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Collecting Duct-Derived Cells Display Mesenchymal
Stem Cell Properties and Retain Selective In Vitro
and In Vivo Epithelial Capacity
Joan Li,* Usukhbayar Ariunbold,* Norseha Suhaimi,* Nana Sunn,† Jinjin Guo,‡
Jill A. McMahon,‡ Andrew P. McMahon,‡ and Melissa Little*
*Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland, Australia; †Diamantina Institute,
University of Queensland, Woolloongabba, Queensland, Australia; and ‡Department of Stem Cell Biology and
Regenerative Medicine, Broad-CIRM Center for Regenerative Medicine and Stem Cell Research, University of
Southern California Keck School of Medicine, Los Angeles, California
ABSTRACT
We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney
that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC–like cells display a
distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice
identified the collecting duct as a source of kidney MSC–like cells, with these cells undergoing an epithelialto-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC–like cells selectively integrated into the aquaporin 2–positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed
after injection of BM-MSCs. Indeed, kidney MSC–like cells retained a capacity to form epithelial structures in
vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate
the origin of kidney MSC–like cells, we further examined Hoxb7+ fractions within the kidney across postnatal development, identifying a neonatal interstitial GFPlo (Hoxb7lo) population displaying an expression
profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4GCE/+:R26tdTomato/+
mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal
collecting duct, suggesting that such intercalation may represent a normal developmental mechanism
giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary
stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and,
possibly, repair.
J Am Soc Nephrol 26: ccc–ccc, 2014. doi: 10.1681/ASN.2013050517
Mesenchymal stromal cells (MSCs; also known as
mesenchymal stem cells)1 were first isolated from
the bone marrow (BM) on the basis of their ability
to adhere to plastic and display a fibroblastic phenotype.2 Although initially proposed to play a critical homeostatic function within the BM, these cells
show panmesodermal potential, including bone,
fat, and cartilage,3–5 suggesting a role as stem cells
for mesenchymal tissues.6 BM-MSCs are immunomodulatory and can also home to damaged tissues,6,7 with their participation in tissue repair at
distant sites of considerable interest. The systemic
delivery of BM-MSCs after a variety of acute renal
J Am Soc Nephrol 26: ccc–ccc, 2014
injuries (glycerol, mercury chloride, cisplatin, and
ischemic injury) elicits a reparative effect in animal
models.8–13 Although it was initially thought to occur by the transdifferentiation of MSCs into renal
Received May 21, 2013. Accepted April 25, 2014.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Prof. Melissa Little, Institute for Molecular
Bioscience, University of Queensland, St. Lucia, QLD 4072,
Australia. Email: [email protected]
Copyright © 2014 by the American Society of Nephrology
ISSN : 1046-6673/2601-ccc
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epithelium,8 the observed improvements in histology and
function are now considered to result from the secretion of
proreparative factors.9,12,14,15
Cells with MSC-like properties have now been isolated from
many solid organs.16 These tissue-derived MSC-like cells apparently represent perivascular cells on the basis of marker
expression (CD146+NG2+CD140b+)16–18 and have been proposed to support local tissue turnover and/or repair. We have
previously reported the isolation of an MSC-like population
from the adult mouse kidney that displayed long-term colonyforming capacity and clongenicity.19 These kidney MSC–like
cells displayed an immunophenotypic profile and functional
properties extremely similar to mouse BM-MSCs. However,
this population also displayed a kidney-specific gene expression signature,19 including the differential expression of the
collecting duct markers natriuretic peptide precursor type B,20
Uroplakin 1b,21 and Hoxb7.22 The expression of such markers
after extensive culture suggested epithelial origin and/or epithelial potential.
In this study, we show that kidney MSC–like cells are enriched
in the renal papilla, can be derived from the mature collecting
duct epithelium, and undergo an epithelial-mesenchymal
transition (EMT), giving rise to a robust, long-term, selfrenewing, and clonogenic MSC-like population. When microinjected into the kidney of a neonatal mouse, kidney MSC–like
cells selectively home and integrate into collecting duct epithelium. Together with in vitro and in vivo epithelial potential,
conditioned media from kidney MSC–like cells display proreparative activity.
RESULTS
Specific In Vivo Epithelial Potential of Kidney MSC–Like
Cells
On the basis of the differential expression of renal epithelial
markers by kidney MSC–like cells, we investigated the fate of
these cells after microinjection into the neonatal kidney at
postnatal day 1 (PND1), a time when nephron formation
and papillary maturation were still active (Figure 1, A and
B). MSCs isolated and cultured from total kidney or BM of
adult ubiquitous green fluorescent protein-positive (GFP+)
mice (Bulk cultures) were used for microinjection at late passage (approximately passage 10 [P10]). After microinjection,
GFP+ BM-MSCs were occasionally detected as rare scattered
single cells within the medullary interstitium but never detected within tubular epithelia (Figure 1C, Supplemental Figure
1A). GFP+ kidney MSC–like cells, although not detected in the
cortex, were detected in the medulla/papilla region (Figure
1D, Supplemental Figure 1A). These GFP+ cells were predominantly within tubular structures surrounded by a Collagen
IV+ basement membrane (Figure 1E), although occasional
interstitial GFP+ cells were also seen, often closely associated
with aquaporin 2–positive (Aqp2+) collecting ducts (Figure
1E). Colocalization with Aqp2, but not Umod, indicated a
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selective homing to and integration into collecting duct epithelium (Figure 1E), with 1260.8% of collecting duct cells
being GFP+ (Supplemental Figure 1B). Coimmunofluorescence with the mitotic marker, pHH3, indicated proliferation
of these integrated GFP+ cells (Figure 1E, Supplemental Figure 2). Nuclei double-positive for GFP and pHH3 represented 1.260.2% of all GFP + cells. By way of comparison,
0.560.2% collecting duct cells were pHH3 + in a normal
PND6 Hoxb7/enhanced GFP+ (EGFP+) kidney (comparable
age) (Supplemental Figure 2B); the incorporating kidney
MSC–like cells showed a statistically higher mitotic rate
than normal collecting duct epithelium (P=0.04). Two weeks
after injection, GFP+ cells could still be detected within Aqp2+
tubules, including some pHH3+ cells (Figure 1F), eliminating the possibility that these cells were being phagocytozed by
the collecting duct epithelium23 and indicating a long-term
contribution to epithelial structures. No GFP signals were
ever detected within F4/80 + macrophages located in the
same region (Supplemental Figure 3).
Enrichment of the Kidney MSC–Like Cells in the
Collecting Duct Epithelium
To identify the origin of MSC-forming cells within the kidney,
three isolation approaches were used and compared with MSClike populations derived from Bulk kidney digests. (1) On the
basis of the previously described stem cell markers, CD24 and
Sca-1,24–26 only the Lin2CD312CD24loSca-1+ fractions could
form MSC colonies in vitro (Supplemental Figure 4A) (termed
the Sorted fraction). (2) Regional isolation was achieved by
enzymatic digestion of manually dissected adult kidney cortex,
medulla, and papilla. (3) Isolation of cells specifically from the
collecting duct was performed using Hoxb7/EGFP mice,27
with both GFP+ and GFP2 fractions being isolated. Freshly
isolated GFP+(Hoxb7+) cells have low levels of CD24 expression (CD24lo) (Supplemental Figure 4B), similar to the Sorted
population.
After establishment and extended culture as MSCs, colonyforming efficiency, clonogenicity from a single colony, and
population doubling time were assessed for these different
isolated populations (Figure 2, Supplemental Figure 5).19 For
colony-forming efficiency, colonies were classified into small
(,25 cells), medium (25–100 cells), or large (.100 cells)
(Figure 2A) as an indicator of stem versus progenitor status.28,29 Cells isolated from papilla showed the highest capacity to form colonies of all sizes at 14 days postplating (Figure
2A), suggesting the strongest enrichment for stem cell properties. GFP+(Hoxb7+) populations displayed a colony-forming
efficiency similar to cells from the papilla, whereas the GFP2
fraction only produced small- and medium-sized colonies at
lower frequencies (Figure 2A). Clonogenicity of the Hoxb7+
fraction was .15-fold higher than Bulk and .3-fold higher
than Sorted populations (Figure 2B). Finally, population doubling times indicate that Hoxb7-derived MSC-like cells proliferate slightly faster than Bulk-cultured cells (Supplemental Figure 5). Together, these results suggest a more stem-like
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phenotype for populations derived from
medulla/papilla or directly isolated from collecting ducts.
Kidney MSC–Like Populations Arise
by an EMT
Because the GFP+ fraction isolated from
adult Hoxb7/EGFP mice is expected to represent mature collecting duct epithelium,
we investigated whether the formation of
kidney MSC-like cells represented an EMT.
After isolation, cells remained GFP+ for up
to 3 weeks while displaying an epithelial
morphology. By passage 4 (approximately
2 months), these cells changed morphology and downregulated Hoxb7 (loss of
GFP) (Figure 2C). Initially, Hoxb7-derived
cultures were immunopositive for collecting duct (Aqp2 and Pax2) and epithelial
(ZO-1) markers (Figure 2D). With passage,
these cells lost Aqp2 and ZO-1 and acquired
the pericyte/mesenchymal marker NG2
(Figure 2D). At passage 2, Hoxb7-derived
cultures were uniformly positive for the
principal cell marker Aqp2+ and showed no
staining for intercalated cell markers Pendrin
(b-intercalated) or AE1 (a-intercalated)
(Figure 2E), suggesting that the MSC-like
cultures arise from mature principal cells
or previously described bipotential Aqp2+
collecting duct progenitors.30
Kidney MSC–Like Cells Show
Panmesodermal Potential
MSCs derived from Bulk, Sorted (CD24lo
Sca1+), and GFP+(Hoxb7+) fractions all
displayed a characteristic murine MSC immunophenotypic profile (CD44, CD49e,
and Sca-1)18 (Figure 3A). Uniquely, by passage 6, 100% of Hoxb7-derived cells were
Figure 1. Specific epithelial integration of the kidney MSC–like cells. (A) Ultrasound
image of PND1 mouse kidney. Dashed line outlines the kidney as viewed under an
Ultrasound Biomicroscope. (B) Fluorescence microscopy showing the detection of
Fluoresbrite Yellow Green microspheres (green), which were mixed with cells and
used to localize the injection site within the nephrogenic zone 24 hours postinjection.
(C and D) Characterization of the location of GFP+ cells in the neonatal kidney 4 days
after injection with (C) BM-MSCs or (D) kidney MSC–like cells isolated from a ubiquitously GFP-expressing mouse. (C) Arrowhead indicates the presence of a single interstitial GFP+ BM-MSC. (D) GFP+ Aqp2+ tubular structures in the medullary region
after injection of kidney MSC–like cells. (E) Coimmunostaining for GFP (green), Collagen
IV (red; basement membrane), Aqp2 (red; collecting duct), Umod (red; loops of Henle),
and pHH3 (red; mitotic cells) in kidney sections injected with GFP+ kidney MSC–like cells
J Am Soc Nephrol 26: ccc–ccc, 2014
at 4 days postdelivery. Arrowheads indicate
occasional interstitial GFP+ cells in all panels
but pHH3 immunofluorescence, where they
indicate pHH3+ cells. Arrows indicate GFP+
cells within the tubular structures. Scale bar,
20 mm. (F) GFP+ tubular cells at 14 days
postdelivery. They can be colocalized with
Aqp2+ collecting duct, with some positive for
the mitosis marker pHH3. Arrowheads indicate a pHH3+ cell within a tubule. Scale bar,
20 mm. All images were captured using an
upright laser-scanning confocal microscope
for bright field and epifluorescence (Carl Zeiss
LSM 710).
Epithelial Potential of Kidney-Derived MSC-Like Cells
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positive for CD24, a marker previously associated with tubular progenitor/stem cells,
including renal progenitors.31,32 Hoxb7+
-derived cultures were also negative for
CD140a and CD140b (PDGFRa and
PDGFRb), known markers of pericytes previously associated with other tissue-derived
MSC populations.19,33 This finding reflects
the active selection against the pericytic
compartment when isolating this population from the Hoxb7/EGFP mouse. Despite
this finding, all three populations displayed
panmesodermal potential (Figure 3B),
which was previously reported for BMMSCs, reinforcing the MSC-like properties
of the Hoxb7-derived cells, despite the absence of classic pericytic marker expression.
Epithelial Potential of Kidney MSC–
Like Cells Derived from Collecting
Duct
Although our data identify a kidney MSC–
like population within the mature collecting duct, it may have represented a distinct
MSC-like population to that isolated from
initial Bulk cultures. We evaluated the epithelial potential of Hoxb7-derived MSClike cells in vitro using three-dimensional
culture in Collagen I.34 These cells formed
E-cadherin+ PECAM2 branching tubular
structures after 9 days, suggesting tubulogenesis but not vasculogenesis (Figure 4A).
No similar structures were observed using
BM-MSCs. To test their in vivo epithelial
integration capacity, Hoxb7-derived kidney
MSC–like cells were cultured for 12–15
passages, labeled with PKH26, and injected
into PND1 neonatal kidney. PKH26-labeled
cells were only detected in Aqp2+ tubular
structures within the medulla/ papilla and
displayed a slightly higher level of integration into the collecting duct than observed
for Bulk GFP + kidney MSC–like cells
(1963.2% versus 1260.8%; P=0.02)
Figure 2. Papillary enrichment and phenotypic changes of kidney MSC–like cells.
(A) Colony-forming efficiency of kidney MSC–like populations isolated by either regional dissection (cortex, medulla, or papilla) or FACS sorting as a CD24loSca-1+
fraction (Sorted) from wild-type mice or GFP+(Hoxb7+) and GFP2(Hoxb72) fractions
from Hoxb7/EGFP mice (n$3). *P,0.01 compared with the Sorted group. (B)
Clonogenicity of kidney MSC–like cultures isolated as Bulk, Sorted, and Hoxb7/GFP+
fractions (n=3). Passage number is indicated in parentheses. *P,0.01 compared with
Bulk culture. (C) Bright-field (BF) and fluorescence images of a GFP+ colony derived
from the Hoxb7+ fraction at passages 0 and 4. Note the loss of GFP on passage
suggesting the downregulation of Hoxb7 in culture. Scale bar, 100 mm. (D) Characterization
of cells derived from Hoxb7+ fractions at passages 2, 6, and 21. Immunofluorescence was
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performed for markers of the collecting duct
(Aqp2 and Pax2) as well as characteristic
markers of mesenchyme/pericyte (NG2) and
epithelium (ZO-1). Scale bar, 50 mm. (E) Expression of the principal cell marker (Aqp2)
and absence of intercalated cell markers
(Pendrin and AE1) on cells derived from
Hoxb7+ fraction at passage 2. Scale bar,
50 mm.
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and accelerate wound healing in vitro.35,36
To test whether kidney MSC–like cells can
facilitate repair, a scratch wound repair assay was performed. Madin–Darby Canine
Kidney (MDCK) cells were cultured to
confluence, subjected to scratch injury,
and continuously cultured in the presence
of normal culture medium or conditioned
medium from Bulk or Hoxb7-derived kidney MSC–like cells. Accelerated scratch
closure ensued in the presence of conditioned media from both kidney MSC–like
populations (Figure 5), suggesting the production of paracrine reparative factors by
these cells.
Detection of Hoxb7lo Interstitial Cells
within the Postnatal Kidney
Although present in adult kidney, we were
unable to isolate kidney MSC–like populations from embryonic kidney (data not
shown), despite the presence of an extensive
Hoxb7+ ureteric epithelium throughout development.37 Although the expression of
Hoxb7 in the embryonic collecting duct is
well characterized,22 the location and identity of Hoxb7+ cells in the postnatal kidney
are less clear. Analyses of Hoxb7/EGFP
transgenic mice between birth and adulthood confirmed that most GFP+ cells were
tubular epithelial cells largely colocalized
with Aqp2+ (Figure 6A). However, rare medullary Hoxb7GFPlo cells were also detected
between PND0 and PND14. They were interstitial as assessed by Collagen IV staining
(Figure 6B). FACS analysis confirmed the
Figure 3. Kidney MSC–like cells show typical MSC immunophenotype and mesoderlo
2
mal differentiation potential. (A) Comparative immunophenotypic analysis of kidney presence of a GFP EpCAM population
+
MSC–like cells derived as Bulk, Sorted, or Hoxb7/GFP cultures. They were analyzed within the neonatal kidney (Figure 6C).
for common MSC markers and the pericytic cell markers (CD140a/b). Passage number Although barely detectable at PND0, this
lo
2
is indicated in parentheses (n=2). (B) Analysis of the mesodermal differentiation ca- nonepithelial GFP EpCAM population
+
+
pacity of kidney MSC–like cells derived from Bulk or Hoxb7/GFP cultures compared reached 4.8% of total GFP cells in the
with BM-MSCs. Light microscopy shows lipid droplets after 21 days of culture in adi- PND5 kidney before disappearing from
pogenic media (scale bar, 100 mm), Alcian blue staining of proteoglycans after 21 day the adult kidney (Figure 6D). Both GFPlo
of pellet culture in chondrogenic media (scale bar, 500 mm), and Alizarin red staining of EpCAM2 (interstitial) and GFP+EpCAM+
osteoid matrix after 21 days of culture in osteogenic media (scale bar, 100 mm).
(epithelial) fractions showed the capacity
to form small, medium, and large MSC-like
(Supplemental Figure 1B). After incorporated into the collecting colonies with equivalent efficiency (Figure 6E). Immunofluoduct, these PKH26-labeled cells re-expressed GFP, suggesting the rescence and FACS for F4/80 confirmed that this interstitial
GFPlo population did not represent granulocyte macrophages
readoption of an Hoxb7+ collecting duct phenotype (Figure 4B).
(Supplemental Figure 6), despite previous reports of Hoxb7 expression in that population.38 Cells isolated from PND5 kidneys
Repair Activity of Kidney-Derived Stromal Cells in
were subsequently sorted into GFP+EpCAM+, GFPloEpCAM2,
Scrape Injury Assay
GFP2EpCAM+, and GFP 2 EpCAM 2 fractions for quantitaTissue-derived MSCs have been proposed to play a role in tissue
turnover, homeostasis, and repair through humoral mechative PCR (qPCR). GFP loEpCAM2 cells displayed differennisms. Indeed, BM-MSC conditioned medium can promote
tial Wnt4 expression similar to neonatal medullary interstitial
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Figure 4. Kidney MSC–like cells isolated as Hoxb7/EGFP+ fraction demonstrate epithelial potential both in vitro and in vivo. (A) Bright-field (BF) and immunofluorescence
images of in vitro three-dimensional cultures containing MDCKs, kidney MSC-like cells
derived from Hoxb7+ fraction, or BM-MSCs stained for E-cadherin (green) and 49,6diamidino-2-phenylindole (blue). Although MDCK cells formed cysts, Hoxb7-derived
MSC cultures formed E-cadherin+ branching epithelial structures. No evidence for
epithelial differentiation was seen after culture of BM-MSCs. Scale bar, 20 mm. (B)
Evidence for in vivo epithelial differentiation of kidney MSC–like cells derived from
Hoxb7+ cultures labeled with PKH26 dye. Detection of PKH26-labeled kidney MSC–
like cells (red) and re-expression of Hoxb7/GFP (green) within Aqp2+ structures 4 days
after neonatal injection is presented. PKH26 signal was excited at 551 nm and detected at emission at 567 nm, and Aqp2 staining was detected with anti-rabbit 647,
excited at 645 nm, and detected at emission at 660 nm using upright laser-scanning confocal microscope for bright field and epifluorescence (LSM 710 up). Scale bar, 20 mm.
cells [GenitoUrinary Development molecular Anatomy Project
(GUDMAP), www.gudmap.org], whereas E-cadherin expression was comparable with other epithelial fractions (Figure
6F). Hence, the GFPloEpCAM2 population displays a metastable phenotype intermediate between interstitium and collecting
duct epithelium.
Possible Origin of Hoxb7-Derived Kidney Stromal Cells
Strong Wnt4 expression has previously been observed in the
renal interstitium as early as embryo day 15.5 (E15.5) and
within occasional collecting duct cells within the papilla at
PND2 and PND6 (GUDMAP accession ID’s: 7156, 14182,
14082, and 14084; www.gudmap.org) (Supplemental Figure
7). We investigated the expression and distribution of Wnt4+
cells using tissue sections from Wnt4 GCE/+:R26 TdTomato/+
mice. Tamoxifen (25 mg/kg) was injected at E17.5 and kidneys
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were collected at either E19.5 (immediately
before birth) or PND49 (adult). A contribution of Wnt4-expressing cells to the developing nephrons was evident at both E19.5 and
PND49, but we will focus on the medulla/
papilla. At E19.5, no Wnt4+ (EGFP+) or
TdTomato+ cells were located within Aqp2+
collecting ducts; however, both EGFP+ and
EGFP+TdTomato+ cells (representing 8.35%
of all GFP+ cells) were present within the interstitium of the medulla/papilla (Figure 7A,
Supplemental Figure 8B). All TdTomato+
cells also expressed EGFP. In the PND49 kidney, as previously reported, active Wnt4 expression (EGFP+) was detected in all Aqp2+
collecting ducts39 (Figure 7, B and C, Supplemental Figure 8A). In addition, approximately 8% of Aqp2+ medullary/papillary
collecting duct cells were double-positive
for TdTomato and EGFP (Figure 7, B and
C, Supplemental Figure 8B), indicating a
population of collecting duct–located cells
derived from the interstitial Wnt4-expressing
cells present at the time of Tamoxifen injection. This finding would suggest a process of
interstitial intercalation during the immediate postnatal kidney maturation, potentially
occurring by a similar mechanism to that observed after the neonatal injection of kidney
MSC–like cells.
DISCUSSION
In this study, we describe the isolation of
MSC-like cells from the adult mouse kidney
collecting duct. These cells are able to
transition between epithelial and mesenchymal states and display robust MSC
characteristics, including long-term expansion (.20 passages), clonogenicity, panmesodermal potential, and a characteristic epitope profile. Most remarkably, even after extensive
culture, these cells retain a capacity to re-epithelialize both in
vitro and in vivo. Indeed, if reintroduced into the neonatal kidney
by microinjection, this phenotypically mesenchymal population
specifically homed to and integrated back into the collecting
duct, thereafter proliferating as an epithelial component of
that compartment.
Although a variety of renal epithelia have been reported to
undergo EMT, including the proximal tubular epithelium,
mesenchymal-epithelial transition is, an unusual response for
a cultured MSC-like population. Indeed, there has been no
evidence of an in vitro or in vivo epithelial potential for BMMSCs.11,40,41 Collecting duct cells can undergo EMT in response to urinary obstruction, resulting in the formation of
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Figure 5. Kidney MSC–like cultures produce factors able to enhance wound repair in a scratch assay. (A) Bright-field images
showing repair of a scratch wound within a lawn of MDCK cells
across a 12-hour period. MDCK cells were cultured with control
medium or conditioned medium from Bulk cultured kidney MSC–
like cells or Hoxb7-derived kidney MSC–like cells. Scale bar,
100 mm. (B) Quantification of the rate of scratch wound closure in all
three conditions measured as wound width at 4- and 12-hour time
points normalized for the wound width at 0 hours. The wound closed
significantly faster in the presence of conditioned medium, especially Hoxb7-derived culture, compared with control medium (n=13).
*P,0.01 compared with control; #P,0.01 compared between Bulk
culture and Hoxb7 culture. cond, conditioned.
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interstitial fibrosis.42,43 However, it is thought to predominantly involve the myofibroblastic transformation of intercalated cells, a cell type not evident in the Hoxb7/GFP+ fraction
at isolation. The selective integration of injected kidney MSC–
like cells into the collecting duct epithelium suggests both an
active homing process and a memory of tissue origin. Although we cannot rule out fusion, the fact that we can show
both EMTand mesenchymal-epithelial transition in these cells
and the re-expression of Aqp2 and Hoxb7 after integration
support genuine epithelial transdifferentiation. This result
poses the question of whether the integration of kidney
MSC–like cells into the neonatal collecting duct represents a
normal process during papillary collecting duct elongation
and maturation. The renal papilla undergo dramatic elongation within a short period of time immediately after birth.44
Although the underlying mechanism is poorly understood,
collecting duct elongation does involve convergent extension45 (Supplemental Figure 9), a process that can also incorporate surrounding cells.46 Curiously, a kidney MSC–like
population could not be derived from embryonic kidney
(J. Li, U. Ariunbold, and M. Little, unpublished data), despite
the presence of abundant Hoxb7+ collecting duct epithelium.
This finding implies that MSC-forming cells within the collecting duct represent a subpopulation rather than it being a
phenotypic option available to all Aqp2+ collecting duct cells.
Indeed, we provide evidence that this subpopulation possibly
arises from the medullary interstitium during the immediate
postnatal period by intercalation of rare interstitial cells.
Several previous reports indicate the presence of stem cells in
the renal papilla. Bromodeoxyuridine, 5-bromo-29-deoxyuridine
pulse chase experiments identified enrichment of long-term
label-retaining cells within the papilla.47 However, such experiments performed after the immediate neonatal period no
longer label a papillary population, instead identifying labelretaining cells within the tubules.43,44 This result suggests the
presence of a dividing neonatal papillary population, which
then becomes relatively quiescent, an observation not inconsistent with our lineage analysis or the appearance and disappearance of the Hoxb7lo population within the papilla. The
human renal papilla has also been reported to contain epithelial Oct4-expressing prominin/CD133+ cells in loops of Henle
that form epithelial structures in vitro.48,49 Our kidney MSC–
like cells are derived from collecting duct and home specifically back to that tubular segment, suggesting no overlap with
these studies.
Lineage-tracing analyses have shown that tubular repair in
the adult kidney after acute injury only involves epithelial cells
within the nephrons.50,51 The integration of a nontubular cell
type into the collecting duct would not have been identified in
these previous lineage-tracing studies, because the lineage
marker used, Six2, only identifies cells derived from the cap
mesenchyme, a compartment that does not give rise to the
collecting duct epithelium. To date, we have no evidence suggesting that continued contribution of interstitial cells into the
collecting duct compartment occurs in the adult mouse.
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Figure 6. Characterization of the location and properties of endogenous Hoxb7+ populations within the postnatal kidney. (A) Detection
of endogenous GFP+(Hoxb7+) and GFPlo populations across kidney development. Immunofluorescence shows the presence of Hoxb7
expression (GFP; green) and staining for Aqp2 (red) on kidney sections at PND0, PND9, and PND14 and adult Hoxb7/EGFP transgenic
mouse kidneys. Arrowheads indicate GFPlo interstitial cells. Scale bar, 20 mm. (B) Immunofluorescence staining for Collagen IV (red)
plus endogenous GFP on sections of PND9 Hoxb7/EGFP transgenic mouse kidney. Arrowheads indicate interstitial GFPlo cells outside
of Collagen IV+ tubule basement membranes. Scale bar, 10 mm. (C) FACS analysis for GFP and EpCAM (marker of epithelium) from
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However, the capacity for kidney MSC–like cells to proliferate
long-term and switch phenotype does raise the prospect that
these cells respond to injury in a reparative fashion, even
within the confines of the collecting duct epithelium. Although more analyses are required, these cells are able to produce growth factors that promote epithelial wound repair in
vitro, which would support a role for such cells in response to
damage in vivo. Indeed, the capacity to switch phenotype may
also endow this population with more substantial roles in collecting duct development, homeostasis, and response to injury, which remain to be investigated.
CONCISE METHODS
Animals
Animal experiments were approved by the University of Queensland
Animal Ethics Committee (Institute for Molecular Bioscience) and
adhered to the Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes. Mice expressing ubiquitous EGFP52
or Hoxb7/EGFP22 were used for cell isolation and FACS sorting.
Wnt4GCE/+:R26tdTomato/+ mice were used for lineage-tracing experiments. Neonates of outbred CD1 mice were used for neonatal injections. Wild-type, EGFP, and Hoxb7/EGFP transgenic mice used for
experimentation were housed within the University of Queensland
Biologic Resources in clean, temperature-controlled mouse facilities
on a 12-hour light/dark cycle and standard diet. Wnt4GCE/+:R26TdTomato/+
transgenic mice were housed in the animal facility at Department of
Stem Cell Biology and Regenerative Medicine, University of Southern
California. Wnt4GCE/+;R26tdTomato/+ mice were generated by crossing
Wnt4GCE/+ mice with R26tdTomato/tdTomato mice to yield bigenic
Wnt4GCE/+;R26tdTomato/+ mice. The Wnt4 locus will have one wildtype allele and one allele with a green fluorescent protein fused to Cre
(GFP-Cre) recombinase–ERT2 fusion protein knocked in downstream of
the Wnt4 promoter. After injection of Tamoxifen, the fusion protein will
translocate to the nucleus and excise a floxed STOP codon upstream of
the tdTomato gene, and all cells expressing Wnt4 at the time of Tamoxifen
injection will be permanently labeled with tdTomato. Tamoxifen injection and tissue harvesting were performed according to the animal experimental guidelines issued by the Animal Care and Use Committee at
University of Southern California. Tamoxifen (25 mg/kg body wt) was
injected into time-mated Wnt4GCE/+:R26tdTomato/+ females at E17.5, and
both kidneys were collected from mice at either E19.5 or PND49. Samples from wild-type littermates were also collected as controls.
BASIC RESEARCH
Isolation of Kidney MSC–Like Cells
Kidney stromal cells were isolated from adult mice (male, 6–8 weeks
old) with or without FACS sorting followed by standard culture as
previously described.28 In brief, kidneys were dissected and subjected
to enzymatic digestion (Collagenase B, 1 mg/ml; Dispase II, 1.2 unit/ml)
followed by two rounds of filtration with a cell strainer (70 and 40 mm;
BD Falcon) to produce single-cells yield. Isolated cells were then either
cultured directly as Bulk or Sorted to collect different fractions. For
Bulk culture, cells were preplated for 24 hours in 20% FCS/a-MEM
(Gibco, Basel, Switzerland). Nonadherent cells were then washed off,
and adherent cells were continuously cultured and expanded over
time. Sorted populations were established through FACS fractionation
on the basis of previously described stem cell markers CD24 and
Sca-1.24–26 Total kidney isolates, excluding Lin+CD31+ cells, were
sorted into CD24+Sca1+, CD242Sca-12, and CD24loSca-1+ fractions.
Only the CD24loSca-1+ fractions could form MSC colonies in vitro
(Supplemental Figure 4A). For regional isolation, kidneys were mechanically dissected into cortex, medulla, and papilla regions and subjected to enzymatic digestion; then, they were plated as total isolates
from that region. Hoxb7-derived cultures were obtained by FACS sorting of GFP+EpCAM+ fractions after single-cell isolation from Hoxb7/
EGFP transgenic mice.
Neonatal Injection Model
Cells isolated from ubiquitous EGFP mice or cells derived from
Hoxb7/GFP+ fraction and labeled with PKH26 (see PKH26 Labeling
and Detection) were resuspended at 0.5–13107/ml in PBS and injected into the neonatal kidney at PND1 using a microinjection pipette under the guidance of Ultrasound Biomicroscope (Vevo770;
VisualSonics). Neonates were anesthetized and mounted on the stage
of a rail injection platform with gauze cushion tapes. The pups were
then moved into the scan plane using the XYZ controls on the platform stage. The kidney was visualized with Ultrasound Biomicroscope fitted with a 55-MHz probe (RMV711; VisualSonics) (Figure
1A). The microinjection needle tip was aligned in the scan plane using
the XYZ controls so that the tip is centered within the focal zone of the
transducer (area of image with the greatest resolution). Using the
Nanoject II, cells were delivered at a regulated pulse (69 nl/pulse)
with three to four pulses per injection, and the final delivery volume
was around 300 nl. Coinjection of Fluoresbrite Yellow Green microspheres (2.0 mm; Polysciences, Inc.) was used to confirm the injection
site within the nephrogenic zone of the neonatal kidney (Figure 1B).
Before injection, cell suspensions were mixed with Fluoresbrite Yellow Green microspheres (2.0 mm; Polysciences, Inc.) at a ratio of 20:1.
Microspheres alone, GFP+ kidney–derived stromal cells, GFP+ BM
PND0, PND9, PND14, and adult Hoxb7/GFP transgenic mouse kidneys highlighting the percentage of the Hoxb7lo population (boxed) at
each time point. (D) Quantitation of GFPloEpCAM2 and GFP+EpCAM+ populations in the postnatal Hoxb7/GFP kidney (PND0, n=3;
PND4, n=5; PND6, n=9; PND14, n=2; adult, n=4). Data show the changes in relative percentage of Hoxb7lo versus Hoxb7+ populations,
illustrating the appearance and decline of an interstitial Hoxb7lo population in the postnatal kidney. (E) Comparison of the colony-forming
efficiency between GFP+EpCAM+ and GFPloEpCAM2 populations isolated from PND5 kidney. There is no significant difference in capacity to form small, medium, or large colonies between these two populations (n=3). (F) Gene expression analysis showing expression of
interstitial (Wnt4) and epithelial (E-cadherin) markers in all four fractions isolated from PND5 Hoxb7/GFP mice kidneys (n=3). *P,0.05
compared with whole kidney.
J Am Soc Nephrol 26: ccc–ccc, 2014
Epithelial Potential of Kidney-Derived MSC-Like Cells
9
BASIC RESEARCH
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stromal cells, or PKH26-labeled Hoxb7-derived
kidney stromal cells, all mixed with microspheres,
were microinjected into the nephrogenic zone
(3000–5000 cells in 300 nl), and kidneys were harvested at 4 and 14 days postinjection. Except for the
beads themselves, no GFP signals were detected in
the beads only control. For the neonatal injection,
six independent experiments have been conducted
with varying numbers of samples collected from
each time point: P4, n=8; P5, n=1; P7, n=6; P9,
n=1; and P14, n=5. Three independent experiments have been conducted using PKH26-labeled
cells derived from Hoxb7 culture, and on average,
three samples were collected from P3, P5, and P8.
For presentation purposes, we only included data
from days 4 and 14 postinjection.
In Vitro Scratch Wound Repair Assay
For the in vitro wound-healing assay, MDCK
cells (1310 6 cells/well; 10% FCS/DMEM)
were plated in a six-well plate and cultured until
confluent. A straight scratch wound was then
created in MDCK cultures using a 200-ml pipette
tip held perpendicular to the plate. After the
scratch, cells were washed two times with PBS
and then provided fresh culture media (20%
FCS/a-MEM) plus 10% FCS/DMEM (1:1 ratio)
or conditional media (1:1 ratio). Cells were imaged
at 0, 4, and 12 hours at the same position using an
inverted bright-field/fluorescence microscope
(Nikon ECLIPSE Ti-U). Scratch area at each
time point was measured using NIS-Elements
BR 4.10 software and used for final quantitation.
+
Figure 7. Expression and distribution of Wnt4 cells in full-term embryo (E19.5) and adult
(PND49) kidney. (A) Analysis of Wnt4 (EGFP; green), TdTomato (red), and Aqp2 (white) in
the cortex, medulla, and papilla of an E19.5 kidney (immediately before anticipated birth)
post-Tamoxifen injection (25 mg/kg body wt) at E17.5. As expected, Wnt4 (EGFP) expression can be detected in renal vesicles within the cortex, and some of the renal
vesicles cells are positive for TdTomato. In the medulla, no collecting ducts were EGFP+
or TdTomato+. All medullary EGFP+ cells appeared to be interstitially located between
Collagen IV+ basement membranes. A small number of these cells was also positive for
TdTomato (arrows indicate a single TdTomato+ cell within the interstitium). Scale bar,
20 mm. (B) Analysis of Wnt4 (EGFP; green), TdTomato (red), and Aqp2 (white) in the
cortex, medulla, and papilla of the PND49 kidney post-Tamoxifen injection (25 mg/kg
body wt at E17.5). As expected, given the expression of Wnt4 in the renal vesicle before
nephron maturation, extensive numbers of TdTomato+ cells are seen along the length of
the renal vesicle-derived nephron, especially in the renal cortex. However, ongoing Wnt4
expression (green) was only detected in Aqp2+ collecting duct cells, with it being
strongest in the papillary collecting duct as previously reported.39 Of note, approximately
8% of Aqp2+ medullary/papillary collecting duct cells were double-positive for TdTomato
and EGFP. Scale bar, 20 mm. (C) High-resolution images of PND49 kidney showing Wnt4
expression (EGFP) and the presence of TdTomato+ cells within Aqp2+ papilla collecting
duct epithelium at PND49. Arrows indicate TdTomato+ cells within the epithelium. DAPI,
49,6-diamidino-2-phenylindole. Scale bars, 20 mm.
10
Journal of the American Society of Nephrology
FACS Analyses and
Immunophenotype Analyses
Freshly isolated cells were sorted into different
fractions on the basis of either specific antibody
stainingorGFP(Hoxb7)expressionusingFACSAria
Cell Sorter (BD Falcon) and then cultured for
additional analysis. For FACS sorting, bioconjugated
Lin cocktail plus Streptavidin-Allophycocyanin and
directly conjugated APC-CD31, FITC-Sca-1, PECD24, and APC-EpCAM (BD Falcon) were used.
Epitope controls conjugated to different fluorochromes were used as nonspecific staining controls
for flow cytometry sorting. For immunophenotyping, cultured cells were stained with directly conjugatedantibodies(PE-CD24,APC-CD44,PE-CD49e,
PE-CD51, PE-CD81, PE-CD140a, PE-CD140b, and
PE-Sca-1; BDFalcon)andanalyzedon FACSCantoII
(BD Falcon). Dead cells were excluded on the basis of
7AAD staining. Data were collected and analyzed
using FACSDiva software (BD Falcon) and FlowJo
(version 7.6.5; Tree Star).
J Am Soc Nephrol 26: ccc–ccc, 2014
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Colony-Forming Efficiency, Clonogenicity, and
Proliferation
Fractionated or total cell isolates were plated at an initial density of 53103
cells/well on a six-well plate in a-MEM (Gibco) with 20% FCS,
100 units/ml Penicillin, and 100 mg/ml Streptomycin and cultured in
5% CO2 in a 37°C incubator. After being cultured for 14 days, cells were
washed with PBS, fixed with ethanol for 5 minutes at room temperature,
and stained with 0.5% Crystal Violet (Sigma-Aldrich) in methanol for
8 minutes at room temperature. The number of colonies was counted
under light microscope. Colonies consisting of .25 cells were scored and
classified into three sizes: large is .100 cells, medium is 50–100 cells, and
small is 25–50 cells. For colony-forming efficiency, the numbers of colonies with different sizes are counted and normalized to the numbers of
cells plated. To evaluate clonogenicity, single cells were sorted directly
into a 96-well plate with one cell per well, and the numbers of colonies
were recorded after 14 days and analyzed. Population-doubling and population-doubling time were calculated to show the proliferation property
of isolated cells in culture using the following equations:
PDs ¼ log2 Ni =N0
and
PDT ¼ Ti =PDs:
PDs is population doubling number, Ni is the accumulated cell
number at the end of incubation time, N0 is the accumulated cell
number at the beginning of the incubation time, PDT is population
doubling time, and Ti is the incubation time in any unit.
BASIC RESEARCH
consisting of a-MEM, 0.1 mM dexamethasone, 2 mM L-ascorbate-2phosphate, 1 mM sodium pyruvate, proline, TGF-b1, 50 mg/ml insulin/
transferrin/selenium, and 40 mg/ml gentamicin. Medium was replaced two
times per week for 3 weeks. Pellets were fixed in 4% PFA and embedded
as paraffin block and sectioned using microtome. Sections were stained
with Alcian blue stain, and nuclei were counterstained with nuclear fast
red.55
PKH26 Labeling and Detection
Cells were labeled with PKH-26 red fluorescence cell linker (SigmaAldrich) following the manufacturer’s instruction and used for neonatal injection or coculture in a three-dimensional system. In brief,
cells were harvested, counted, and resuspended at desired density,
and then, they were washed one time with PBS. The cell pellet was
then resuspended in Solution C provided in the kit, and PKH26 dye
was added to achieve the desired final concentration. Cells were incubated at room temperature for 5 minutes. The dye was then washed
off, and the cells were washed one more time with PBS to get rid of
any residue; then, it is ready to be used. Labeling efficacy was .98%.
Cell viability was evaluated by Trypan blue exclusion and was .96%.
PKH26 signal was directly detected using either an upright fluorescence microscope (Olympus BX-51) or a confocal microscope (Carl
Zeiss LSM 510 Meta UV or Carl Zeiss LSM 710 upright).
Immunofluorescence of Kidney Sections
Cells were seeded onto coverslips as above, grown to 100% confluence,
and cultured for 3 weeks in osteogenic differentiation media consisting of
a-MEM, 10% FCS, 0.1 mM dexamethasone, 100 mM b-glycerol phosphate, 10 mM L-ascorbate-2-phosphate, and 40 mg/ml gentamicin. Medium was replaced two times per week for 3 weeks, and cells were fixed
as above and stained with Alizarin red-S solution.18,56
For immunofluorescence staining, kidney samples were fixed in 4% PFA,
infiltrated with 30% sucrose/PBS overnight, then embedded in TissueTek OCT Compound (Sakura Finetek, Torrance, CA), and frozen in
liquid nitrogen. Ten-micrometer-thick sections were cut and postfixed in
4% PFA for 10 minutes at room temperature and then incubated with
various primary antibodies, including anti-GFP (Sapphire Bioscience),
Aqp2 (EMD Millipore, Australia), Collagen IV (Chemicon International), Umod (Chemicon International), pHH3 (Upstate Cell Signaling
Solutions), and F4/80 (Serotec). For immunocytochemistry, cultured
cells were fixed with ice-cold methanol at 4C° for 10 minutes and stained
with GFP (Sapphire Bioscience), Pax2 (Zymed Laboratories Inc.), NG2
(Chemicon International), ZO-1 (Invitrogen), Pendrin (Santa Cruz
Biotechnology), AE-1 (Alpha Diagnostic), and E-cadherin (BD Biosciences) followed by secondary antibody. All images were captured
on a fluorescence microscope (Olympus BX-51) or a confocal microscope (Carl Zeiss LSM 710 upright).
Kidneys collected from Wnt4GCE/+:R26TdTomato/+ time-mated females,
which were injected with Tamoxifen at E17.5, were fixed for 1 hour at 4°C
in 4% PFA, infiltrated with 30% sucrose/PBS overnight, then embedded
in Tissue-Tek OCT Compound (Sakura Finetek), and frozen in an ethanol/
dry ice bath. Samples were shipped as cryopreserved blocks in dry ice. Tenmicrometer-thick sections were cut and postfixed in 4% PFA for 10
minutes at room temperature and then stained with various primary
antibodies, including anti-GFP (Sapphire Bioscience), Aqp2 (EMD
Millipore), and Collagen IV (Chemicon International). TdTomato was
detected directly using a confocal microscope (Carl Zeiss LSM 710 upright).
Chondrogenic Differentiation
Three-Dimensional Collagen Gel Culture
Cells were centrifuged at 1503g for 5 minutes to pellet the cells. Cells were
then cultured as a pellet in chondrogenic differentiation medium
MDCK cells (strain II) were harvested from confluent culture using
trypsin-EDTA and resuspended at a concentration of 43104 cells/ml
Mesodermal Differentiation Assays
Mesodermal differentiation was carried out as per the work by Barlow
et al.53
Adipogenic Differentiation
Cells were seeded into six-well plates at a density of 83104 cells/well.
When cells reached 80% confluence, they were washed in PBS and
cultured for 7–21 days in adipogenic differentiation medium consisting of a-MEM, 10% FCS, 0.5 mM 3-isobutyl-1-methylxanthine, 60
mM indomethacin, 10 mg/ml insulin, 1 mM dexamethasone, and 40
mg/ml gentamicin.54,55 Medium was replaced two times per week,
and after the formation of intracellular lipid droplets was observed,
cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes at
room temperature and stained with oil red-O stain (Sigma-Aldrich).
Osteogenic Differentiation
J Am Soc Nephrol 26: ccc–ccc, 2014
Epithelial Potential of Kidney-Derived MSC-Like Cells
11
BASIC RESEARCH
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in ice-cold Collagen I solution prepared as described previously.34
Aliquots of the cell suspension were dispensed into 24-well plates
(250 ml/well; Nunc; Kampstrup, Roskilde, Denmark) and allowed
to gel for 30 minutes at 37°C before adding 500 ml culture medium
(either 10% FCS/MEM or 20% FCS/a-MEM; Gibco). Culture medium was changed every 2–3 days. The cultures were monitored and
photographed using an inverted fluorescence microscope (Nikon Ti-U)
every 2–3 days, fixed on day 14 with 4% PFA, and subjected to immunofluorescence for additional analysis.
V. Nink and Dr. G. Osborne at the Flow Cytometry Facility, Queensland
Brain Institute, for FACS sorting, and the staff at the Australian Cancer
Research Foundation Imaging Facility for microscopy support. M.L. is a
Senior Principal Research Fellow of the National Health and Medical
Research Council of Australia. This work was funded by the Australian
Stem Cell Centre, National Health and Medical Research Council Grant
APP1054895, National Institutes of Health Grant DK054364 (to A.P.M.),
and Center for Regenerative Medicine and Stem Cell Research Grant
C10-06536 (to A.P.M.).
Quantification
For quantification of the proportion of medullary collecting duct cells
derived from injected kidney MSC–like cells, kidney samples were examined from three independent neonatal injection experiments, in
which cells of interest were delivered into CD1 neonates at PND1 and
then collected at PND6 for analysis. Two to three sections were chosen
from each sample and stained with antibody to detect injected cells (antiGFP) and collecting duct epithelial cells (anti-Aqp2). Staining with 49,6diamidino-2-phenylindole was also used to detect individual nuclei.
When injecting PKH26-labeled cells, they were by direct fluorescence
without any staining. Under the microscope, the field with the most
significant incorporation events within the medullary/papillary region
was chosen to be imaged. All images (300 dpi at 320) were captured
with a fluorescence microscope (Olympus BX-51) and uploaded into
Imaris software (version 7.2; BitPlane AG) for quantification. Imaris
software was then used to create masks on the basis of specific staining
(Aqp2, GFP, or PKH26), and spot counting was performed. The numbers of nuclei (identified using 49,6-diamidino-2-phenylindole) within
each specific mask were counted separately and used to calculate integration efficiency (GFP+Aqp2+/Aqp2+ or PKH26+Aqp2+/Aqp2+). For
the quantification of the relative proliferation rate of incorporated kidney
MSC–like cells, kidney samples were again examined from three independent neonatal injection experiments, in which GFP+ kidney MSC–
like cells were delivered into CD1 neonates at PND1 and then collected at
PND6. In this instance, immunofluorescence was performed for antiGFP and anti-pHH3, a marker of mitosis. Imaris software was again used
to spot count nuclei that were GFP+ or pHH3+, such that the ratio of
GFP+pHH3+/GFP+ could be determined. To investigate the relative proliferation within normal collecting duct, samples were collected from
PND6 Hoxb7/EGPF mice, immunofluorescence was performed for
anti-pHH3, and the ratio of EGFP+pHH3+/EGFP+ cells was quantified.
Gene Expression Analyses
Total RNA was extracted from cells using TRIzol (Life Technologies),
and cDNA was synthesized from .100 ng RNA using Super Script III
reverse transcription (Life Technologies). qPCR analyses were performed with Syber Green (Applied Biosystems) by an ABI PRISM
7500 Real-Time PCR Machine. The sequences of primers used for
qPCR are as listed (Supplemental Table 1).
ACKNOWLEDGMENTS
We thank N. Martel for his assistance with timed mating and animal
maintenance, T. Hitchcock and her staff at the University of Queensland
Biological Resources facility for their assistance with animal models,
12
Journal of the American Society of Nephrology
DISCLOSURES
None.
REFERENCES
1. Horwitz EM, Le Blanc K, Dominici M, Mueller I, Slaper-Cortenbach I,
Marini FC, Deans RJ, Krause DS, Keating A; International Society for
Cellular Therapy: Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7: 393–395, 2005
2. Friedenstein AJ, Latzinik NW, Grosheva AG, Gorskaya UF: Marrow
microenvironment transfer by heterotopic transplantation of freshly
isolated and cultured cells in porous sponges. Exp Hematol 10: 217–
227, 1982
3. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD,
Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147,
1999
4. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause
D, Deans R, Keating A, Prockop D, Horwitz E: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8: 315–317,
2006
5. Kassem M: Mesenchymal stem cells: Biological characteristics and
potential clinical applications. Cloning Stem Cells 6: 369–374, 2004
6. Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic
tissues. Science 276: 71–74, 1997
7. Reinders ME, Fibbe WE, Rabelink TJ: Multipotent mesenchymal stromal cell therapy in renal disease and kidney transplantation. Nephrol
Dial Transplant 25: 17–24, 2010
8. Herrera MB, Bussolati B, Bruno S, Fonsato V, Romanazzi GM, Camussi
G: Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury. Int J Mol Med 14: 1035–1041, 2004
9. Bi B, Schmitt R, Israilova M, Nishio H, Cantley LG: Stromal cells protect
against acute tubular injury via an endocrine effect. J Am Soc Nephrol
18: 2486–2496, 2007
10. Tögel F, Yang Y, Zhang P, Hu Z, Westenfelder C: Bioluminescence
imaging to monitor the in vivo distribution of administered mesenchymal stem cells in acute kidney injury. Am J Physiol Renal Physiol 295:
F315–F321, 2008
11. Tögel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C: Administered
mesenchymal stem cells protect against ischemic acute renal failure
through differentiation-independent mechanisms. Am J Physiol Renal
Physiol 289: F31–F42, 2005
12. Imberti B, Morigi M, Tomasoni S, Rota C, Corna D, Longaretti L, Rottoli
D, Valsecchi F, Benigni A, Wang J, Abbate M, Zoja C, Remuzzi G: Insulin-like growth factor-1 sustains stem cell mediated renal repair. J Am
Soc Nephrol 18: 2921–2928, 2007
13. Rookmaaker MB, Smits AM, Tolboom H, Van ’t Wout K, Martens AC,
Goldschmeding R, Joles JA, Van Zonneveld AJ, Gröne HJ, Rabelink
J Am Soc Nephrol 26: ccc–ccc, 2014
www.jasn.org
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
TJ, Verhaar MC: Bone-marrow-derived cells contribute to glomerular
endothelial repair in experimental glomerulonephritis. Am J Pathol
163: 553–562, 2003
Tögel F, Isaac J, Hu Z, Weiss K, Westenfelder C: Renal SDF-1 signals
mobilization and homing of CXCR4-positive cells to the kidney after
ischemic injury. Kidney Int 67: 1772–1784, 2005
Tögel F, Zhang P, Hu Z, Westenfelder C: VEGF is a mediator of the
renoprotective effects of multipotent marrow stromal cells in acute
kidney injury. J Cell Mol Med 13[8B]: 2109–2114, 2009
Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G,
Sun B, Zheng B, Zhang L, Norotte C, Teng PN, Traas J, Schugar R,
Deasy BM, Badylak S, Buhring HJ, Giacobino JP, Lazzari L, Huard J,
Péault B: A perivascular origin for mesenchymal stem cells in multiple
human organs. Cell Stem Cell 3: 301–313, 2008
da Silva Meirelles L, Chagastelles PC, Nardi NB: Mesenchymal stem
cells reside in virtually all post-natal organs and tissues. J Cell Sci 119:
2204–2213, 2006
Meirelles LS, Nardi NB: Murine marrow-derived mesenchymal stem
cell: Isolation, in vitro expansion, and characterization. Br J Haematol
123: 702–711, 2003
Pelekanos RA, Li J, Gongora M, Chandrakanthan V, Scown J, Suhaimi N,
Brooke G, Christensen ME, Doan T, Rice AM, Osborne GW, Grimmond
SM, Harvey RP, Atkinson K, Little MH: Comprehensive transcriptome and
immunophenotype analysis of renal and cardiac MSC-like populations
supports strong congruence with bone marrow MSC despite maintenance of distinct identities. Stem Cell Res (Amst) 8: 58–73, 2012
Costello-Boerrigter LC, Boerrigter G, Cataliotti A, Harty GJ, Burnett JC
Jr.: Renal and anti-aldosterone actions of vasopressin-2 receptor antagonism and B-type natriuretic peptide in experimental heart failure.
Circ Heart Fail 3: 412–419, 2010
Yu J, Valerius MT, Duah M, Staser K, Hansard JK, Guo JJ, McMahon J,
Vaughan J, Faria D, Georgas K, Rumballe B, Ren Q, Krautzberger AM,
Junker JP, Thiagarajan RD, Machanick P, Gray PA, van Oudenaarden A,
Rowitch DH, Stiles CD, Ma Q, Grimmond SM, Bailey TL, Little MH,
McMahon AP: Identification of molecular compartments and genetic
circuitry in the developing mammalian kidney. Development 139:
1863–1873, 2012
Srinivas S, Goldberg MR, Watanabe T, D’Agati V, al-Awqati Q,
Costantini F: Expression of green fluorescent protein in the ureteric bud
of transgenic mice: A new tool for the analysis of ureteric bud morphogenesis. Dev Genet 24: 241–251, 1999
Kim J, Cha JH, Tisher CC, Madsen KM: Role of apoptotic and nonapoptotic cell death in removal of intercalated cells from developing rat
kidney. Am J Physiol 270: F575–F592, 1996
Dekel B, Zangi L, Shezen E, Reich-Zeliger S, Eventov-Friedman S,
Katchman H, Jacob-Hirsch J, Amariglio N, Rechavi G, Margalit R,
Reisner Y: Isolation and characterization of nontubular sca-1+lin- multipotent stem/progenitor cells from adult mouse kidney. J Am Soc
Nephrol 17: 3300–3314, 2006
Bussolati B, Bruno S, Grange C, Buttiglieri S, Deregibus MC, Cantino D,
Camussi G: Isolation of renal progenitor cells from adult human kidney.
Am J Pathol 166: 545–555, 2005
Challen GA, Bertoncello I, Deane JA, Ricardo SD, Little MH: Kidney
side population reveals multilineage potential and renal functional
capacity but also cellular heterogeneity. J Am Soc Nephrol 17: 1896–
1912, 2006
Watanabe T, Costantini F: Real-time analysis of ureteric bud branching
morphogenesis in vitro. Dev Biol 271: 98–108, 2004
Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF, Rudakowa
SF, Luriá EA, Ruadkow IA: Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay
method. Exp Hematol 2: 83–92, 1974
Louis SA, Rietze RL, Deleyrolle L, Wagey RE, Thomas TE, Eaves AC,
Reynolds BA: Enumeration of neural stem and progenitor cells in the
neural colony-forming cell assay. Stem Cells 26: 988–996, 2008
J Am Soc Nephrol 26: ccc–ccc, 2014
BASIC RESEARCH
30. Wu H, Chen L, Zhou Q, Zhang X, Berger S, Bi J, Lewis DE, Xia Y, Zhang
W: Aqp2-expressing cells give rise to renal intercalated cells. J Am Soc
Nephrol 24: 243–252, 2013
31. Swetha G, Chandra V, Phadnis S, Bhonde R: Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with
traits of renal progenitors. J Cell Mol Med 15: 396–413, 2011
32. Challen GA, Martinez G, Davis MJ, Taylor DF, Crowe M, Teasdale RD,
Grimmond SM, Little MH: Identifying the molecular phenotype of renal
progenitor cells. J Am Soc Nephrol 15: 2344–2357, 2004
33. Chong JJ, Chandrakanthan V, Xaymardan M, Asli NS, Li J, Ahmed I,
Heffernan C, Menon MK, Scarlett CJ, Rashidianfar A, Biben C, Zoellner
H, Colvin EK, Pimanda JE, Biankin AV, Zhou B, Pu WT, Prall OW, Harvey
RP: Adult cardiac-resident MSC-like stem cells with a proepicardial
origin. Cell Stem Cell 9: 527–540, 2011
34. Montesano R, Schaller G, Orci L: Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell 66: 697–711, 1991
35. Smith AN, Willis E, Chan VT, Muffley LA, Isik FF, Gibran NS, Hocking
AM: Mesenchymal stem cells induce dermal fibroblast responses to
injury. Exp Cell Res 316: 48–54, 2010
36. Walter MN, Wright KT, Fuller HR, MacNeil S, Johnson WE: Mesenchymal stem cell-conditioned medium accelerates skin wound healing:
An in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell
Res 316: 1271–1281, 2010
37. Davies J, Lyon M, Gallagher J, Garrod D: Sulphated proteoglycan is
required for collecting duct growth and branching but not nephron
formation during kidney development. Development 121: 1507–1517,
1995
38. Fuller JF, McAdara J, Yaron Y, Sakaguchi M, Fraser JK, Gasson JC: Characterization of HOX gene expression during myelopoiesis: Role of HOX A5
in lineage commitment and maturation. Blood 93: 3391–3400, 1999
39. DiRocco DP, Kobayashi A, Taketo MM, McMahon AP, Humphreys BD:
Wnt4/b-catenin signaling in medullary kidney myofibroblasts. J Am
Soc Nephrol 24: 1399–1412, 2013
40. Duffield JS, Bonventre JV: Kidney tubular epithelium is restored without replacement with bone marrow-derived cells during repair after
ischemic injury. Kidney Int 68: 1956–1961, 2005
41. Li L, Truong P, Igarashi P, Lin F: Renal and bone marrow cells fuse after
renal ischemic injury. J Am Soc Nephrol 18: 3067–3077, 2007
42. Butt MJ, Tarantal AF, Jimenez DF, Matsell DG: Collecting duct epithelial-mesenchymal transition in fetal urinary tract obstruction. Kidney
Int 72: 936–944, 2007
43. Trnka P, Hiatt MJ, Ivanova L, Tarantal AF, Matsell DG: Phenotypic
transition of the collecting duct epithelium in congenital urinary tract
obstruction. J Biomed Biotechnol 2010: 696034, 2010
44. Wilkinson L, Kurniawan ND, Phua YL, Nguyen MJ, Li J, Galloway GJ,
Hashitani H, Lang RJ, Little MH: Association between congenital defects in papillary outgrowth and functional obstruction in Crim1 mutant
mice. J Pathol 227: 499–510, 2012
45. Karner CM, Chirumamilla R, Aoki S, Igarashi P, Wallingford JB, Carroll
TJ: Wnt9b signaling regulates planar cell polarity and kidney tubule
morphogenesis. Nat Genet 41: 793–799, 2009
46. Oda-Ishii I, Ishii Y, Mikawa T: Eph regulates dorsoventral asymmetry of
the notochord plate and convergent extension-mediated notochord
formation. PLoS ONE 5: e13689, 2010
47. Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q: The renal
papilla is a niche for adult kidney stem cells. J Clin Invest 114: 795–804,
2004
48. Ward HH, Romero E, Welford A, Pickett G, Bacallao R, Gattone VH 2nd,
Ness SA, Wandinger-Ness A, Roitbak T: Adult human CD133/1(+)
kidney cells isolated from papilla integrate into developing kidney tubules. Biochim Biophys Acta 1812: 1344–1357, 2011
49. Bussolati B, Moggio A, Collino F, Aghemo G, D’Armento G, Grange C,
Camussi G: Hypoxia modulates the undifferentiated phenotype of
human renal inner medullary CD133+ progenitors through Oct4/miR145 balance. Am J Physiol Renal Physiol 302: F116–F128, 2012
Epithelial Potential of Kidney-Derived MSC-Like Cells
13
BASIC RESEARCH
www.jasn.org
50. Humphreys BD, Czerniak S, DiRocco DP, Hasnain W, Cheema R,
Bonventre JV: Repair of injured proximal tubule does not involve
specialized progenitors. Proc Natl Acad Sci U S A 108: 9226–9231,
2011
51. Humphreys BD, Valerius MT, Kobayashi A, Mugford JW, Soeung S,
Duffield JS, McMahon AP, Bonventre JV: Intrinsic epithelial cells repair
the kidney after injury. Cell Stem Cell 2: 284–291, 2008
52. Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A: Generating green fluorescent mice by germline transmission of green
fluorescent ES cells. Mech Dev 76: 79–90, 1998
53. Barlow S, Brooke G, Chatterjee K, Price G, Pelekanos R, Rossetti T,
Doody M, Venter D, Pain S, Gilshenan K, Atkinson K: Comparison of
human placenta- and bone marrow-derived multipotent mesenchymal
stem cells. Stem Cells Dev 17: 1095–1107, 2008
14
Journal of the American Society of Nephrology
54. Erices A, Conget P, Minguell JJ: Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109: 235–242, 2000
55. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH: Isolation of
multipotent mesenchymal stem cells from umbilical cord blood. Blood
103: 1669–1675, 2004
56. Javazon EH, Colter DC, Schwarz EJ, Prockop DJ: Rat marrow stromal
cells are more sensitive to plating density and expand more rapidly
from single-cell-derived colonies than human marrow stromal cells.
Stem Cells 19: 219–225, 2001
This article contains supplemental material online at http://jasn.asnjournals.
org/lookup/suppl/doi:10.1681/ASN.2013050517/-/DCSupplemental.
J Am Soc Nephrol 26: ccc–ccc, 2014