingleQuotes (hydrocortisone, hEGF, FBS, epinephrine, in-

sulin, triiodothyronine, transferrin, and gentamicin/amphotericin-B;

2444 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 24432456, 2006


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Cambrex Bio Science) and was supplemented with 50 ng/ml hepato-

cyte growth factor (HGF) (Peprotech, Rocky Hill, NJ) for 30 d.

Osteogenic, adipogenic, or neurogenic differentiation of

CD24CD133 PECderived clones was induced as described else-

where (24,26,27). For osteogenic induction, CD24CD133 PEC were

cultured in -MEM and 10% horse serum that contained 100 nM

dexamethasone, 50 M ascorbic acid, and 2 mM -glycero-phosphate

(all from Sigma-Aldrich). The medium was changed twice a week for 3

wk. For adipogenic differentiation, CD24CD133 PEC were incubatedin DMEM high glucose (hg; Invitrogen, Carlsbad, CA) that contained

10% FBS, 1 M dexamethasone, 0.5 M 1-methyl-3-isobutylxanthine, 10

g/ml insulin, and 100 M indomethacin (all from Sigma-Aldrich).

After 72 h, the medium was changed to DMEM hg, 10% FBS, and 10

g/ml insulin for 24 h. These treatments were repeated three times.

The cells then were maintained in DMEM hg, 10% FBS, and 10 g/ml

insulin for one additional week. For neurogenic differentiation,

CD24CD133 PEC were plated in DMEM hg and 10% FBS. After 24 h,

medium was replaced with DMEM hg, 10% FBS that contained B27

(Invitrogen), 10 ng/ml EGF (Peprotech), and 20 ng/ml basic fibroblast

growth factor (Peprotech). Five days later, cells were washed and

incubated with DMEM that contained 5 g/ml insulin, 200 M indo-

methacin, and 0.5 mM 1-methyl-3-isobutylxanthine in the absence ofFBS for 5 h. Alizarin red, Oil-Red O, and alkaline phosphatase (AP)

staining was performed as described (24,26,27).

Electrophysiologic AnalysisThe whole-cell patch-clamp technique was performed in voltage-

clamp conditions, as described in detail previously (28). Cells in the

recording chamber were superfused at a rate of 1.8 ml/min at 22 to

24C with the following bath solution: 122.5 mM NaCl, 2 mM CaCl2, 10

mM HEPES, and 20 mM TEA-OH as K channel blocker. For blocking

of Na and L-type Ca2 channels, 1 M Tetrodotoxin (TTX), 10 M

nifedipine, and 100 M Cd2 (added as CdSO4) were used. Pipette

solution contained 150 mM CsBr, 5 mM MgCl2, 10 mM EGTA, and 10

mM HEPES. For bath and pipette solution, pH was titrated to 7.4 withNaOH and to 7.2 with TEA-OH, respectively. Pipettes resistance was 2

to 3 M.

Determination of [Ca2]i[Ca2]i was determined by a laser confocal microscope (LSM 510

META, Zeiss), as described (29).

Xenograft in SCID Mice Model of ARFModels of rhabdomyolysis-induced ARF were performed in female

SCID mice (Harlan, S. Pietro al Natisone, Italy), as described previously

(30,31), by intramuscular injection with hypertonic glycerol (8 ml/kg

body wt of a 50% glycerol solution; Sigma-Aldrich) into the inferiorhind limbs. Animal experiments were performed in accordance with

institutional, regional, and state guidelines and in adherence to the

National Institutes of Health Guide for the Care and Use of Laboratory

Animals. Two groups of mice on days 3 and 4 after glycerol received an

intravenous injection into the tail vein as follows: Group 1, saline (n

32); and group 2, CD24CD133 PEC (n 32; 0.75 106 on day 3 and

0.75 106 on day 4) obtained from five different human donors (two

men and 3 women). Mice were killed at different time intervals (days 3,

7, 11, and 14), and samples for blood urea nitrogen (BUN) determina-

tion were collected. BUN levels were measured in heparinized blood by

the Aeroset c8000 test (Abbott, Wiesbaden, Germany). BUN levels that

exceeded 40 mg/dl were considered abnormal. Normal range in our

experiments was between 30 and 37 mg/dl, as calculated in 16 addi-

tional untreated mice that were killed when the other mice received

Figure 1. Coexpression of the stem cell (SC) markers CD24 andCD133 identifies a subset of parietal epithelial cells (PEC) in theBowmans capsule of adult human kidney. (A) Double-labelimmunofluorescence showing expression of CD24 (red) and

CD133 (green) by PEC in the Bowmans capsule of an adulthuman kidney. Merged image (yellow) demonstrates coexpres-sion of CD24 and CD133 by a subset of PEC localized at theurinary pole (UP; bar 50 m). To-pro-3 counterstains nuclei(blue). (B) High-power magnification of a double-label immu-nofluorescence showing expression of CD24 (red) and CD133(green) by PEC. Merged image demonstrates co-localization ofCD24 and CD133 in the cytoplasm and on the membrane ofPEC facing the glomerulus (G), whereas only CD24 is ex-pressed on the basal membrane of the cells (bar 10 m).To-pro-3 counterstains nuclei (blue). (C) CD133 detection withtwo different anti-CD133 mAb. Both 293C3 (red) and AC133(green) mAb stain a subset of PEC in the Bowmans capsule.Merged image demonstrates co-staining of the same cells (yel-low; bar 50 m). To-pro-3 counterstains nuclei (blue). (D)Detection of CD24 (red), CD133 (green), and CD29 (blue) atkidney glomerular level. CD29 staining allows identification ofthe afferent arteriola (AA). Merged image shows that CD24 andCD133 selectively co-stain a subset of PEC localized opposite tothe vascular pole (yellow; bar 50 m). (E) High-power mag-nification of a triple-label immunofluorescence showing ex-pression by PEC of CD24 (red), CD133 (green), and CD106(blue). Merged image demonstrates co-localization of CD24and CD133 (yellow) in the cytoplasm and on the membrane ofPEC facing the glomerulus, whereas CD24 and CD106 (purple)are coexpressed on the basal membrane. Apical membrane ofPEC is indicated by the arrow. Areas of coexpression among

CD24, CD133, and CD106 appear white (bar 10 m).

J Am Soc Nephrol 17: 24432456, 2006 Identification of Multipotent Progenitors in the Bowmans Capsule 2445


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injection of glycerol (day 0). Two additional groups of mice were

treated as follows: Group 3 (n 8) received an intravenous injection of

CD24CD133 cells (0.75 106 on day 3 and 0.75 106 on day 4) that

were labeled with the PKH26 Red Fluorescence Cell Linker Kit (Sigma-

Aldrich); and group 4 (n 8) received an intravenous injection of

CD24CD133 cells (0.75 106 on day 3 and 0.75 106 on day 4) that

were labeled with the red fluorescent dye PKH26 and obtained from

three female human donors. Mice were killed 10 d later (day 14). BUN

levels were measured as described above. Kidneys were collected fromall groups of animals.

Immunohistochemistry and Fluorescence In SituHybridization Analysis

Immunohistochemical analysis was performed as described previ-

ously (25). Sections for fluorescence in situ hybridization analysis were

prepared using the Paraffin Pretreatment Reagent Kit (Vysis, Olympus,

Milan, Italy), and Y chromosome was revealed with whole chromo-

some paint Y spectrum orange probe (Vysis, Olympus), following the

manufacturers instructions.

Statistical AnalysesThe results are expressed as mean SD. Comparison between

groups was performed by the Mann-Whitney test. P 0.05 was con-

sidered to be statistically significant.

ResultsSubset of PEC of the Bowmans Capsule Coexpresses CD24and CD133

Analysis by confocal microscopy of tissue specimens from

normal adult human kidneys revealed that CD24, a marker of

the renal embryonic progenitor population, was expressed not

only by a subset of distal tubules that localized mainly in the

medulla but also by a subset of PEC of the Bowmans capsule

(Figure 1, A and B). Of note, an anti-CD133 mAb (clone 293C3),which recognizes an epitope that is selectively expressed by

adult SC, co-stained the same subset of PEC (Figure 1, A and B),

in addition to some interstitial cells, and rare tubular structures.

Such a distribution of CD133 was confirmed by using another

anti-CD133 mAb (clone AC133; Figure 1C). Only PEC that

localized in close proximity to the tubule/glomerular junction

at the urinary pole showed the co-staining for CD24 and CD133Figure 2. Isolation and characterization of CD24CD133 PEC.(A) Light microscopy image of cells outgrowing from seededcapsulated glomeruli. (B) Laser confocal microscopy demon-strates CD24 expression by all cells outgrowing from glomeruli(green). To-pro-3 counterstains nuclei (blue; bar 100 m). (C)Laser confocal microscopy demonstrates absence of green sig-nal in all cells outgrowing from glomeruli when stained with anisotype-matched control antibody. To-pro-3 counterstains nu-clei (blue; bar 100 m). (D) CD24CD133 PEC that werederived from glomerular outgrowth represent a homogeneouspopulation that is composed of virtually 100% of cells thatexpress CD24, CD133, CD106, CD105, and CD44, but all arenegative for the endothelial markers CD31 and CD34. Flowcytometry analysis of a representative bulk culture is shown.(E) CD24CD133 PEC that were derived from glomerularoutgrowth do not express the podocyte marker CD35, as as-sessed by flow cytometry. (F) Confocal microscopy demon-strates that CD24CD133 do not express the podocyte mark-ers synaptopodin and WT-1 (bar 100 m). (G) Primary

cultures of podocytes express high levels of synaptopodin at

the cytoplasmic level (red) and WT-1 at the nuclear level (light

blue; bar

100

m). (H) CD24

CD133

PEC that were derivedfrom glomerular outgrowth do not express the distal tubules/collecting ducts marker epithelial membrane antigen-1 (EMA-1), as assessed by flow cytometry. (I) CD24CD133 PEC thatwere derived from glomerular outgrowth lack the distal tubulemarker Tamm-Horsfall glycoprotein (THG; left), as well asfluorescence staining for the proximal tubule markers LotusTetragonolobus lectin (LTA; middle; bar 100 m), as assessedby confocal microscopy. Negative histochemical staining foralkaline phosphatase (AP; right). A representative bulk cultureis shown. (J) Comparison by quantitative reverse transcriptasePCR (RT-PCR) of mRNA levels for markers of differentiatedtubular cells in CD24CD133 versus CD24CD133 renalcells. Columns represent mean values SD as obtained from

three different donors. Magnification, 40 in A.



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(Figure 1A). A triple staining for CD24, CD133, and CD29

(which allowed clear definition of glomerular morphology)

confirmed that CD24CD133 PEC localized opposite to the

vascular pole (Figure 1D). CD24CD133 PEC also coex-

pressed CD106 (Figure 1E), CD105, CD54, and CD44 (data not

shown). Triple-label immunofluorescence demonstrated co-

localization of CD24 and CD133 in the cytoplasm and on the

membrane of PEC facing the glomerulus (G), whereas only

CD24 was expressed on the basal membrane of the cells (Figure

1B), where it co-localized with CD106 (Figure 1E).

Isolation and Characterization of CD24CD133 PECTo obtain CD24CD133 PEC free from any other renal cell

type, we plated isolated capsulated glomeruli in fibronectin-

coated dishes. After 5 d of culture, cellular outgrowth from

scattered adherent glomeruli was observed (Figure 2A). Con-

focal microscopy demonstrated that these cells originated from

the outgrowing of CD24 PEC of the Bowmans capsule (Fig-

ure 2B). Cells that grew out from plated glomeruli that stained

with an isotype control yielded negative results (Figure 2C).

FACS analysis showed that the recovered population homoge-

neously exhibited the presence of CD24, CD133, CD106, CD105,

and CD44 (Figure 2D), whereas surface molecules that were

expressed by endothelial cells (CD31, CD34) were not detect-

able (Figure 2D). FACS analysis for the podocyte marker CD35

yielded negative results (Figure 2E). Expression of the podocyte

markers WT-1 and synaptopodin was very weak or absent

(Figure 2F), whereas the same markers strongly stained cul-

tured podocytes (Figure 2G), as assessed by confocal micros-

copy. The absence of contaminating cells that originated from

distal tubules or collecting ducts was confirmed at the protein

level by the lack of EMA-1 (Figure 2H) and THG expression

(Figure 2I, left). Furthermore, the negative staining with LTA(Figure 2I, middle) and for AP excluded the possible contami-

nation by proximal tubules (Figure 2I, right).

To address further the nature of CD24CD133 cells, we also

assessed and compared mRNA levels of several lineage-specific

renal cell markers in freshly isolated CD24CD133 and

CD24CD133 cells by real-time RT-PCR. To this aim, total

cell suspensions of digested cortical renal tissue were sorted

with immunomagnetic beads into CD24CD133 and

CD24CD133 cells. CD24CD133 cells represented 0.5 to 4%

of cortical renal cells, whereas CD24CD133 cells were on

average 95 to 99% of them. As expected, in agreement with

their nature of fully differentiated cells, CD24CD133 cells

Figure 3. Growth properties of CD24CD133 cells and comparison with CD24CD133 cells. (A) Representative growth curvesof the CD24CD133 cells (F) or CD24CD133 () cells that were obtained through immunomagnetic sorting from total renalcell suspensions. The results represent mean values SD of cell counts that were obtained in four different experiments from fourdifferent donors in the first 10 d of culture. (B) CD24CD133 cells in culture were expanded for 60 to 90 population doublings(PD) during a 4-mo period. Results are mean SD obtained from four different donors. (C) Flow cytometric analysis of DNAcontent performed on bulk cultures of CD24CD133 cells at 50 PD, demonstrating 100% diploid cells. One representative of fourseparate experiments is shown. (D) Assessment of mRNA levels for BmI-1 by real-time quantitative RT-PCR in cultures of humanmicrovascular endothelial cells (HMVEC), human renal proximal tubular cells (HRPTEC), human mesangial cells (HMC), humanglomerular visceral epithelial cells (HGVEC), human aortic smooth muscle cells (HASMC), CD24CD133 cells, CD24CD133

cells, and HEK cells. Results are expressed as mean SD of triplicate assessment in primary cultures from five different donors.(E) Assessment of mRNA levels for Oct-4 by real-time quantitative RT-PCR in cultures of HMVEC, HRPTEC, HMC, HGVEC,HASMC, CD24CD133 cells, CD24CD133 cells, and HEK cells. Results are expressed as mean SD of triplicate assessmentin primary cultures from five different donors.



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AP-positive colonies (Figure 6A, left) that, during differentia-

tion, transformed into mineralized nodules, as assessed by

Alizarin Red staining (Figure 6A, left). Accordingly, osteogen-

esis associated with upregulation of Runx2 mRNA levels (Fig-

ure 6A, right). Adipogenic differentiation of CD24CD133

PEC was demonstrated by Oil Red O staining of lipid vacuoles,

which were completely absent from undifferentiated cells (Fig-

ure 6B, left), and upregulation of adiponectin mRNA levels(Figure 6B, right).

CD24CD133 PEC that were cultured under neurogenic

conditions acquired the expression of NF-200, neurofilament

M, choline acetyl-transferase, MAP-2 (Figure 7, A and B), and a

neuron-like morphology (Figure 7C). Furthermore, differenti-

ated cells exhibited strong upregulation of mRNA levels of

-protein, MAP-2, necdin, neural enolase, nestin, and -tubulin

III (Figure 7D). Patch-clamp recordings that were performed on

CD24CD133 PEC derived neural cells put in evidence a

slow high-voltage activated inward current (Figure 7E). The

voltage threshold of its occurrence (approximately 40 mV)

and its typical time course suggested the activation of the

high-voltage operated L-type Ca2 channel. Accordingly, the

specific channel blockers nifedipine and Cd2 completely abol-

ished such a current. Moreover, the I-V relation showed max-

imal current amplitude at 20 mV and could be fitted best by a

Boltzmann function with parameters in agreement with the

presence of L-type Ca2 channel of neuronal type (Figure 7F).

In the presence of L-type Ca2

channel blockers, a fast, tran-sient inward current could be recorded in the first 10 ms of the

test pulse (Figure 7G). This current resolved in approximately 7

ms at10 mV, peaking at 0.7 ms. It activated from40 mV and

showed a maximum at 5 mV. Addition of 1 M TTX com-

pletely but reversibly abolished this current (Figure 7G, red

line). Figure 7I shows current traces that were recorded during

inactivation. The I-V activation and inactivation curve (Figure

7, H and J) were fitted by Boltzmann function with parameters

in agreement with TTX-sensitive Na channels of neuronal

type.

Intravenously Injected CD24

CD133

PEC RegenerateTubular Cells in SCID Mice with ARF

To test the ability of CD24CD133 PEC to participate to

renal repair, we used an in vivo model of rhabdomyolysis-

induced ARF in SCID mice, generated by intramuscular injec-

tion of glycerol. Compared with normal renal tissue (Figure

8A), kidneys from glycerol-treated mice showed vacuolization,

widespread necrosis of tubular epithelial cells, and tubular

hyaline cast formation (Figure 8B). Proximal and distal tubules

displayed loss of brush border and flattening of epithelial cells

(Figure 8B). At the peak of tubular injury, CD24CD133 PEC

as well as CD24CD133 cells were labeled with the red fluo-

rescent dye PKH26, and each of the two cell populations wasinjected into the tail vein of glycerol-treated SCID mice. Ten

days later, both kidneys were harvested from each mouse, and

sections were analyzed for the presence of labeled cells. La-

beled cells were never detected in control mice that received

injections of CD24CD133 cells (Figure 8C) or of saline solu-

tion (data not shown), whereas in mice that received injections

of CD24CD133 PEC, they spread to the cortex and the me-

dulla (Figure 8D, red). Most injected PEC localized inside the

tubules, where they expressed specific markers of different

portions of the nephron (Figure 8, D through F, arrows), even

when some cells also were observed in the interstitium (Figure

8D) and a very few in the glomeruli (data not shown). Quan-

tification of the number of PKH26-positive cells that expressed

markers of differentiated tubular cells was performed on sec-

tions that were stained with LTA or DBA. The number of

PKH26-labeled/LTA-stained tubular cells was equal to 6.48

3.4% of all LTA-stained proximal tubular cells, whereas pro-

portions of PKH26-labeled/DBA-stained cells corresponded to

5.8 2.6% of all DBA-stained distal tubules/collecting ducts. In

SCID mice that received injections of CD24CD133 cells (Fig-

ure 8G, left) or saline (data not shown), HLA-I human antigen

expression was never found, whereas human HLA-I antigen

was detected consistently in mice that received injections of

CD24CD133 cells (Figure 8G, middle and right). Double-

label immunohistochemistry for human HLA-I antigen and

Figure 6. Differentiation of CD24

CD133

PECderived clonesin osteoblasts and adipocytes. (A, left) Representative micro-graphs of histochemical staining for Alizarin red and AP before(day 0) and after (21 d) CD24CD133 PEC culture in osteo-genic differentiation medium. (Right) Assessment of mRNAlevels of Runx2 before (day 0) and after (21 d) culture in thesame medium. Columns represent mean values SD obtainedfrom 50 different clones. (B, left) Representative micrographs ofhistochemical staining for Oil Red-O before (day 0) and after(21 d) CD24CD133 PEC culture in adipogenic differentiationmedium. (Inset) High-power magnification of some differenti-ated cells. (Right) Assessment of mRNA levels of adiponectin at0 d and after 21 d of culture in the same medium. Columnsrepresent mean values SD obtained from 50 different clones.

Magnifications: 100 in A; 200 in B; 320 in B, inset.



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cytokeratin confirmed the engraftment of CD24CD133 PEC

into tubular structures (Figure 8G, middle and right). In addi-

tion, whereas kidney cells of mice that received injections of

saline (Figure 8H, left) never exhibited the presence of the Y

chromosome (Figure 8H, left), this could be detected clearly in

kidney cells from mice that received injections of

CD24CD133 PEC that were derived from male human do-

nors (Figure 8H, middle and right, red).

Effects of CD24CD133 PEC on Renal Function andStructure

To determine whether CD24CD133 PEC also could influ-

ence the renal function of mice with ARF, we measured BUN

levels in mice that had glycerol-induced ARF and received

injections of CD24CD133 PEC or saline. In our setting, in-

jection of glycerol induced significant increases in serum BUN,

which peaked at day 3, declined at day 7, and stabilized at days

11 and 14 to values that were significantly higher than those in

healthy mice (Figure 9, A and B), as described previously

(34,35). Intravenous injection of CD24CD133 PEC on days 3

and 4 seemed to be protective of renal function, as reflected by

significantly lower BUN values on days 11 and 14 in compar-

ison with mice that received injections of saline or

CD24CD133 cells (Figure 9, A and B). It is interesting that on

day 14, mice that were treated with CD24CD133 PEC dis-

played completely restored renal function, with BUN levels

that were not significantly different from those of healthy mice,

whereas mice that were treated with saline or CD24CD133

cells showed significantly higher levels of BUN in comparison

Figure 7. Acquisition by CD24CD133 PECderived clones ofphenotypic and functional properties of neural cells. (A) Ab-sence of the neural markers neurofilament 200 (NF200), neuro-filament M (NFM), choline acetyl-transferase (ChAT), and mi-crotubule-associated protein-2 (MAP-2) before culturing PEC inneurogenic differentiation medium, as assessed by confocalmicroscopy. To-pro-3 counterstains nuclei (bar 100 m). Onerepresentative clone is shown. (B) Strong expression of theneural markers NF200, NFM, ChAT, and MAP-2 after differen-tiation in the same medium (green). To-pro-3 counterstainsnuclei (bar 100 m). One representative clone is shown. (C)High-power magnification of a representative image showing

acquisition of a typical neuronal morphology and staining forChAT (green) by CD24CD133 PEC cultured under neuro-genic conditions (bar 100 m). (D) Assessment by real-timequantitative RT-PCR of mRNA levels fold increase of severalneural markers after differentiation under neurogenic condi-tions compared with values that were obtained in the same cellsbefore differentiation. Columns represent mean values SDobtained from 50 different clones. (E through H) Inward Ca2

and Na currents in CD24CD133 PECderived neurons.Representative current traces recorded at a holding potential of90 mV; 1-s step pulses from 80 to 50 mV were applied in10-mV increments. Data were acquired with different samplingtime (50 s in the first 100 ms and 1 ms for the remainingduration of the test pulse) to highlight fast or slow phenomena.

(E) Time course of L-type Ca2 current (ICa); for clarity, only

current traces that were recorded at 60, 40, 20, 0, 20, 30,and 40 mV are presented. (F) ICa-V curve determined at thecurrent peak (n 26). (G) Time course of Na current (INa);only current traces that were recorded at 60, 40, 30, 20,10, 0, 20, and 30 mV are presented; red line is INa elicited at 0mV in the presence of Tetrodotoxin (1 M). (H) INa-V curvedetermined at the current peak (n 26). (F and H) Continuousline superimposed through the data are the fitted Boltzmannfunction for activation: Ia(V) Gmax(V Vrev)/{1 exp[(Va V)/ka]}, where Gmax is the maximal conductance, Vrev is theapparent reversal potential, Va is the potential that elicits the

half-maximal increase in conductance and ka is the slope factor.The best-fit parameters for ICa were Gmax 6 1 nS, Va 0 2 mV, ka 8.4 2 mV, and Vrev 79 8 mV; those for INawere Gmax 28 7 pS, Va 18 2 mV, ka 6.0 1 mV,and Vrev 47 4 mV. (I) INa inactivation evoked from holdingpotential of90 mV; test pulse to 0 mV prepulsed from 90 to30 mV in 10-mV increments, only traces without prepulse (90mV) and prepulsed at 70, 60, 30, 40, and 30 mV aredepicted. (J) Normalized inactivation curve for INa; continuousline superimposed through the data are the fitted Boltzmannfunction for inactivation: Ih(V) 1/{1 exp[(Vh V)/kh]},where Vh is the potential eliciting the half-maximal current andkh is the slope factor for inactivation. The best-fit parameterswere Vh 58 5 mV and kh 6.0 6 mV. For comparison,

the curve reported on the right is that for activation.



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with both CD24CD133 PECtreated mice or healthy mice

(Figure 9B).

Next, we investigated whether the improvement of renal

function by CD24CD133 PEC treatment was also associated

with a better preservation of renal structure. Assessment of

renal histology on day 14 after injury provided clear evidence

for tubular repair, although it was not uncommon to observe a

small proportion of tubules with abnormal morphology, to-gether with areas of tubulointerstitial and periglomerular fibro-

sis. Fibrotic areas therefore were quantified through direct mea-

surement of green fluorescence area for -SMA and TGF-1

expression by image analysis. In Figure 9, C and D, represen-

tative confocal micrographs of a single optical section of kidney

parenchyma from the two groups of mice are depicted. Kidney

tissue from the CD24CD133-treated group was normal (Fig-

ure 9D). By contrast, kidneys from mice that were treated with

glycerol and received saline (Figure 9C) were characterized by

focal areas of interstitial and periglomerular fibrosis, as illus-

trated by -SMA staining (Figure 9C). In mice that were treated

with CD24

CD133

cells, there was a significant decrease in-SMAstained tissue area (181.8 54.4 versus 3124 808.7

m2; P 0.001) and in TGF-1stained tissue area (1233.9

205.3 versus 4608.9 1311.6 m2; P 0.01) compared with mice

that were treated with saline, as quantified by confocal micros-

copy.

DiscussionThere is increasing evidence that cells that show at least

multipotentiality (24) and possibly pluripotentiality (20) exist in

different adult organs. We demonstrate here that in adult nor-

mal kidney, a subset of PEC in the Bowmans capsule is the

only cell type that shows coexpression of the SC markers CD24

and CD133 and of the SC-specific transcription factors Oct-4

and BmI-1 but a lack of lineage-specific markers. Differently

from all other types of renal cells, CD24CD133 PEC also

expressed CD106, a surface molecule that together with CD105,

CD54, and CD44 is usually coexpressed by adult SC types that

grow adherent, such as mesenchymal SC or multipotent adultFigure 8. Engraftment of CD24CD133 PEC in kidneys of

SCID mice with acute renal failure (ARF) and generation ofdifferent types of renal tubular cells. (A) Light micrographshowing normal mouse renal tissue stained with hematoxylinand eosin (H&E; left) or with phalloidin (green, right; bar 50m). (B) Tubulonecrotic injury observed after an intramuscularinjection of glycerol, as assessed with H&E staining (left) or

with phalloidin (right); the latter reveals the loss of brushborder and the flattening of epithelial cells (green; bar 50m). (C) Representative micrograph of kidney sections of con-trol SCID mice that received injections of CD24CD133 cellsand stained with LTA showing the absence of red-stained cells,as assessed by confocal microscopy (bar 20 m). (D) Repre-sentative micrograph of kidney sections of mice that had ARFand received injections of PKH26-labeled CD24CD133 PEC(red) and stained with LTA (green), as assessed by confocalmicroscopy. Small arrows point to multiple red cells. The largerarrow points to a proximal tubule (bar 20 m). (E) High-power magnification of the kidney section shown in D, whichdemonstrates regeneration of a proximal tubule structure(bar 20 m). (F) High-power magnification of another kidney

section obtained from a SCID mouse that had ARF and received

an injection of PKH26-labeled CD24CD133 PEC (red) andstained with Dolichos Biflorus Agglutinin (DBA) on the basalsurface of two tubular structures (green), which demonstrates

regeneration of a collecting duct structure (arrow). Other tubu-lar structures that are stained with PKH26 but not with thecollecting ducts marker DBA are visible (bar 20 m). (G)Double-label immunohistochemistry for cytokeratin (blue) andHLA-I human antigen (red) in kidneys of SCID mice withglycerol-induced ARF. (Left) Absence of red signal in tubules ofa kidney section from a mouse that received an injection ofCD24CD133 cells. (Middle and right) Human HLA classIpositive cells (red, arrows) in cytokeratin-expressing (blue)tubules of SCID mice with glycerol-induced ARF after injectionof CD24CD133 PEC. (H) Detection of chromosome Y by thefluorescence in situ hybridization technique in control mice thatreceived injections of saline solution (left) and in kidneys fromfemale mice that received injections of CD24CD133 PEC

from human men (red, middle and right; bar 20 m).



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progenitor cells (MAPCS) (20,27). Purified CD24CD133 PECcould be obtained directly from outgrowth of isolated glomer-

ular structures and exhibited high clonogenic efficiency and

self-renewal potential. Moreover, under appropriate culture

conditions, clones that were derived from single

CD24CD133 PEC could be induced to differentiate into tu-

bular epithelial cells that showed markers of cells from differ-

ent portions of the nephron. Differentiation toward tubular

cells resulted in the acquisition of high mRNA levels of markers

that are characteristic of fully differentiated tubular epithelia,

such as aminopeptidase A and Na/Gluc1, -GT, the Na/H

exchanger, AQP1, AQP3, or the thiazide-sensitive Na/Cl trans-

porter, with a prominent increase in AQP1 levels, consistently

with the high proportions of cells in differentiated clones that

acquire protein markers of proximal tubular cells. Importantly,

cells that were derived from the same clones also could be

differentiated in extrarenal cell types, such as adipocytes, os-

teoblasts, or cells that show phenotypic markers and functional

properties of neurons, as it has already been shown for other

types of adult human SC (36,37). Taken together, these results

strongly suggest that CD24

CD133

PEC represent a popula-tion of multipotent progenitor cells. Accordingly,

CD24CD133 but not CD24CD133 human renal cells en-

grafted into the kidney of SCID mice that had glycerol-induced

ARF and also improved the morphologic and functional kidney

damage.

To our knowledge, this is the first report to show that a

population of resident renal cells of human origin ameliorate

the structural recovery of the kidney after the induction of ARF

and, more important, that they exert therapeutic effects on

renal function. Acute tubular necrosis is the most common form

of ARF and is considered a potentially reversible process. How-

ever, high percentages of patients (approximately 40%) fail torecover their renal function completely, and at discharge, they

show mild to moderate renal failure (38,39). It is interesting that

recent follow-up studies also demonstrated that approximately

10% of these patients require renal replacement therapy for

ESRD after 5 yr because of progressive renal fibrosis and

chronic dysfunction (40). Therefore, the observation that treat-

ment of mice that were affected by acute tubular necrosis with

CD24CD133 cells led to a complete recovery of renal func-

tion and to a significant reduction of renal fibrosis whereas

control mice did not completely recover renal function and

developed large areas of interstitial and periglomerular fibrosis

is of potential clinical relevance. Functional protection byCD24CD133 cells probably is due to the capacity of these

cells to engraft the damaged kidney and to integrate/differen-

tiate within tubules, as shown by the demonstration that

CD24CD133 but not CD24CD133 renal cells repopulated

the tubule, exploiting their potential to generate tubular epithe-

lial cells of different portions of the nephron.

One possible explanation for this phenomenon is that it is the

result of a cell fusion. Recently, indeed, cell fusion between

transplanted cells and recipient tissue has been claimed as an

alternative novel mechanism to differentiation, which can occur

in vivo and produce functional cells (4143). However, in other

experimental systems, the cell fusion process has been excludedas a way to explain bone marrow SC plasticity (44,45). Whether

in our setting CD24CD133 celldriven regeneration of tubu-

lar cells also might result from the fusion with resident cells

cannot be ruled out completely, at least in vivo. However, our in

vitro results strongly suggest that CD24CD133 PEC are plas-

tic and acquire phenotypic and functional properties of renal

and extrarenal cell types through differentiation and not

through cell fusion, because multidifferentiation was achieved

in clonal progenies that were derived from single

CD24CD133 cells. Taken together, these results suggest that

CD24CD133 cells represent a previously unidentified popu-

lation of resident renal multipotent progenitors and thus can be

Figure 9. CD24CD133 cells protect glycerol-treated micefrom renal structure and function deterioration. (A) Blood ureanitrogen (BUN) levels as measured in untreated (E) or in glyc-erol-treated mice that received saline (red-filled circle) orCD24CD133 cells (F). Arrows point to the days of injectionof saline or CD24CD133 cells. Data are expressed as meanvalues SD. *P 0.01 and **P 0.001 versus glycerolsaline

at the same time. (B) Comparison of BUN levels among healthymice (), mice that were treated with saline (light gray), micethat were treated with CD24CD133 cells (dark gray), andmice that were treated with CD24CD133 cells (f) at day 14.Data are expressed as mean values SD. (C) Representativemicrographs of kidneys that were treated with saline andstained for -smooth muscle actin (-SMA; green). Nuclei arestained with To-pro-3 (bar 100 m). (D) Representative mi-crographs of kidneys that were treated with CD24CD133

cells and stained for -SMA (green). Nuclei are stained withTo-pro-3 (bar 100 m).



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named adult parietal epithelial multipotent progenitors

(APEMP).

The results of this study not only demonstrate the existence

of a population of renal multipotent progenitors within glomer-

ular structures but also suggest that the urinary pole of the

Bowmans capsule may represent a renal SC niche (46). This

hypothesis is supported by the observation that when renal

tubular cells are differentiated from embryoid bodies in thepresence of nephrogenic factors and injected in developing

kidney rudiments, they selectively localize to the glomerular/

proximal tubule junction (47). Another study suggested the

possible existence of SC niche in the renal papilla (5). Given the

complex embryologic origin of the kidney, it is possible that

two distinct SC niches exist in the medulla and in the cortex and

that proliferating amplifying renal cell progenitors likely local-

ize to the proximal and distal tubules. In agreement with this

hypothesis, possible tubular progenitors that express lineage

markers and show limited differentiation potential (4,5,9) were

identified recently in normal kidneys at the level of proximal

and distal tubular structures or at the interstitial level (49).The demonstration that APEMP represent a population of

multipotent progenitors may provide an important contribu-

tion to the understanding of the pathogenesis of nephron loss.

As known, the majority of diseases that progress to chronic

renal failure start at the glomerular level, in the endocapillary

compartment, where the inflammatory process involves the

capillaries and/or the mesangium. As long as a glomerular

disease remains restricted to the endocapillary compartment,

restitution or repair is possible, even in the case of massive

lesions. By contrast, spreading of the inflammation to the ext-

racapillary compartment (Bowmans space and Bowmans cap-

sule) results in dramatic kidney injury (48,49). The glomerulusmost likely dies, and the nephron is lost. On the basis of the

results of this study, we suggest that these irreversible pro-

cesses might reflect the loss of the renal SC niche that occurs

only when the extracapillary compartment is affected, thus

impairing the possibility of repair that may be provided by

APEMP. By contrast, when the injury is limited to the endo-

capillary compartment and the SC niche is not affected, the

glomerular damage can be repaired.

The identification of a subset of multipotent progenitors in

the Bowmans capsule also may provide an intriguing expla-

nation for the genesis of crescents, which are known to reflect

uncontrolled proliferation of PEC and their transdifferentiation

into mesenchymal and myeloid cells during rapidly progres-

sive glomerulonephritis (48,49). We suggest that crescent for-

mation might reflect a dysregulated activation of APEMP in

response to chronic inflammatory stimulation. The nature of a

multipotent progenitor of a subset of PEC also may provide a

reasonable explanation for another renal disorder, such as em-

bryonal hyperplasia of Bowmans capsular epithelium

(EHBCE) (50). EHBCE is a lesion that occurs in kidneys of

patients who are maintained on chronic dialysis, which consists

of poorly differentiated cells that proliferate around sclerosed

or obsolescent glomeruli (50). EHBCE is considered a reversion

of Bowmans capsular PEC to the state of embryonic cell (50).

APEMP might represent such a previously unidentified popu-

lation of embryonic progenitor-like cells. Taken together, the

results of this study provide the first description of a multipo-

tent progenitor cell in adult human glomeruli, thus opening

new avenues for the development of autologous cell therapies

in renal disorders.

AcknowledgmentsThis study was supported by the Tuscany Ministry of Health, by

Ministero dellIstruzione, dellUniversita e della Ricerca (MIUR), and

by the Research Institute BIOAGROMED of the University of Foggia.

B.M. is the recipient of a Fondazione Italiana per la Ricerca sul Cancro

(FIRC) fellowship.

We thank Melissa Poggesi for assistance.

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