NZ713875B2 - Organoids comprising isolated renal cells and uses thereof - Google Patents
Organoids comprising isolated renal cells and uses thereof Download PDFInfo
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- NZ713875B2 NZ713875B2 NZ713875A NZ71387514A NZ713875B2 NZ 713875 B2 NZ713875 B2 NZ 713875B2 NZ 713875 A NZ713875 A NZ 713875A NZ 71387514 A NZ71387514 A NZ 71387514A NZ 713875 B2 NZ713875 B2 NZ 713875B2
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- cells
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- renal
- cell population
- kidney
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/25—Urinary tract cells, renal cells
- C12N2502/256—Renal cells
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
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- C12N2513/00—3D culture
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- C12N2533/54—Collagen; Gelatin
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
Abstract
Described herein are organoids comprising admixtures of selected bioactive primary renal cells and a bioactive cell population, e.g., an endothelial cell populations, e.g. HUVEC cells, and methods of treating a subject in need thereof with such organoids. Further, the isolated renal cells, which may include tubular and erythropoietin {EPO}- producing kidney cell populations, and/or the endothelial cell populations may be of autologous, syngeneic, allogeneic or xenogeneic origin, or any combination thereof. Further provided are methods of treating a subject in need with the organoids. include tubular and erythropoietin {EPO}- producing kidney cell populations, and/or the endothelial cell populations may be of autologous, syngeneic, allogeneic or xenogeneic origin, or any combination thereof. Further provided are methods of treating a subject in need with the organoids.
Description
ORGANOIDS COMPRISING ISOLATED RENAL CELLS AND USES THEREOF
Field of the Invention
The present disclosure is directed to admixtures of selected bioactive primary renal
cells and r bioactive cell populations, and methods of treating a subject in need
thereof. The disclosure is further directed to organoids comprising isolated renal cells,
including tubular and erythropoietin (EPO)-producing kidney cell populations, and methods
of treating a subject in need with the organoids.
Background of the ion
Chronic Kidney Disease (CKD) affects over 19M people in the United States and is
frequently a consequence of metabolic disorders ing obesity, diabetes, and
hypertension. Examination of the data reveals that the rate of increase is due to the
development of renal failure secondary to hypertension and non-insulin dependent diabetes
mellitus (NIDDM) (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda,
MD, National Institutes of , National Institute of Diabetes and Digestive and Kidney
es, 2007 pp 223-238) – two diseases that are also on the rise worldwide. Obesity,
hypertension, and poor glycemic control have all been shown to be independent risk factors
for kidney damage, causing glomerular and tubular lesions and leading to nuria and
other systemically-detectable alterations in renal filtration function (Aboushwareb, et al.,
World J Urol, 26: 295-300, 2008; Amann, K. et al., Nephrol Dial Transplant, 13: 1958-66,
1998). CKD ts in stages 1-3 of progression are d by lifestyle changes and
pharmacological interventions aimed at controlling the underlying disease s), while
ts in stages 4-5 are managed by dialysis and a drug regimen that lly includes
anti-hypertensive agents, erythropoiesis stimulating agents (ESAs), iron and vitamin D
supplementation. ing to the United States Renal Data Service (USRDS), the average
end-stage renal disease (ESRD) patient expends >$600 per month on injectable
erythropoiesis-stimulating agents (ESAs), Vitamin D supplements, and iron supplements
(United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, MD, National
Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007
pp 223-238). When paired with the annual average cost of dialysis ($65,405), the healthcare
cost for nance of a single patient rises to >$72,000/yr (United States Renal Data
System: Costs of CKD and ESRD. ed. Bethesda, MD, National Institutes of Health, National
Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 8) – a number that
reflects only standard procedural costs and does not include treatment of other
cations, emergency procedures, or ancillary procedures such as the placement of
vascular grafts for dialysis access. Combined re costs for CKD and ESRD in 2005
totaled $62B – representing 19% of all medicare spending for that year (United States Renal
Data : Costs of CKD and ESRD. ed. Bethesda, MD, National Institutes of Health,
National Institute of Diabetes and ive and Kidney es, 2007 pp 223-238). Kidney
transplantation is an effective option for stage 4-5 patients as a pre-emptive measure to
avoid dialysis or when dialysis is no longer sufficient to manage the disease state, but the
number of stage 5 CKD patients in the US (>400,000) who could t from whole kidney
transplant far exceeds the number of suitable donor kidneys ble in any given year
(~16,000) (Powe, NR et al., Am J Kidney Dis, 53: S37-45, 2009). Thus, new treatment
paradigms are needed to delay or reduce dependency on dialysis and to fill the void left by
the shortage of donor kidneys.
Progressive renal disease results from a combination of the initial disease injury (e.g,
hypertension), followed by a maladaptive renal response to that injury. Such a response
includes the production of pro-inflammatory and pro-fibrotic cytokines and growth factors.
Therefore, one strategy to slow CKD progression is to ameliorate the inflammatory and
fibrotic response as well as te or reverse renal degeneration through the repair
and/or regeneration of renal tissue.
Chronic renal failure is prevalent in humans as well as some domesticated s.
ts with renal failure experience not only the loss of kidney function (uremia), but also
develop anemia due to the inability of the bone marrow to produce a sufficient number of
red blood cells (RBCs) via opoiesis. Erythroid homeostasis is dependent on both the
production of erythropoietin (EPO) by specialized interstitial fibroblasts that reside in the
kidney and the ability of targeted erythroid progenitors in the bone marrow to respond to
EPO and manufacture more RBCs. The anemia of renal failure is due to both reduced
production of EPO in the kidney and the ve effects of uremic factors on the s of
EPO in the bone marrow.
To date, clinical approaches to the treatment of chronic renal failure involve dialysis
and kidney transplantation for restoration of renal filtration and urine production, and the
systemic delivery of recombinant EPO or EPO analogs to restore erythroid mass. Dialysis
offers survival benefit to patients in -late stage renal e, but causes significant
quality-of-life issues. Kidney transplant is a highly desired (and often the only) option for
patients in the later stages of renal failure, but the supply of high-quality donor s does
not meet the demand for the renal failure population. Bolus dosing with recombinant EPO
to treat anemia has now been associated with serious ream health risks, leading to
black box warnings from the FDA for the drug, and necessitating further investigation into
alternative treatments to restore erythroid homeostasis in this population. nical
investigations have examined in vivo efficacy and safety of EPO-producing cells that have
been generated via gene therapy approaches. These studies have shown that it is possible
to ently stimulate erythropoiesis and RBC number by in vivo delivery of epo-producing
cells. However, to date, none of these ches have offered regulated erythroid
homeostasis or long-term in vivo functionality. Consequently, HCT and RBC number are
often increased beyond normal values, leading to polycythemia vera and other
complications. Delivery of EPO-producing cells that are eutically-relevant and provide
advantages over delivery of recombinant EPO must not only increase HCT, but should
restore erythroid homeostasis, with both positive and negative regulatory mechanisms
intact. It is important to note that EPO-deficient s, while ent in patients with
kidney disease, can also develop as a result of other disease states, including heart failure,
multi-organ system failure, and other chronic es.
Regenerative medicine technologies provide next-generation therapeutic options for
chronic kidney disease (CKD). Presnell et al. WO/2010/056328 and Ilagan et al.
describe isolated ive renal cells, including tubular and
erythropoietin (EPO)-producing kidney cell populations, and methods of isolating and
culturing the same, as well as methods of treating a subject in need with the cell
populations.
There is a need for improved and more targeted regenerative medicine therapeutic
options for subjects in need.
SUMMARY OF THE INVENTION
Accordingly, in one aspect the invention es a method of forming a spheroid
comprising:
suspension culturing a heterogenerous renal cell population and a non-renal
bioactive cell population in a three-dimensional (3D) culture system
wherein the 3D culture system comprises a spinner and lacks an exogenous
scaffold to which cells of the heterogeneous renal cell population and the bioactive cell
population attach;
wherein the heterogenous renal cell population is enriched for renal erythropoietin
(EPO)-producing cells, and
wherein the non-renal bioactive cell population is a non-renal elial cell
population, a non-renal endothelial progenitor population, a non-renal mesenchymal
stem cell population, or a non-renal adipose-derived progenitor population.
[008a] In another , the invention provides a id formed according to the
method of the invention.
[008b] In another aspect, the invention es an isolated cluster of cells comprising a
heterogeneous renal cell population and a nal bioactive cell population,
wherein the geneous renal cell population is enriched for renal EPO-producing
cells,
wherein the nal bioactive cell population is a non-renal endothelial cell
population, a non-renal endothelial progenitor cell population, a non-renal mesenchymal
stem cell population, or a non-renal adipose-derived progenitor population; and
wherein the cluster of cells is cultured in media in suspension without attachment to
a scaffold.
[008c] In another aspect, the invention provides an injectable ation comprising at
least one cluster of cells according to the invention and a liquid .
[008d] In another aspect, the invention provides an injectable formulation sing at
least one cluster of cells according to the invention and a hydrogel.
[008e] In another aspect, the invention realtes to the use of at least one cluster of cells
according to the invention for the manufacture of a medicament for treating kidney disease
in a subject.
[008f] In another aspect, the invention relates to the use of at least one cluster of cells
according to the invention and a liquid medium in the manufacture of a medicament for
treating kidney disease in a subject.
[008g] In another aspect, the invention s to the use of at least one cluster of cells
according to the invention and a hydrogel in the cture of a medicament for treating
kidney disease in a subject.
[008h] Certain statements that appear below are broader than what appears in the
statements of the invention above. These statements are provided in the sts of
providing the reader with a better understanding of the invention and its practice. The
reader is directed to the accompanying claim set which defines the scope of the invention.
[008i] Described herein are organoids, s for their preparation and use.
Organoids as described herein provide a therapeutic benefit to a subject in need
without the use of a scaffold; and/or at least provide the public with a useful choice.
Also described is a method of forming an id comprising a heterogeneous renal
cell tion and a bioactive cell population. In one ent, the metheod comprises
culturing the heterogenerous renal cell population and a bioactive cell tion in a
culture system selected from the group consisting of i) 2D culture; ii) 3D e: COL(I) gel;
iii) 3D e: Matrigel; iv) 3D culture: spinners, followed by COL(I)/Matrigel; and v) 3D
culture: ) gel. In some embodiments, the heterogeneous renal cell population
comprises a bioactive renal cell population. In certain embodiments, the heterogeneous
renal cell population ses a B2 cell population comprising an enriched population of
tubular cells, and wherein the heterogeneous renal cell population is depleted of a B1 cell
population and/or a B5 cell population or combination thereof. In some embodiments, the
heterogeneous renal cell population comprises a cell population selected from B2, B2/B3,
B2/B4, and B2/B3/B4. In embodiments, the heterogeneous renal cell population comprises
erythropoetin (EPO)-producing cells.
In another embodiment, the bioactive cell population is an endothelial cell
population. In certain embodiments, the endothelial cell population is a cell line. In some
ments, the endothelial cell population is derived from human umbilical cord. In
some ments, the bioactive cell population comprise endothelial progenitor cells. In
some ments, the bioactive cell population comprise mesenchymal stem cells. In
some embodiments, the endothelial cell population is adult-sourced. In some
embodiments, the cell populations are selected from xenogeneic, syngeneic, allogeneic,
autologous and combinations thereof.
In another embodiment, the heterogeneous renal cell population and the bioactive
cell tion are cultured separately for a first time period, combined and cultured for a
second time period. In certain ments, the renal cell population and bioactive cell
tion are ed at a ratio of 1:1. In most embodiments, the renal cell population
and bioactive cell population are combined, e.g., suspended, in growth medium. In some
embodiments, the second time period is between 24 and 72 hours in length, preferably 24
hours.
Also bed is an organoid. In some embodiments, the organoids are made
according to the methods described herein. In all embodiments, the organoids comprise a
heterogeneous renal cell population and a bioactive cell population. In some embodiments,
the bioactive cell population is an endothelial cell population. In some embodiments, the
endothelial cell population is a cell line. In certain embodiments, the endothelial cell
population comprises HUVEC cells.
In some embodiments, the heterogeneous renal cell population comprises a B2 cell
population comprising an enriched population of tubular cells, and n the
heterogeneous renal cell population is depleted of a B1 cell population. In certain
embodiments, the heterogeneous renal cell population is further depleted of a B5 cell
population. In select embodiments, the geneous renal cell population comprises a
cell population selected from B2, B2/B3, B2/B4, and B2/B3/B4. In some embodiments, the
heterogeneous renal cell population comprises erythropoetin (EPO)-producing cells.
Also described is an able formulation comprising at least one organoid and a
liquid medium. In one ment, the the liquid medium is ed from a cell growth
medium, DPBS and combinations f. In another embodiment, the organoids are
suspended in the liquid medium.
In a second embodiment, the injectable formulation comprises organoids and a
temperature-sensitive cell-stabilizing biomaterial that maintains (i) a substantially solid state
at about 8°C or below, and (ii) a substantially liquid state at about ambient temperature or
above. In one other embodiment, the bioactive cells comprise renal cells, as described
herein. In another embodiment, the bioactive cells are substantially uniformly dispersed
hout the volume of the cell-stabilizing biomaterial. In other embodiments, the
biomaterial has a solid-to-liquid transitional state between about 8°C and about ambient
temperature or above. In one embodiment, the substantially solid state is a gel state. In
another ment, the cell-stabilizing biomaterial comprises a hydrogel. In one other
embodiment, the hydrogel comprises gelatin. In other embodiments, the gelatin is t
in the formulation at about 0.5% to about 1% (w/v). In one embodiment, the gelatin is
present in the formulation at about 0.75% (w/v).
Also bed is a method of treating kidney disease in a subject in need comprising
administering at least one organoid sing a heterogeneous renal cell population and a
bioactive cell population. In some embodiments, the bioactive cell population is an
endothelial cell population. In certain ments, the endothelial cell population is a cell
line. In one embodiment, the endothelial cell population comprises HUVEC cells.
In most embodiments, the heterogeneous renal cell population comprises a B2 cell
tion comprising an enriched population of tubular cells, and wherein the
heterogeneous renal cell population is depleted of a B1 cell population. In select
embodiments, the heterogeneous renal cell population is further depleted of a B5 cell
tion. In some embodiments, the geneous renal cell population comprises a
cell tion selected from B2, B2/B3, B2/B4, and B2/B3/B4. In most ments, the
heterogeneous renal cell population comprises erythropoetin (EPO)-producing cells.
In an embodiment, the method of treating kidney disease in a subject in need
comprising administering an injectable formulation as described herein. In all
embodiments, the t is a mammal selected from dogs, cats, horses, rabbits, zoo
animals, cows, pigs, sheep, and primates. In a specific embodiment, the mammal is a
human. In all embodiments, the t has a kidney disease. In embodiments, an
improvement in any one of the following measures of anemia (Hct, Hgb, RBC), inflammation
(WBC), urine concentration (spGrav) and azotemia (BUN) is observed.
In another embodiment, the use of an organoid in the preparation of a medicament
for the treatment of kidney disease is described.
BRIEF DESCRIPTION OF THE DRAWINGS
depicts cell culture morphology of primary ZSF1 cells on fibronectin-coated
plates. A. p0 unfractionated presort viewed at 5x magnification; B. p1 CD31+ viewed at 5x
magnification; C. primary culture of unfractionated kidney cells at the end of passage 0
grown in 21%O2 on fibronectin-treated flask in KGM; D. negative flow through (CD31-) from
Miltenyi micro-beads ion;and E. 90% CD31+ cells ed at the end of passage 1
grown on fibronectin coated flasks in EGM2 fully mented .
shows human cell culture logy viewed at 5x. A. Unfractionated kidney
cells at the end of p0 that were grown in 21%O2 on ated flask in KGM; B.
Unfractionated kidney cells at the end of passage 0 grown exposed to 2%O2 O/N on TC-
treated flask in KGM; C. Unfractionated kidney cells at the end of passage 0 on fibronectin
coated flasks in EGM2 fully mented medium; D. CD31+ positively selected cells at the
end of passage 1 grown on fibronectin coated flasks in fully supplemented EGM2 media.
depicts FACS analysis showing percentage positive CD31 elial cells in
selected samples during culture period. The unfractionated (UNFX) endothelial ition
was <3% at p0 and was enriched ~ 15 fold at p1 when plated on fibronectin and ed in
EGM-2 media.
1 shows Bigeneic Cre/loxP reporters for lineage tracing studies.
A. Six2-Cre x R26td Tomato Red cross traces epithelial cells: parietal, proximal and distal
epithelial cells, but not interstitial or collecting duct. B. Unlabeled control. C. Six-2 positivity
from p1 culture (3 day normoxic followed by 1 day hypoxic culture).
shows spinner flasks (A) and low bind plates on rotator (B) used for SRC
organoid formation.
depicts phase imaging (10x) of A. rat and B. human SRC showing uniformity in
size. C. Organoids expressing pan-cadherin phenotype (green), nuclear (blue) (20x).
shows human organoids cultured within a 3D collagen I/IV gel. A. Phase image
showing low magnification of id tube formation (ringed in white). B. Phase image at
higher magnification (ringed in white) along with the remnant organoids. C. GGT-1
phenotypic expression (green), nuclear (blue) D. CK18 expression (green), nuclear ( blue) at
20x magnification.
depicts ne dye labeled organoids. A. SRC only labeled with DiL (red) at
x100 maginification. B. Organoid plus, SRC labeled with DiL (red), HuVEC labeled with DiO
(green) at x100 maginification.
shows 3D tubulogenesis assay in Col I/IV gel of self-generated organoids plus
in panels A, B ,and C showing the presence of both SRC tion (red) and endothelial
cells (green). When , tions appear yellow. Nuclear ng (blue) at 20x
magnification.
depicts SPIO Rhodamine labeled organoid (red) prior to injection.
shows magnetic resonance imaging (MRI) of organoid retention post-
implantation green= cells. A. 24hrs. B. 48hrs post-injection.
depicts prussian blue staining of implanted organoids g cell retention
and bio-distribution at low and high magnification. A. Implanted left kidney 24hrs post
implant. B. Implanted left kidney 48hrs post-implant.
is a panel of photographs g the HLA1 staining of human cells in a rat
kidney that had been administered the organoids described herein. A. Normal human
kidney; B. Human kidney cells in rat kidney. C. Untreated Nephrectomized rat kidney. D.
NKO treated (low power micoscopy). E. NKO treated (high power microscopy). F. A second
NKO treated animal. Panels A and B demonstrate staining (brown) of normal human kidney
tissue as well as human kidney cells (green arrows) acutely delivered to a rodent kidney.
Background ng is present in end of study non-treated diseased rat kidneys presumably
due to “sticky” nature of proteinaceous casts and damaged tubules as identified in panels C
and F by yellow arrows. This staining is lly lighter in color but is sometimes dark and
of size smaller than cells. Panels D, E and F demonstrate lower magnification utilized for
screening to fy dark staining cells, then higher magnification confirmation of HLA1
staining cells (green arrows).
The patent or application file contains at least one drawing in color. Copies of this
patent or patent publication with color drawing(s) will be provided by the Office upon
request and payment of the necessary fee.
DETAILED DESCRIPTION OF THE INVENTION
Many cell types make up the nephron, the functional unit within the kidney
parenchyma. The ability to isolate therapeutically bioactive renal cells from both normal
and diseased animals and humans has been recently established 1-3. The method used in
these studies is dependent on the isolation of a mixture of renal cells that exist in a diseased
tissue biopsy, using buoyant y gradient centrifugation.
Described is the isolation, identification and expansion of selected dual cell
populations of the various nephron compartments or niches, such that novel, le
enriched cell types can be combined as selected admixtures. The novel selected admixutres
as described herein provide improved targeting of specific structural and functional
deficiencies associated with the clinical and pathophysiologic basis of renal disease. The
isolation and enrichment of multiple, individual cell types that make up the nephron and
combined as selected admixtures enables improved targeted treatment of specific renal
disease cohorts; and/or at least provides the public with a useful choice.
Cell types such as the vascular endothelium, tubular and collecting duct lium,
titial cells, ular cells, mesenchymal stem cells, etc., can be isolated, identified
and expanded ex-vivo. While each cell type may require a unique method and media
formulation for sub-culture, they can be added back in selective combinations or admixtures
as id clusters to provide enhanced ry and improved treatment for an
underlying renal tissue/ cell deficiency associated with a specific acute and/or chronic
kidney disease patient syndrome/cohort 8.
The present description contemplates methods of treating renal impairment
associated with diseases of the vasculature (e.g., hypertension, microangiopathic anemias)
using a defined ratio of renal tubular epithelial to endothelial cells. The y to isolate,
characterize and expand resident renal endothelial cells using selective culture systems and
magnetic sorting allows for enrichment of a usly characterized ive regenerative
renal epithelial cell (SRC) population 2, 4 with a specific percentage of purified renal
endothelial cells . The SRC+ cell tions as described herein are comprised of
selected renal cells (SRCs or BRCs), as previously bed and also desribed herein, and
further bioactive cell populations, including but not limted to, endothelial cells, endothelial
progenitors, mesenchymal stem cells, and/or adipose-derived progenitors. In one
embodiment, the further bioactive cell populations are sourced from renal sources. In other
embodiments, the further bioactive cell populations are sourced from non-renal sources.
Also described are organoids comprising and/or formed from heterogenous mixtures
or fractions of ed or bioactive renal cells (SRCs or BRCs) or SRC+ cell poulations,
methods of isolating and ing the same, as well as methods of treatment using the
organoids as bed herein. Directed delivery of SRC or SRC+ populations to the kidney
as organoids is expected to improve cell retention, thereby leading to improved overall
therapeutic outcomes. Also bed are methods of treatment using SRC+ cell
populations.
Definitions
Unless defined ise, technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. Principles of Tissue Engineering, 3rd Ed. d by R Lanza, R Langer, & J Vacanti),
2007 provides one skilled in the art with a general guide to many of the terms used in the
present application. One skilled in the art will recognize many methods and materials
similar or equivalent to those bed herein, which could be used in the practice of the
present invention. Indeed, the present invention is in no way limited to the s and
materials described.
The term “cell population” as used herein refers to a number of cells obtained by
isolation directly from a suitable tissue source, usually from a mammal. The isolated cell
population may be uently cultured in vitro. Those of ordinary skill in the art will
appreciate that various methods for isolating and culturing cell populations for use with the
present ion and various numbers of cells in a cell population that are suitable for use
in the present invention. A cell population may be an unfractionated, geneous cell
population derived from the kidney. For example, a heterogeneous cell population may be
isolated from a kidney biopsy or from whole kidney tissue. Alternatively, the heterogeneous
cell population may be derived from in vitro es of mammalian cells, established from
kidney biopsies or whole kidney tissue. An unfractionated heterogeneous cell population
may also be referred to as a non-enriched cell tion.
The term “native kidney” shall mean the kidney of a living subject. The subject may
be healthy or lthy. An unhealthy subject may have a kidney disease.
The term “regenerative effect” shall mean an effect which provides a benefit to a
native . The effect may include, without limitation, a reduction in the degree of injury
to a native kidney or an improvement in, restoration of, or stabilization of a native kidney
function. Renal injury may be in the form of fibrosis, inflammation, glomerular hypertrophy,
etc. and related to kidney disease in the subject.
The term “regenerative potential” or “potential regenerative bioactivity” as used
herein refers to the potential of the organoids comprising bioactive cell preparations and/or
admixtures described herein to e a regenerative effect.
The term “admixture” as used herein refers to a combination of two or more
isolated, enriched cell populations derived from an unfractionated, heterogeneous cell
population. According to certain embodiments, the cell populations of the present
ion are renal cell populations.
An “enriched” cell population or ation refers to a cell population d from
a starting kidney cell population (e.g., an unfractionated, heterogeneous cell population)
that contains a greater percentage of a specific cell type than the tage of that cell
type in the starting population. For example, a starting kidney cell population can be
enriched for a first, a second, a third, a fourth, a fifth, and so on, cell population of interest.
As used herein, the terms “cell population”, “cell preparation” and “cell prototype” are used
interchangeably.
The term “enriched” cell population as used herein refers to a cell population
derived from a starting kidney cell population (e.g., a cell sion from a kidney biopsy or
cultured mammalian kidney cells) that contains a percentage of cells capable of ing
EPO that is greater than the percentage of cells capable of producing EPO in the starting
population. For e, the term “B4” is a cell population derived from a starting kidney
cell population that contains a greater percentage of EPO-producing cells, glomerular cells,
and vascular cells as compared to the starting population. The cell populations of the
present ion may be enriched for one or more cell types and depleted of one or more
other cell types. For example, an enriched EPO-producing cell population may be enriched
for interstitial lasts and depleted of tubular cells and collecting duct epithelial cells
relative to the interstitial fibroblasts and tubular cells in a non-enriched cell population, i.e.
the ng cell population from which the enriched cell population is derived. In all
embodiments citing EPO-enriched or “B4” populations, the ed cell populations are
heterogeneous populations of cells containing cells that can produce EPO in an oxygenregulated
manner, as demonstrated by oxygen-tunable EPO expression from the
endogenous native EPO gene.
In r embodiment, an enriched cell population, which contains a greater
percentage of a specific cell type, e.g., vascular, glomerular, or endocrine cells, than the
percentage of that cell type in the starting population, may also lack or be deficient in one
or more specific cell types, e.g., vascular, glomerular, or endocrine cells, as compared to a
starting kidney cell population derived from a healthy individual or subject. For example,
the term “B4’,” or B4 prime,” in one embodiment, is a cell population derived from a
ng kidney cell population that lacks or is ent in one or more cell types, e.g.,
vascular, ular or endocrine, depending on the disease state of the starting specimen,
as compared to a healthy individual. In one embodiment, the B4’ cell population is derived
from a subject having chronic kidney disease. In one ment, the B4’ cell population is
d from a subject having focal segmental ulosclerosis (FSGS). In another
ment, the B4’ cell population is derived from a subject having autoimmune
glomerulonephritis. In another embodiment, B4’ is a cell population derived from a starting
cell population including all cell types, e.g., vascular, glomerular, or endocrine cells, which is
later depleted of or made deficient in one or more cell types, e.g., vascular, glomerular, or
endocrine cells. In yet another embodiment, B4’ is a cell population d from a starting
cell population including all cell types, e.g., vascular, glomerular, or ine cells, in which
one or more ic cell types e.g., vascular, glomerular, or endocrine cells, is later
enriched. For example, in one embodiment, a B4’ cell population may be enriched for
vascular cells but depleted of glomerular and/or endocrine cells. In another embodiment, a
B4’ cell population may be enriched for glomerular cells but depleted of ar and/or
ine cells. In r embodiment, a B4’ cell population may be enriched for
endocrine cells but depleted of vascular and/or glomerular cells. In another embodiment, a
B4’ cell population may be enriched for vascular and endocrine cells but depleted of
ular cells. In preferred embodiments, the B4’ cell population, alone or admixed with
another enriched cell population, e.g., B2 and/or B3, retains therapeutic properties. A B4’
cell population, for example, is described herein in the Examples, e.g., Examples 7-9.
In another ment, an enriched cell population may also refer to a cell
population derived from a starting kidney cell population as discussed above that contains a
percentage of cells expressing one or more vascular, glomerular and proximal tubular
markers with some oducing cells that is greater than the tage of cells
expressing one or more vascular, ular and proximal tubular markers with some EPO-
producing cells in the starting population. For example, the term “B3” refers to a cell
population derived from a starting kidney cell population that contains a greater percentage
of proximal tubular cells as well as vascular and glomerular cells as compared to the starting
tion. In one embodiment, the B3 cell population contains a greater percentage of
proximal tubular cells as compared to the starting population but a lesser percentage of
proximal tubular cells as ed to the B2 cell tion. In another embodiment, the
B3 cell tion contains a greater percentage of vascular and glomerular cells markers
with some EPO-producing cells as compared to the starting population but a lesser
tage of vascular and glomerular cells markers with some EPO-producing cells as
compared to the B4 cell population.
In another embodiment, an ed cell population may also refer to a cell
population d from a starting kidney cell population as discussed above that ns a
percentage of cells expressing one or more tubular cell markers that is greater than the
percentage of cells expressing one or more tubular cell markers in the starting population.
For example, the term “B2” refers to a cell population derived from a starting kidney cell
population that contains a greater percentage of tubular cells as compared to the starting
population. In addition, a cell population enriched for cells that express one or more tubular
cell markers (or “B2”) may contain some epithelial cells from the collecting duct system.
Although the cell population enriched for cells that express one or more tubular cell markers
(or “B2”) is relatively depleted of EPO-producing cells, glomerular cells, and vascular cells,
the enriched population may contain a smaller percentage of these cells (EPO-producing,
glomerular, and vascular) in comparison to the starting population. In general, a
heterogeneous cell tion is depleted of one or more cell types such that the depleted
cell population contains a lesser tion of the cell ) relative to the proportion of
the cell type(s) contained in the heterogeneous cell population prior to depletion. The cell
types that may be depleted are any type of kidney cell. For example, in certain
embodiments, the cell types that may be depleted include cells with large granularity of the
ting duct and tubular system having a density of < about 1.045 g/ml, ed to as
“B1”. In certain other embodiments, the cell types that may be depleted include debris and
small cells of low granularity and viabilty having a density of > about 1.095 g/ml, referred to
as “B5”. In some embodiments, the cell population enriched for tubular cells is vely
depleted of all of the following: “B1”, “B5”, oxygen-tunable EPO-expressing cells, glomerular
cells, and vascular cells.
The term “hypoxic” culture conditions as used herein refers to culture conditions in
which cells are subjected to a reduction in available oxygen levels in the culture system
ve to standard culture conditions in which cells are cultured at atmospheric oxygen
levels (about 21%). Non-hypoxic conditions are referred to herein as normal or ic
culture conditions.
The term n-tunable” as used herein refers to the ability of cells to te
gene expression (up or down) based on the amount of oxygen available to the cells.
“Hypoxia-inducible” refers to the upregulation of gene expression in response to a reduction
in oxygen n (regardless of the pre-induction or starting oxygen tension).
The term “spheroid” refers to an aggregate or assembly of cells cultured to allow 3D
growth as opposed to growth as a monolayer. It is noted that the term oid” does not
imply that the aggregate is a geometric sphere. The ate may be highly organized with
a well defined morphology or it may be an unorganized mass; it may include a single cell
type or more than one cell type. The cells may be primary isolates, or a permanent cell line,
or a combination of the two. ed in this definition are organoids and organotypic
cultures.
The term “organoid” as used herein refers to a heterogeneous 3D eration of
cells that recapitulates aspects of cellular self-organization, architecture and signaling
interactions present in the native organ. The term “organoid” includes spheroids or cell
clusters formed from suspension cell es.
The term “biomaterial” as used here refers to a natural or synthetic biocompatible
material that is suitable for uction into living tissue. A natural biomaterial is a material
that is made by a living system. Synthetic biomaterials are materials which are not made by
a living system. The biomaterials disclosed herein may be a combination of natural and
synthetic biocompatible materials. As used herein, biomaterials include, for example,
polymeric matrices and scaffolds. Those of ordinary skill in the art will appreciate that the
erial(s) may be configured in various forms, for example, as liquid hydrogel
suspensions, porous foam, and may comprise one or more natural or synthetic
biocompatible materials.
The term “construct” refers to one or more cell populations deposited on or in a
e of a ld or matrix made up of one or more synthetic or naturally-occurring
biocompatible materials. The one or more cell populations may be coated with, deposited
on, embedded in, attached to, seeded, or entrapped in a biomaterial made up of one or
more synthetic or naturally-occurring biocompatible polymers, proteins, or peptides. The
one or more cell populations may be combined with a biomaterial or scaffold or matrix in
vitro or in vivo. In general, the one or more biocompatible materials used to form the
scaffold/biomaterial is selected to direct, facilitate, or permit the ion of multicellular,
three-dimensional, organization of at least one of the cell populations deposited thereon.
The one or more biomaterials used to generate the construct may also be ed to direct,
tate, or permit dispersion and/or integration of the construct or cellular components of
the construct with the nous host tissue, or to direct, facilitate, or permit the al,
engraftment, tolerance, or functional performance of the construct or cellular components
of the construct.
The term “marker” or “biomarker” refers generally to a DNA, RNA, protein,
carbohydrate, or glycolipid-based molecular marker, the expression or presence of which in
a cultured cell population can be detected by standard methods (or methods disclosed
herein) and is consistent with one or more cells in the cultured cell population being a
particular type of cell. The marker may be a polypeptide expressed by the cell or an
identifiable physical location on a chromosome, such as a gene, a restriction clease
ition site or a nucleic acid encoding a polypeptide (e.g., an mRNA) expressed by the
native cell. The marker may be an expressed region of a gene referred to as a “gene
expression marker”, or some segment of DNA with no known coding function. The
biomarkers may be cell-derived, e.g., secreted, products.
The terms rentially sed gene,” “differential gene expression” and their
synonyms, which are used interchangeably, refer to a gene whose expression is activated to
a higher or lower level in a first cell or cell population, relative to its expression in a second
cell or cell population. The terms also e genes whose expression is activated to a
higher or lower level at different stages over time during passage of the first or second cell
in culture. It is also understood that a differentially expressed gene may be either activated
or inhibited at the nucleic acid level or protein level, or may be subject to ative splicing
to result in a different polypeptide product. Such differences may be evidenced by a change
in mRNA levels, surface expression, secretion or other partitioning of a ptide, for
example. Differential gene expression may include a comparison of expression n
two or more genes or their gene products, or a comparison of the ratios of the expression
between two or more genes or their gene products, or even a comparison of two differently
processed products of the same gene, which differ between the first cell and the second
cell. Differential expression includes both quantitative, as well as qualitative, differences in
the temporal or cellular expression n in a gene or its expression products among, for
example, the first cell and the second cell. For the purpose of this description, “differential
gene expression” is considered to be present when there is a difference between the
sion of a given gene in the first cell and the second cell. The differential expression of
a marker may be in cells from a t before administration of a cell population,
admixture, or uct (the first cell) ve to expression in cells from the t after
administration (the second cell).
The terms “inhibit”, “down-regulate”, “under-express” and “reduce” are used
interchangeably and mean that the expression of a gene, or level of RNA les or
equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of
one or more proteins or protein subunits, is reduced relative to one or more controls, such
as, for example, one or more positive and/or negative controls. The expression may
be in cells from a patient before administration of a cell population, admixture, or construct
relative to cells from the patient after administration.
The term “up-regulate” or “over-express” is used to mean that the expression of a
gene, or level of RNA molecules or equivalent RNA molecules encoding one or more
proteins or protein subunits, or activity of one or more proteins or protein subunits, is
elevated relative to one or more controls, such as, for example, one or more positive and/or
ve controls. The over-expression may be in cells from a patient after administration
of a cell population, admixture, or construct relative to cells from the patient before
administration.
The term “anemia” as used herein refers to a deficit in red blood cell number and/or
hemoglobin levels due to inadequate tion of functional EPO protein by the EPO-
producing cells of a subject, and/or inadequate release of EPO protein into systemic
circulation, and/or the inability of erythroblasts in the bone marrow to respond to EPO
protein. A subject with anemia is unable to maintain erythroid homeostasis. In l,
anemia can occur with a decline or loss of kidney function (e.g., chronic renal failure),
anemia associated with relative EPO deficiency, anemia associated with tive heart
e, anemia associated with myelo-suppressive therapy such as chemotherapy or antiviral
therapy (e.g., AZT), anemia associated with non-myeloid cancers, anemia associated
with viral infections such as HIV, and anemia of chronic diseases such as mune
diseases (e.g., rheumatoid arthritis), liver disease, and organ system failure.
The term “EPO-deficiency” refers to any condition or disorder that is treatable with
an erythropoietin receptor agonist (e.g., recombinant EPO or EPO analogs), including
anemia.
The term “organ-related disease” as used herein refers to disorders associated with
any stage or degree of acute or c organ failure that results in a loss of the organ’s
ability to perform its function.
The term y disease” as used herein refers to disorders associated with any
stage or degree of acute or chronic renal e that results in a loss of the kidney’s ability
to perform the function of blood filtration and elimination of excess fluid, electrolytes, and
wastes from the blood. Kidney disease also includes endocrine dysfunctions such as anemia
(erythropoietin-deficiency), and mineral imbalance (Vitamin D deficiency). Kidney disease
may originate in the kidney or may be secondary to a variety of conditions, including (but
not limited to) heart failure, hypertension, diabetes, autoimmune disease, or liver disease.
Kidney disease may be a condition of chronic renal e that develops after an acute
injury to the kidney. For example, injury to the kidney by ischemia and/or exposure to
toxicants may cause acute renal failure; incomplete ry after acute kidney injury may
lead to the development of chronic renal failure.
The term “treatment” refers to both therapeutic treatment and prophylactic or
preventative measures for kidney disease, anemia, EPO deficiency, tubular transport
deficiency, or glomerular tion deficiency wherein the object is to reverse, prevent or
slow down (lessen) the ed disorder. Those in need of treatment include those already
having a kidney disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular
filtration deficiency as well as those prone to having a kidney e, anemia, EPO
deficiency, tubular transport ency, or glomerular filtration deficiency or those in whom
the kidney disease, anemia, EPO deficiency, tubular transport deficiency, or ular
filtration deficiency is to be prevented. The term “treatment” as used herein includes the
stabilization and/or improvement of kidney on.
The term “subject” shall mean any single human subject, including a patient, eligible
for treatment, who is experiencing or has experienced one or more signs, symptoms, or
other indicators of a kidney disease, anemia, or EPO deficiency. Such subjects include
without limitation subjects who are newly diagnosed or previously diagnosed and are now
experiencing a recurrence or relapse, or are at risk for a kidney disease, anemia, or EPO
ency, no matter the cause. The subject may have been previously treated for a kidney
disease, anemia, or EPO deficiency, or not so treated.
The term “patient” refers to any single animal, more preferably a mammal ding
such non-human animals as, for example, dogs, cats, horses, rabbits, zoo s, cows,
pigs, sheep, and non-human primates) for which treatment is d. Most preferably, the
patient herein is a human.
The term “sample” or “patient sample” or “biological sample” shall generally mean
any ical sample obtained from a subject or patient, body fluid, body tissue, cell line,
tissue e, or other source. The term includes tissue biopsies such as, for example,
kidney biopsies. The term includes cultured cells such as, for example, cultured mammalian
kidney cells. s for obtaining tissue biopsies and cultured cells from mammals are
well known in the art. If the term “sample” is used alone, it shall still mean that the
“sample” is a “biological sample” or “patient sample”, i.e., the terms are used
interchangeably.
The term “test sample” refers to a sample from a t that has been treated by a
method as described herein. The test sample may originate from various s in the
mammalian subject including, without limitation, blood, semen, serum, urine, bone marrow,
mucosa, tissue, etc.
The term “control” or “control ” refers a negative or positive control in which
a negative or positive result is expected to help correlate a result in the test sample.
Controls that are suitable for the present ption include, without limitation, a sample
known to exhibit indicators characteristic of normal erythroid homeostasis, a sample known
to exhibit indicators characteristic of anemia, a sample obtained from a subject known not
to be anemic, and a sample obtained from a subject known to be anemic. Additional
controls suitable for use in the methods described herein include, t limitation,
samples derived from subjects that have been treated with pharmacological agents known
to modulate erythropoiesis (e.g., recombinant EPO or EPO analogs). In addition, the control
may be a sample obtained from a subject prior to being treated by a method as bed.
An additional suitable control may be a test sample obtained from a subject known to have
any type or stage of kidney disease, and a sample from a subject known not to have any
type or stage of kidney disease. A control may be a normal healthy matched control. Those
of skill in the art will appreciate other controls suitable for use as described herein.
“Regeneration sis”, “regenerative prognosis”, or “prognostic for
regeneration” generally refers to a forecast or tion of the probable rative
course or outcome of the administration or tation of a cell population, admixture or
uct described herein. For a regeneration prognosis, the st or prediction may be
informed by one or more of the following: ement of a functional organ (e.g., the
kidney) after implantation or administration, development of a functional kidney after
implantation or administration, development of improved kidney function or capacity after
implantation or administration, and expression of certain markers by the native kidney
following implantation or stration.
“Regenerated organ” refers to a native organ after implantation or administration of
a cell population, admixture, or construct as described herein. The regenerated organ is
characterized by various indicators including, without tion, development of function or
capacity in the native organ, improvement of on or capacity in the native organ, and
the expression of certain markers in the native organ. Those of ordinary skill in the art will
appreciate that other indicators may be suitable for characterizing a regenerated organ.
“Regenerated ” refers to a native kidney after implantation or administration
of a cell population, admixture, or construct as described herein. The regenerated kidney is
characterized by various indicators including, without limitation, development of function or
capacity in the native kidney, improvement of function or capacity in the native kidney, and
the expression of certain markers in the native kidney. Those of ordinary skill in the art will
appreciate that other indicators may be suitable for terizing a regenerated .
[0073a] The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When interpreting statements in this specification and claims which include
the term ising”, other features besides the features prefaced by this term in each
statement can also be present. Related terms such as “comprise” and “comprised” are to be
interpreted in similar manner.
[0073b] In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless ically stated
otherwise, reference to such external documents is not to be construed as an admission
that such nts, or such s of information, in any jurisdiction, are prior art, or
form part of the common general knowledge in the art.
SRC+ cell populations
Described are cell populations comprising isolated, heterogeneous
populations of kidney cells, enriched for specific bioactive components or cell types and/or
depleted of specific inactive or undesired components or cell types and r bioactive cell
populations, ing but not limted to, endothelial cells, endothelial progenitors,
mesenchymal stem cells, adipose-derived progenitors, for use in the treatment of acute or
c kidney disease. The isolated, heterogeneous populations of kidney cells, enriched
for specific bioactive components or cell types and/or depleted of specific ve or
undesired components or cell types may include any of the cell populations as described
herein. In one embodiment, the further bioactive components, e.g., endothelial cells,
endothelial progenitors, mesenchymal stem cells, adipose-derived progenitors, are admixed
with the isolated, heterogeneous populations of kidney cells, enriched for specific bioactive
components or cell types and/or depleted of specific inactive or undesired components or
cell types. Also described are methods for preparing the SRC+ cell populations, as bed
herein. The further bioactive components, e.g., endothelial cells, endothelial progenitors,
mesenchymal stem cells, adipose-derived progenitors, comprising the cell tion, may
be present in any percentage sufficient to improve a cell or tissue deficiency.
The cell populations of the invention may se one or more further bioactive
ents, for e but not d to, endothelial cells, endothelial progenitors,
hymal stem cells, adipose-derived progenitors, comprising the cell population. In
n embodiments, the cell population comprises two further bioactive components. In
certain embodiments, the cell population comprises three further bioactive components. In
n embodiments, the cell population comprises four further bioactive components. In
n embodiments, the cell population comprises five further ive components. In
certain embodiments, the cell population comprises six further bioactive components. In
certain embodiments, the cell population comprises seven further bioactive components. In
certain embodiments, the cell population comprises eight further ive components. In
certain ments, the cell population comprises nine further ive components. In
certain embodiments, the cell population ses ten further bioactive components.
In one embodiment, a further bioactive component is an epithelial cell. In one
embodiment, the epithelial cell is a proximal r epithelial cell. In another embodiment,
the epithelial cell is a distal tubular epithelial cell. In another ment, the epithelial cell
is a parietal lial cell.
In another embodiment, a further bioactive component is an epithelial cell. In one
embodiment, the epithelial cell is a venous endothelial cell. In one embodiment, the
lial cell is an arterial endothelial cell. In one embodiment, the epithelial cell is a
capillary endothelial cell. In one embodiment, the epithelial cell is a tic endothelial
cell.
In another embodiment, a further bioactive component is a collecting duct cell. In
yet another embodiment, a further bioactive component is a smooth muscle cell. In
another embodiment, a further bioactive ent is a mesenchymal stem cell. In
another embodiment, a further bioactive component is a progenitor of endothelial,
mesenchymal, epithelial or hematopoietic lineage. In another embodiment, a further
bioactive component is a progenitor of endodermal, ectodermal or mesenchymal embryonic
origin. In certain embodiments, a further bioactive component is a stem cell of any origin or
derivation, including but not limited to embryonic (ES) and induced pluripotent (iPS) stem
cell. In some embodiments, a further ive component is a derivative of a stem cell of
any origin or derivation, including but not limited to embryonic (ES) and d pluripotent
(iPS) stem cell, wherein the derivative may be generated by the directed entiation of
the stem cell by defined combinations or cocktails of small molecules and/or protein and/or
nucleic acid molecules. In one embodiment, a further bioactive component is a derivative of
a progenitor of endothelial, mesenchymal, epithelial or hematopoietic lineage wherein the
derivative may generated by the directed differentiation of the stem cell by defined
combinations or cocktails of small molecules and/or protein and/or nucleic acid molecules.
In one embodiment, a r bioactive component is a derivative of a progenitor of
endodermal, ectodermal or mesenchymal embryonic origin wherein the derivative may be
generated by the directed differentiation of the stem cell by defined combinations or
cocktails of small molecules and/or protein and/or nucleic acid molecules. In one
embodiment, a further bioactive component is a genetically ed cell of any lineage or
derivation.
In still another embodiment, a further bioactive ent is an intersitial
cell. In one embodiment, the interstitial cell is a supportive fibroblast. In another
embodiment, the interstitial cell is a specialized cortical erythropoietin-producing fibroblast.
In one embodiment, the further bioactive component is derived from a
source that is autologous to the subject. In one other embodiment, the further bioactive
component is derived from a source that is allogeneic to the subject. In certain
embodiments, a further bioactive component is d from a source that is gous to
the t, which further ive component is a ed admixture with a still further
bioactive components which are derived from a source that is allogeneic to the subject.
Also described are methods for the targeted regeneration of renal mass and
functionality by directed delivery of the cell tions and/or organoids and/or
biomaterials described herein. Also described are methods method for the rescue and/or
recovery of renal functionality in patients having acute or chronic renal e by
administration of cell populations and/or organoids and/or biomaterials bed herein.
Therapeutic Organoids
Also described are organoids comprising and/or formed from the bioactive
ents described herein, e.g., B2, B4, and B3, which are depleted of inactive or
undesired components, e.g., B1 and B5, alone or admixed for use in the treatment of acute
and/or chronic kidney diseaseAlso described are ods comprising and/or formed from
a specific subfraction, B4, depleted of or deficient in one or more cell types, e.g., vascular,
endocrine, or endothelial, i.e., B4’, retains eutic properties, e.g., stabilization and/or
improvement and/or regeneration of kidney function, alone or when admixed with other
bioactive subfractions, e.g., B2 and/or B3. In a preferred embodiment, the bioactive cell
population is B2. In certain embodiments, the B2 cell population is admixed with B4 or B4’.
In other ments, the B2 cell population is admixed with B3. In other embodiments,
the B2 cell population is admixed with both B3 and B4, or specific cellular components of B3
and/or B4. In all embodiments, the organoids described herein are formed and cultured ex
vivo. In all embodiments, the ids may further comprise and/or be formed from
r bioactive cell populations, including but not limted to, endothelial cells, endothelial
itors, mesenchymal stem cells, adipose-derived progenitors. In one embodiment,
the r bioactive cell tions, including but not limted to, endothelial cells,
endothelial progenitors, mesenchymal stem cells, adipose-derived progenitors are admixed
with the isolated, heterogeneous populations of kidney cells, enriched for specific bioactive
components or cell types and/or depleted of specific inactive or undesired components or
cell types.
In one embodiment, the organoids as described comprise or are formed from a B2
cell population, wherein the B2 cell population comprises an enriched population of tubular
cells. In another embodiment, the heterogenous renal cell population further ses a
B4 cell population. In yet another embodiment, the heterogeneous renal cell population
r comprises a B3 population. In still another embodiment, the heterogeneous renal
cell population further ses a B5 population.
In certain embodiments, the cell population comprises a B2 cell population, wherein
the B2 cell population comprises an enriched population of tubular cells, and is depleted of
a B1 cell population, and/or a B5 cell population.
Also described are methods of forming organoids comprising and/or formed from
bioactive components as described herein, e.g., B2, B4, and B3, which are depleted of
inactive or undesired components, e.g., B1 and B5, alone or admixed. Also described are
ods comprising and/or formed from a specific ction, B4, depleted of or ent
in one or more cell types, e.g., vascular, endocrine, or endothelial, i.e., B4’, retains
therapeutic properties, e.g., stabilization and/or improvement and/or regeneration of
kidney function, alone or when admixed with other bioactive subfractions, e.g., B2 and/or
B3. In a preferred embodiment, the bioactive cell population is B2. In certain embodiments,
the B2 cell population is admixed with B4 or B4’. In other embodiments, the B2 cell
population is admixed with B3. In other embodiments, the B2 cell population is admixed
with both B3 and B4, or ic cellular components of B3 and/or B4.
In one embodiment, the organoids bed herein comprise and/or are formed
from a B2 cell population, wherein the B2 cell population comprises an enriched tion
of tubular cells. In another ment, the heterogenous renal cell population further
comprises a B4 cell population. In yet another embodiment, the heterogeneous renal cell
population further comprises a B3 population. In still another embodiment, the
heterogeneous renal cell population further comprises a B5 population.
In certain embodiments, the cell population comprises a B2 cell population, wherein
the B2 cell tion comprises an enriched population of tubular cells, and is depleted of
a B1 cell population, and/or a B5 cell population.
Also described are methods of forming organoids using the bioactive cell
preparations and/or admixtures described . General metho ds for generating tubules
from primary renal cell tions using 3D COL(I) gel culture are known in the art, for
e, as in Joraku et al., Methods. 2009 Feb;47(2):129-33. In all embodiments, the
ids described herein are formed and cultured ex vivo.
In some embodiments, formation of organoids and tubules from the bioactive cell
preparations and/or admixtures described herein may be induced, for example and without
limitation, using the following culture methods or systems: i) 2D culture; ii) 3D culture:
COL(I) gel; iii) 3D culture: Matrigel; iv) 3D e: spinners, then COL(I)/Matrigel; and v) 3D
culture: COL(IV) gel. Specific examples of formation of organoids and tubules from NKA are
provided in 2 and 4 below.
In one embodiment, organoids formed from the bioactive cell preparations and/or
admixtures bed herein may be induced in 2D culture. In one embodiment, the
bioactive cell preparations and/or ures bed herein are seeded on standard 2D
c-ware. In one embodiment, cells are seeded at a density of about 5000 cells/cm2.
Cells may be seeded in an appropriate medium, such as, for example Renal Cell Complete
Growth Media (RCGM). In l, cell populations may be grown past confluence for
about 7, 8, 9, 10, 11, 12, 13, 14, 15 days or more, with regular changes of media about every
3-4 days. In one embodiment, cells demonstrate spontaneous self-organization into
spheroidal structures, i.e., organoids, and tubules between about 7 to about 15 days.
In another embodinment, organoids formed from the bioactive cell ations
and/or admixtures described herein may be induced in 3D culture. In one embodiment, the
organoid is generated with the cell populations as described herein together with a
biomaterial scaffold of l or synthetic origin. In one embodiment, formulated the
bioactive cell preparations and/or admixtures described herein may be incorporated into a
en (I) gel, collagen (IV) gel, Matrigel or a e of any of these as previously
described (see aes-Souza et al., 2012. In vitro reconstitution of human kidney
structures for renal cell therapy. Nephrol Dial Transplant 0: 1-9). The liquid gel may be
brought to a neutral pH and the bioactive cell preparations and/or admixtures described
herein mixed in at about 500-2500 cells/ul. In one embodiment, about 1000 cells/ul are
mixed in. The cell/gel mixture may be aliquoted into a well of a 24 well plate, for example,
(about 200 to about 400ul/well) and allowed to solidify at 37 degrees C for several hours.
Cell culture media may then added and the cultures allowed to mature for about 4, about 5,
about 6, about 7, about 8, about 9, or about 10 days with r changes of media. In one
embodiment ks of tubular structures ze as lattices and rings form throughout
the gel matrix by the bioactive cell preparations and/or admixtures described herein.
In another embodiment, organoids may be formed by suspension culture of the
bioactive cell preparations and/or admixtures described herein in r flasks or low-bind
plasticware. In one embodiment, cells may be ed in media in r flasks for up to 4
days at about 80rpm. Spheroids may then be further cultured for about 7, about 8, about 9,
or about 10 days on Matrigel coated plates, for example. In one embodiment, spheroids
formed from the bioactive cell preparations and/or admixtures described herein show
tubulogenic potential as shown by de novo budding of tubular structures from cultured
ids.
Cell populations
The SRC+ cell populations and/or organoids described herein may contain and/or be
formed from isolated, heterogeneous tions of kidney cells, and admixtures thereof,
enriched for specific bioactive components or cell types and/or depleted of specific inactive
or undesired components or cell types for use in the treatment of kidney e, i.e.,
providing stabilization and/or improvement and/or ration of kidney function, were
previously described in Presnell et al. U.S. 2011-0117162 and Ilagan et al.
, the entire contents of which are incorporated herein by reference.
The organoids may contain isolated renal cell fractions that lack cellular components as
compared to a healthy individual yet retain therapeutic properties, i.e., provide stabilization
and/or improvement and/or regeneration of kidney function. The cell populations, cell
ons, and/or admixtures of cells described herein may be derived from healthy
individuals, individuals with a kidney disease, or subjects as described herein.
Bioactive cell populations
The present ption contemplates SRC+ cell populations and therapeutic
organoids comprising bioactive cell populations that are to be administered to target organs
or tissue in a subject in need. A bioactive cell population generally refers to a cell
tion potentially having therapeutic properties upon stration to a subject. For
example, upon administration to a subject in need, a organoid comprising a bioactive renal
cell population can provide stabilization and/or improvement and/or regeneration of kidney
function in the subject. The therapeutic properties may include a regenerative effect.
Bioactive cell populations include, without limitation, stem cells (e.g., pluripotent,
multipotent, oligopotent, or unipotent) such as embryonic stem cells, amniotic stem cells,
adult stem cells (e.g., poietic, mammary, intestinal, mesenchymal, placental, lung,
bone marrow, blood, umbilical cord, endothelial, dental pulp, adipose, neural, olfactory,
neural crest, testicular), d pluripotent stem cells; genetically modified cells; as well as
cell populations or tissue explants derived from any source of the body. The bioactive cell
populations may be isolated, enriched, purified, homogeneous, or geneous in nature.
Those of ry skill in the art will appreciate other bioactive cell populations that are
suitable for use in generating the organoids as described herein.
In one embodiment, the source of cells is the same as the intended target organ or
tissue. For example, renal cells may be sourced from the kidney to generate an organoid to
be administered to the kidney. In another embodiment, the source of cells is not the same
as the intended target organ or tissue. For e, erythropoietin-expressing cells may be
sourced from renal adipose to generate an organoid to be administered to the kidney.
Also described are organoids comprising certain subfractions of a heterogeneous
tion of renal cells, enriched for bioactive components and depleted of inactive or
undesired components provide or therapeutic and regenerative es than the
starting tion. For example, ive renal cells described herein, e.g., B2, B4, and B3,
which are depleted of inactive or undesired components, e.g., B1 and B5, alone or admixed,
can be used to generate an id to be used for the stabilization and/or improvement
and/or regeneration of kidney function.
In another embodiment, the organoids contain a specific subfraction, B4, depleted of
or deficient in one or more cell types, e.g., vascular, endocrine, or endothelial, i.e., B4’, that
retain therapeutic properties, e.g., stabilization and/or improvement and/or regeneration of
kidney function, alone or when d with other bioactive subfractions, e.g., B2 and/or
B3. In a preferred embodiment, the bioactive cell population is B2. In n embodiments,
the B2 cell population is admixed with B4 or B4’. In other embodiments, the B2 cell
population is admixed with B3. In other embodiments, the B2 cell tion is admixed
with both B3 and B4, or specific cellular components of B3 and/or B4.
The B2 cell population is characterized by expression of a tubular cell marker
ed from the group consisting of one or more of the ing: megalin, cubilin,
hyaluronic acid synthase 2 (HAS2), Vitamin D3 25-Hydroxylase 25), N-cadherin
(Ncad), E-cadherin (Ecad), Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS
oncogene family (Rab17), GATA binding n 3 (Gata3), FXYD domain-containing ion
transport regulator 4 (Fxyd4), solute carrier family 9 m/hydrogen exchanger), member
4 4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), de
dehydrogenase 1 , member A3 (Aldh1a3), and Calpain-8 (Capn8), and collecting duct
marker Aquaporin-4 (Aqp4). B2 is larger and more granulated than B3 and/or B4 and thus
having a buoyant density between about 1.045 g/ml and about 1.063 g/ml (rodent),
between about 1.045 g/ml and 1.052 g/ml (human), and between about 1.045 g/ml and
about 1.058 g/ml (canine).
The B3 cell population is characterized by the expression of vascular,
glomerular and proximal tubular markers with some EPO-producing cells, being of an
intermediate size and granularity in comparison to B2 and B4, and thus having a buoyant
density between about 1.063 g/ml and about 1.073 g/ml (rodent), between about 1.052
g/ml and about 1.063 g/ml (human), and between about 1.058 g/ml and about 1.063 g/ml
(canine). B3 is characterized by expression of markers selected from the group consisting of
one or more of the following: aquaporin 7 (Aqp7), FXYD domain-containing ion transport
regulator 2 (Fxyd2), solute carrier family 17 (sodium phosphate), member 3 (Slc17a3), solute
carrier family 3, member 1 (Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase (Napsa),
solute carrier family 2 (facilitated e transporter), member 2 (Slc2a2), alanyl
(membrane) aminopeptidase (Anpep), embrane protein 27 (Tmem27), acyl-CoA
synthetase medium-chain family member 2 (Acsm2), glutathione peroxidase 3 ,
fructose-1,6- biphosphatase 1 , and alanine-glyoxylate aminotransferase 2 (Agxt2).
B3 is also characterized by the vascular expression marker Platelet endothelial cell adhesion
molecule (Pecam) and the glomerular sion marker podocin (Podn).
The B4 cell population is characterized by the expression of a vascular marker
set containing one or more of the following: PECAM, VEGF, KDR, HIF1a, CD31, CD146; a
glomerular marker set containing one or more of the ing: Podocin (Podn), and
Nephrin (Neph); and an oxygen-tunable EPO ed population compared to
unfractionated (UNFX), B2 and B3. B4 is also characterized by the expression of one or
more of the following markers: chemokine (C-X-C motif) receptor 4 (Cxcr4), endothelin
or type B (Ednrb), collagen, type V, alpha 2 (Col5a2), in 5 (Cdh5), plasminogen
activator, tissue (Plat), angiopoietin 2 (Angpt2), kinase insert domain protein receptor (Kdr),
secreted protein, acidic, cysteine-rich (osteonectin) (Sparc), cin (Srgn), TIMP
metallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless-type MMTV
integration site family, member 4 (Wnt4), regulator of G-protein signaling 4 (Rgs4), Platelet
endothelial cell adhesion molecule (Pecam), and Erythropoietin (Epo). B4 is also
characterized by smaller, less granulated cells compared to either B2 or B3, with a buoyant
density between about 1.073 g/ml and about 1.091g/ml t), between about 1.063
g/ml and about 1.091 g/mL (human and canine).
The B4’ cell population is d as having a t density of between
1.063 g/mL and 1.091 g/mL and expressing one or more of the following markers: PECAM,
vEGF, KDR, HIF1a, podocin, nephrin, EPO, CK7, CK8/18/19. In one embodiment, the B4’ cell
population is characterized by the expression of a vascular marker set containing one or
more of the following: PECAM, vEGF, KDR, HIF1a, CD31, CD146. In another embodiment,
the B4’ cell population is characterized by the expression of an ine marker EPO. In
one embodiment, the B4’ cell population is characterized by the expression of a glomerular
marker set containing one or more of the following: Podocin (Podn), and Nephrin (Neph). In
certain embodiments, the B4’ cell tion is characterized by the expression of a vascular
marker set containing one or more of the following: PECAM, vEGF, KDR, HIF1a and by the
expression of an endocrine marker EPO. In another embodiment, B4’ is also characterized
by r, less granulated cells compared to either B2 or B3, with a buoyant density
n about 1.073 g/ml and about 1.091g/ml (rodent), between about 1.063 g/ml and
about 1.091 g/mL (human and canine).
Also described are organoids containing an isolated, enriched B4’ population
of human renal cells comprising at least one of erythropoietin (EPO)-producing cells,
vascular cells, and glomerular cells having a density between 1.063 g/mL and 1.091 g/mL. In
one embodiment, the B4’ cell population is characterized by sion of a vascular
marker. In certain embodiments, the B4’ cell population is not characterized by expression
of a glomerular marker. In some embodiments, the B4’ cell population is capable of oxygentunable
erythropoietin (EPO) expression.
In one embodiment, the organoid described herein contains the B4’ cell
population but does not include a B2 cell population comprising tubular cells having a
density between 1.045 g/mL and 1.052 g/mL. In another embodiment, the B4’ cell
population-containing organoid does not include a B1 cell population comprising large
granular cells of the collecting duct and tubular system having a density of < 1.045 g/ml. In
yet another embodiment, the B4’ cell population organoid does not include a B5 cell
population comprising debris and small cells of low granularity and viability with a density >
1.091 g/ml.
In one embodiment, the B4’ cell population-containing organoid does not
include a B2 cell population comprising tubular cells having a density between 1.045 g/mL
and 1.052 g/mL; a B1 cell population sing large granular cells of the collecting duct
and tubular system having a density of < 1.045 g/ml; and a B5 cell population sing
debris and small cells of low granularity and ity with a density > 1.091 g/ml. In some
embodiments, the B4’ cell tion may be derived from a subject having kidney disease.
Also described are organoids containing admixtures of human renal cells
sing a first cell population, B2, comprising an isolated, enriched population of tubular
cells having a y between 1.045 g/mL and 1.052 g/mL, and a second cell population,
B4’, comprising erythropoietin (EPO)-producing cells and vascular cells but depleted of
ular cells having a density between about 1.063 g/mL and 1.091 g/mL, wherein the
admixture does not include a B1 cell population comprising large granular cells of the
collecting duct and r system having a y of < 1.045 g/ml, or a B5 cell population
comprising debris and small cells of low granularity and viability with a y > 1.091 g/ml.
In certain embodiment, the B4’ cell population is characterized by sion of a vascular
marker. In one ment, the B4’ cell population is not characterized by expression of a
glomerular marker. In certain embodiments, B2 further comprises collecting duct epithelial
cells. In one embodiment, the id contains or is formed from an admixture of cells
that is capable of receptor-mediated albumin uptake. In another embodiment, the
ure of cells is capable of -tunable erythropoietin (EPO) expression. In one
embodiment, the admixture contains HASexpressing cells capable of producing and/or
stimulating the production of high-molecular weight species of hyaluronic acid (HA) both in
vitro and in vivo. In all embodiments, the first and second cell populations may be derived
from kidney tissue or cultured kidney cells (Basu et al. Lipids in Health and Disease, 2011,
:171).
In one embodiment, the id contains an admixture that is capable of
providing a regenerative stimulus upon in vivo delivery. In other embodiments, the
admixture is capable of reducing the decline of, stabilizing, or improving glomerular
filtration, tubular resorption, urine production, and/or endocrine function upon in vivo
delivery. In one embodiment, the B4’ cell population is derived from a t having
kidney disease.
Also described are organoids containing an isolated, enriched B4’ population
of human renal cells comprising at least one of erythropoietin (EPO)-producing cells,
vascular cells, and glomerular cells having a density between 1.063 g/mL and 1.091 g/mL. In
one embodiment, the B4’ cell population is characterized by sion of a vascular
marker. In certain embodiments, the B4’ cell population is not characterized by expression
of a ular . The glomerular marker that is not expressed may be podocin (see
Example 10). In some embodiments, the B4’ cell population is capable of oxygen-tunable
erythropoietin (EPO) expression.
In one embodiment, the B4’ cell population-containing organoid does not
include a B2 cell population comprising tubular cells having a density between 1.045 g/mL
and 1.052 g/mL. In another embodiment, the B4’ cell population organoid does not include
a B1 cell tion comprising large granular cells of the collecting duct and tubular system
having a density of < 1.045 g/ml. In yet another embodiment, the B4’ cell population
id does not include a B5 cell population comprising debris and small cells of low
granularity and viability with a y > 1.091 g/ml.
In one embodiment, the B4’ cell tion-containing id does not
include a B2 cell tion comprising tubular cells having a density between 1.045 g/mL
and 1.052 g/mL; a B1 cell population comprising large granular cells of the collecting duct
and r system having a density of < 1.045 g/ml; and a B5 cell population comprising
debris and small cells of low granularity and viability with a density > 1.091 g/ml. In some
embodiments, the B4’ cell population may be derived from a subject having kidney disease.
Also described are organoids containing an admixture of human renal cells
comprising a first cell population, B2, sing an isolated, enriched population of tubular
cells having a density between 1.045 g/mL and 1.052 g/mL, and a second cell population,
B4’, comprising erythropoietin (EPO)-producing cells and vascular cells but depleted of
glomerular cells having a density between about 1.063 g/mL and 1.091 g/mL, wherein the
admixture does not include a B1 cell population comprising large granular cells of the
collecting duct and tubular system having a density of < 1.045 g/ml, or a B5 cell population
comprising debris and small cells of low granularity and ity with a density > 1.091 g/ml.
In certain embodiment, the B4’ cell population is characterized by expression of a vascular
. In one embodiment, the B4’ cell population is not characterized by expression of a
glomerular marker. In certain embodiments, B2 further comprises collecting duct epithelial
cells. In one ment, the admixture of cells is capable of receptor-mediated albumin
uptake. In another embodiment, the admixture of cells is capable of oxygen-tunable
erythropoietin (EPO) expression. In one embodiment, the admixture contains HAS
expressing cells capable of producing and/or stimulating the production of high-molecular
weight species of hyaluronic acid (HA) both in vitro and in vivo. In all embodiments, the first
and second cell tions may be derived from kidney tissue or cultured kidney cells.
Also described are organiods ning a geneous renal cell
tion comprising a combination of cell fractions or enriched cell populations (e.g., B1,
B2, B3, B4 (or B4’), and B5). In one embodiment, the combination has a buoyant density
n about 1.045 g/ml and about 1.091 g/ml. In one other ment, the
combination has a buoyant density between less than about 1.045 g/ml and about 1.099
g/ml or about 1.100 g/ml. In another embodiment, the combination has a buoyant density
as determined by separation on a density gradient, e.g., by centrifugation. In yet another
embodiment, the combination of cell ons contains B2, B3, and B4 (or B4’) depleted of
B1 and/or B5. In some embodiments, the combination of cell fractions contains B2, B3, B4
(or B4’), and B5 but is depleted of B1. Once depleted of B1 and/or B5, the combination may
be subsequently cultured in vitro prior to the preparation of an organoid comprising the
combination of B2, B3, and B4 (or B4’) cell fractions.
The inventors of the present ion have surprisingly discovered that in
vitro culturing of a B1-depleted combination of B2, B3, B4, and B5 results in depletion of B5.
In one embodiment, B5 is depleted after at least one, two, three, four, or five passages. In
one other embodiment, the B2, B3, B4, and B5 cell fraction combination that is passaged
under the conditions described herein provides a passaged cell population having B5 at a
percentage that is less than about 5%, less than about 4%, less than about 3%, less than
about 2%, less than about 1%, or less than about 0.5% of the passaged cell population.
In another embodiment, B4’ is part of the combination of cell fractions. In
one other embodiment, the in vitro culturing depletion of B5 is under c conditions.
In one embodiment, the organoid contains an admixture that is capable of
providing a regenerative stimulus upon in vivo delivery. In other embodiments, the
admixture is capable of reducing the decline of, stabilizing, or improving glomerular
tion, tubular resorption, urine production, and/or endocrine function upon in vivo
delivery. In one embodiment, the B4’ cell tion is derived from a subject having
kidney disease.
In a preferred embodiment, the organoid contains and/or is formed from an
admixture that comprises B2 in combination with B3 and/or B4. In another red
ment, the ure comprises B2 in ation with B3 and/or B4’. In other
preferred embodiments, the admixture ts of or consists essentially of (i) B2 in
combination with B3 and/or B4; or (ii) B2 in ation with B3 and/or B4’.
The admixtures that contain a B4’ cell population may contain B2 and/or B3
cell populations that are also obtained from a non-healthy subject. The non-healthy t
may be the same subject from which the B4’ on was obtained. In st to the B4’
cell population, the B2 and B3 cell populations obtained from non-healthy subjects are
typically not deficient in one or more specific cell types as compared to a starting kidney cell
population derived from a healthy individual.
As described in Presnell et al. WO/2010/056328, it has been found that the
B2 and B4 cell preparations are capable of expressing higher molecular weight species of
hyaluronic acid (HA) both in vitro and in vivo, through the actions of hyaluronic acid
synthase-2 (HAS-2) – a marker that is enriched more specifically in the B2 cell population.
Treatment with B2 in a 5/6 Nx model was shown to reduce fibrosis, concomitant with strong
expression HAS-2 expression in vivo and the ed production of high-molecular-weight
HA within the treated tissue. Notably, the 5/6 Nx model left untreated resulted in fibrosis
with limited detection of HAS-2 and little production of high-molecular-weight HA. Without
wishing to be bound by theory, it is esized that this anti-inflammatory high-molecular
weight species of HA ed predominantly by B2 (and to some degree by B4) acts
synergistically with the cell preparations in the reduction of renal fibrosis and in the aid of
renal regeneration. Accordingly, the instant description includes organoids containing the
bioactive renal cells described herein along with a biomaterial comprising hyaluronic acid.
Also contemplated by the instant description is the provision of a biomaterial component of
the regenerative stimulus via direct production or stimulation of production by the
ted cells.
Also described are organoids containing and/or generated from isolated,
heterogeneous tions of EPO-producing kidney cells for use in the treatment of kidney
disease, anemia and/or EPO deficiency in a subject in need. In one ment, the cell
populations are derived from a kidney biopsy. In another embodiment, the cell populations
are derived from whole kidney tissue. In one other embodiment, the cell populations are
derived from in vitro cultures of mammalian kidney cells, established from kidney biopsies
or whole kidney tissue. In all embodiments, these populations are tionated cell
populations, also referred to herein as non-enriched cell populations.
Also described are organoids containing and/or generated from isolated
tions of erythropoietin (EPO)-producing kidney cells that are further enriched such
that the tion of EPO-producing cells in the ed subpopulation is greater relative
to the proportion of EPO-producing cells in the starting or l cell population. In one
embodiment, the enriched EPO-producing cell fraction contains a greater proportion of
interstitial fibroblasts and a lesser proportion of tubular cells relative to the interstitial
fibroblasts and tubular cells contained in the unenriched initial tion. In certain
embodiments, the enriched EPO-producing cell fraction contains a greater proportion of
glomerular cells and vascular cells and a lesser proportion of collecting duct cells relative to
the glomerular cells, vascular cells and collecting duct cells contained in the unenriched
initial population. In such embodiments, these populations are referred to herein as the
“B4” cell tion.
] Also described are organoids containing and/or ted from an EPO-
producing kidney cell population that is admixed with one or more additional kidney cell
populations. In one embodiment, the EPO-producing cell tion is a first cell population
enriched for oducing cells, e.g., B4. In r embodiment, the EPO-producing cell
population is a first cell population that is not enriched for oducing cells, e.g., B2. In
another embodiment, the first cell population is admixed with a second kidney cell
population. In some embodiments, the second cell population is enriched for tubular cells,
which may be demonstrated by the presence of a tubular cell phenotype. In r
embodiment, the tubular cell phenotype may be indicated by the presence of one tubular
cell marker. In another embodiment, the tubular cell phenotype may be indicated by the
presence of one or more tubular cell markers. The tubular cell markers include, without
limitation, megalin, cubilin, hyaluronic acid synthase 2 (HAS2), Vitamin D3 25-Hydroxylase
(CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2),
RAB17, member RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD
domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9
(sodium/hydrogen exchanger), member 4 (Slc9a4), de dehydrogenase 3 family,
member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and
Calpain-8 (Capn8). In another embodiment, the first cell population is admixed with at least
one of several types of kidney cells including, without limitation, interstitium-derived cells,
tubular cells, collecting duct-derived cells, glomerulus-derived cells, and/or cells derived
from the blood or ature.
The organoids of the present description may include or be formed from
oducing kidney cell populations ning B4 or B4’ in the form of an ure with
B2 and/or B3, or in the form of an enriched cell population, e.g., B2+B3+B4/B4’.
] In one embodiment, the organoids contain and/or are generated from EPO-
producing kidney cell populations that are characterized by EPO expression and
bioresponsiveness to oxygen, such that a ion in the oxygen tension of the culture
system results in an induction in the expression of EPO. In one embodiment, the EPO-
producing cell populations are enriched for EPO-producing cells. In one ment, the
EPO expression is induced when the cell population is ed under conditions where the
cells are subjected to a reduction in available oxygen levels in the culture system as
compared to a cell population cultured at normal atmospheric (~21%) levels of ble
oxygen. In one embodiment, EPO-producing cells cultured in lower oxygen conditions
express greater levels of EPO relative to EPO-producing cells cultured at normal oxygen
conditions. In l, the culturing of cells at reduced levels of available oxygen (also
referred to as hypoxic culture conditions) means that the level of d oxygen is reduced
relative to the culturing of cells at normal atmospheric levels of available oxygen (also
referred to as normal or normoxic culture conditions). In one embodiment, hypoxic cell
culture conditions include culturing cells at about less than 1% oxygen, about less than 2%
, about less than 3% oxygen, about less than 4% oxygen, or about less than 5%
oxygen. In another embodiment, normal or normoxic culture conditions include ing
cells at about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% , about
% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% ,
about 20% oxygen, or about 21% oxygen.
In one other embodiment, ion or increased expression of EPO is
obtained and can be observed by culturing cells at about less than 5% available oxygen and
comparing EPO expression levels to cells cultured at atmospheric (about 21%) oxygen. In
another embodiment, the induction of EPO is obtained in a e of cells capable of
sing EPO by a method that includes a first culture phase in which the e of cells is
cultivated at atmospheric oxygen (about 21%) for some period of time and a second culture
phase in which the available oxygen levels are reduced and the same cells are ed at
about less than 5% available oxygen. In another embodiment, the EPO expression that is
sive to hypoxic conditions is ted by HIF1α. Those of ordinary skill in the art will
appreciate that other oxygen manipulation culture conditions known in the art may be used
for the cells described herein.
In one embodiment, the organoid contains and/or is formed from enriched
populations of EPO-producing mammalian cells characterized by bio-responsiveness (e.g.,
EPO expression) to perfusion conditions. In one embodiment, the perfusion conditions
include transient, intermittent, or continuous fluid flow (perfusion). In one embodiment,
the EPO expression is mechanically-induced when the media in which the cells are cultured
is intermittently or continuously circulated or agitated in such a manner that dynamic forces
are transferred to the cells via the flow. In one embodiment, the cells subjected to the
transient, ittent, or continuous fluid flow are cultured in such a manner that they are
present as three-dimensional structures in or on a material that provides framework and/or
space for such three-dimensional structures to form. In one embodiment, the cells are
cultured on porous beads and subjected to intermittent or continuous fluid flow by means
of a rocking platform, orbiting platform, or spinner flask. In another embodiment, the cells
are cultured on three-dimensional scaffolding and placed into a device whereby the scaffold
is stationary and fluid flows ionally through or across the scaffolding. Those of
ry skill in the art will appreciate that other perfusion culture conditions known in the
art may be used for the cells described herein.
Inactive cell populations
As described herein, the present invention is based, in part, on the surprising
g that ids comprising and/or formed from certain subfractions of a
heterogeneous population of renal cells, enriched for bioactive components and depleted of
inactive or undesired components, provide superior therapeutic and regenerative outcomes
than the starting population. In preferred embodiments, the organoids as described herein
contain cellular populations that are depleted of B1 and/or B5 cell populations. For
instance, the following may be depleted of B1 and/or B5: admixtures of two or more of B2,
B3, and B4 (or B4’); an enriched cell population of B2, B3, and B4 (or B4’).
The B1 cell tion ses large, granular cells of the collecting duct
and r system, with the cells of the population having a buoyant density less than
about 1.045 g/m. The B5 cell population is comprised of debris and small cells of low
granularity and viability and having a t density greater than about 1.091 g/ml.
s of isolating and culturing cell populations
In one embodiment, the SRC+ cell populations and/or organoids described
herein contain and/or are formed from cell populations that have been ed and/or
cultured from kidney tissue. Methods are described herein for separating and ing the
renal cellular components, e.g., enriched cell populations that are contained in the
organoids for therapeutic use, ing the treatment of kidney disease, anemia, EPO
deficiency, r ort deficiency, and glomerular filtration deficiency. In one
embodiment, the cell populations are isolated from freshly digested, i.e., mechanically or
enzymatically digested, kidney tissue or from heterogeneous in vitro cultures of mammalian
kidney cells. Methods for isolating the further bioactive cell populations which comprise the
SRC+ cell populations described herein are further described in the Examples.
The organoids may contain and/or are formed from heterogeneous mixtures
of renal cells that have been cultured in hypoxic culture conditions prior to separation on a
density gradient provides for enhanced distribution and composition of cells in both B4,
including B4’, and B2 and/or B3 fractions. The enrichment of oxygen-dependent cells in B4
from B2 was observed for renal cells isolated from both diseased and non-diseased kidneys.
Without wishing to be bound by theory, this may be due to one or more of the following
phenomena: 1) selective survival, death, or proliferation of ic ar ents
during the hypoxic e period; 2) alterations in cell granularity and/or size in response to
the hypoxic culture, thereby effecting tions in buoyant density and subsequent
localization during density gradient separation; and 3) alterations in cell gene / protein
expression in response to the hypoxic e period, thereby resulting in differential
characteristics of the cells within any given fraction of the gradient. Thus, in one
embodiment, the organoids contain and/or are formed from cell populations enriched for
tubular cells, e.g., B2, are hypoxia-resistant.
Exemplary techniques for separating and ing the cell populations of the
invention include separation on a density gradient based on the differential specific gravity
of different cell types contained within the population of interest. The ic gravity of any
given cell type can be influenced by the degree of granularity within the cells, the
intracellular volume of water, and other factors. Also bed are optimal gradient
conditions for ion of the cell preparations of the instant description, e.g., B2 and B4,
including B4’, across multiple species including, but not limited to, human, canine, and
rodent. In a preferred embodiment, a density gradient is used to obtain a novel enriched
population of tubular cells fraction, i.e., B2 cell population, derived from a heterogeneous
population of renal cells. In one embodiment, a density gradient is used to obtain a novel
enriched population of EPO-producing cells fraction, i.e., B4 cell population, d from a
heterogeneous population of renal cells. In other ments, a density nt is used
to obtain enriched subpopulations of tubular cells, glomerular cells, and elial cells of
the kidney. In one embodiment, both the EPO-producing and the tubular cells are
separated from the red blood cells and cellular debris. In one embodiment, the EPO-
producing, glomerular, and vascular cells are separated from other cell types and from red
blood cells and cellular debris, while a subpopulation of tubular cells and collecting duct
cells are concomitantly separated from other cell types and from red blood cells and cellular
. In one other embodiment, the endocrine, glomerular, and/or vascular cells are
separated from other cell types and from red blood cells and cellular debris, while a
subpopulation of r cells and collecting duct cells are concomitantly separated from
other cell types and from red blood cells and cellular debris.
In one embodiment, the the ids as described herein n and/or
are formed from cell tions ted by using, in part, the OPTIPREP® (Axis-Shield)
density gradient medium, comprising 60% nonionic iodinated compound iodixanol in water,
based on certain key features described below. One of skill in the art, however, will
recognize that any density gradient or other means, e.g., immunological separation using
cell surface markers known in the art, comprising necessary features for isolating the cell
populations of the instant invention may be used in accordance with the invention. It
should also be ized by one skilled in the art that the same cellular features that
contribute to separation of cellular subpopulations via density gradients (size and
granularity) can be exploited to te cellular subpopulations via flow try
(forward scatter = a reflection of size via flow cytometry, and side scatter = a reflection of
granularity). Importantly, the density gradient medium should have low toxicity towards
the specific cells of interest. While the density gradient medium should have low toxicity
toward the ic cells of interest, the instant description plates the use of gradient
mediums which play a role in the selection process of the cells of interest. Without wishing
to be bound by theory, it appears that the cell tions of the instant invention
recovered by the gradient comprising iodixanol are iodixanol-resistant, as there is an
appreciable loss of cells between the g and recovery steps, suggesting that exposure
to iodixanol under the conditions of the gradient leads to elimination of certain cells. The
cells appearing in the specific bands after the nol gradient are resistant to any
untoward effects of iodixanol and/or density gradient exposure. Accordingly, the use of
additional contrast media which are mild to moderate nephrotoxins in the isolation and/or
selection of the cell populations for the organoids described herein is also contemplated. In
addition, the y gradient medium should also not bind to proteins in human plasma or
adversely affect key functions of the cells of interest.
Also described are organoids containing and/or formed from cell populations
that have been enriched and/or ed of kidney cell types using fluorescent activated cell
sorting (FACS). In one embodiment, kidney cell types may be enriched and/or depleted
using BD FACSAria™ or equivalent.
In another embodiment, the organoids contain and/or are formed from cell
tions that have been enriched and/or depleted of kidney cell types using ic
cell g. In one embodiment, kidney cell types may be enriched and/or depleted using
the Miltenyi autoMACS® system or equivalent.
In another embodiment, the organoids may include and/or may be formed
from renal cell populations that have been subject to three-dimensional culturing. In one
embodiment, the methods of culturing the cell populations are via continuous perfusion. In
one embodiment, the cell populations cultured via three-dimensional culturing and
continuous perfusion demonstrate greater cellularity and interconnectivity when compared
to cell tions ed statically. In another embodiment, the cell populations ed
via three dimensional culturing and continuous perfusion demonstrate greater expression of
EPO, as well as enhanced expression of renal tubule-associate genes such as e-cadherin
when compared to static cultures of such cell populations. In yet another embodiment, the
cell populations cultured via continuous perfusion trate greater levels of glucose
and glutamine consumption when compared to cell populations cultured statically.
] As bed herein, low or hypoxic oxygen conditions may be used in the
methods to e the cell populations for the organoids described herein. However, the
methods of preparing cell populations may be used without the step of low oxygen
conditioning. In one embodiment, normoxic conditions may be used.
Those of ordinary skill in the art will appreciate that other methods of
isolation and culturing known in the art may be used for the cells bed herein.
Biomaterials
As described in Bertram et al. U.S. Published Application 20070276507,
polymeric matrices or scaffolds may be shaped into any number of desirable configurations
to satisfy any number of overall system, geometry or space ctions. In one
embodiment, the matrices or scaffolds useful in the present invention may be imensional
and shaped to conform to the dimensions and shapes of an organ or tissue
structure. For example, in the use of the polymeric scaffold for treating kidney disease,
anemia, EPO deficiency, r transport deficiency, or glomerular filtration deficiency, a
three-dimensional (3-D) matrix may be used. A variety of differently shaped 3-D scaffolds
may be used. Naturally, the polymeric matrix may be shaped in different sizes and shapes
to conform to differently sized patients. The polymeric matrix may also be shaped in other
ways to accommodate the l needs of the patient. In another ment, the
polymeric matrix or scaffold may be a biocompatible, porous polymeric scaffold. The
scaffolds may be formed from a variety of synthetic or naturally-occurring materials
including, but not limited to, open-cell polylactic acid (OPLA®), cellulose ether, cellulose,
cellulosic ester, fluorinated polyethylene, phenolic, polymethylpentene, polyacrylonitrile,
polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate,
polycyanoarylether, ter, polyestercarbonate, polyether, polyetheretherketone,
polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin,
polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide,
polypropylene, polystyrene, polysulfide, lfone, trafluoroethylene,
polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated
cellulose, silicone, urea-formaldehyde, collagens, laminins, fibronectin, glycosaminoglycans,
silk, n, te, hyaluronic acid, agarose, or copolymers or physical blends f.
Scaffolding configurations may range from liquid el suspensions to soft porous
scaffolds to rigid, shape-holding porous scaffolds.
] The scaffold may be composed of any material form of biomaterial including
but not limited to diluents, cell carriers, micro-beads, material fragments, scaffolds of
synthetic ition including but not limited to PGA, PLGA, PLLA, OPLA, electrospun
nanofibers and foams of synthetic composition including but not limited to PGA, PLGA,
PLLA, OPLA, electrospun nanofibers.
Hydrogels may be formed from a variety of polymeric materials and are
useful in a variety of biomedical applications. Hydrogels can be described physically as
dimensional networks of hydrophilic polymers. Depending on the type of hydrogel,
they contain varying percentages of water, but altogether do not dissolve in water. Despite
their high water content, hydrogels are capable of additionally binding great volumes of
liquid due to the presence of hydrophilic residues. Hydrogels swell extensively without
changing their gelatinous structure. The basic physical features of hydrogel can be
ically ed, according to the properties of the polymers used and the additional
special equipments of the products.
Preferably, the hydrogel is made of a polymer, a biologically derived material,
a tically derived al or combinations thereof, that is biologically inert and
physiologically compatible with ian tissues. The hydrogel material preferably does
not induce an inflammatory response. Examples of other materials which can be used to
form a hydrogel include (a) ed alginates, (b) ccharides (e.g. gellan gum and
carrageenans) which gel by exposure to monovalent cations, (c) polysaccharides (e.g.,
hyaluronic acid) that are very viscous liquids or are ropic and form a gel over time by
the slow evolution of structure, and (d) polymeric hydrogel precursors (e.g., polyethylene
oxide-polypropylene glycol block copolymers and proteins). U.S. Pat. No. 6,224,893 B1
provides a detailed description of the various polymers, and the chemical properties of such
polymers, that are suitable for making hydrogels for use in accordance with the present
invention.
Scaffolding or biomaterial characteristics may enable cells to attach and
interact with the lding or biomaterial material, and/or may provide porous spaces into
which cells can be entrapped. In one embodiment, the porous scaffolds or erials
useful in the present invention allow for the addition or deposition of one or more
populations or admixtures of cells on a biomaterial configured as a porous scaffold (e.g., by
attachment of the cells) and/or within the pores of the scaffold (e.g., by entrapment of the
cells). In another embodiment, the scaffolds or biomaterials allow or promote for cell:cell
and/or cell:biomaterial ctions within the scaffold to form ucts as bed
herein.
In one ment, the biomaterial useful in accordance with the present
invention is comprised of hyaluronic acid (HA) in hydrogel form, containing HA molecules
ranging in size from 5.1 kDA to >2 x 106 kDa. In another embodiment, the biomaterial useful
in accordance with the t invention is comprised of hyaluronic acid in porous foam
form, also containing HA les ranging in size from 5.1 kDA to >2 x 106 kDa . In yet
another embodiment, the biomaterial useful in accordance with the present ion is
comprised of of a poly-lactic acid (PLA)-based foam, having an ell structure and pore
size of about 50 microns to about 300 microns. In yet another embodiment, the specific cell
populations, preferentially B2 but also B4, provide directly and/or stimulate synthesis of
high molecular weight Hyaluronic Acid through Hyaluronic Acid Synthase-2 (HAS-2),
especially after intra-renal implantation.
The biomaterials described herein may also be designed or adapted to
d to certain external conditions, e.g., in vitro or in vivo. In one embodiment, the
biomaterials are temperature-sensitive (e.g., either in vitro or in vivo). In another
embodiment, the biomaterials are adapted to respond to exposure to enzymatic
degradation (e.g., either in vitro or in vivo). The biomaterials’ response to external
conditions can be fine tuned as described herein. Temperature sensitivity of the organoid
bed can be varied by adjusting the percentage of a biomaterial in the organoid.
Alternatively, erials may be chemically cross-linked to provide r resistance to
enzymatic degradation. For instance, a carbodiimide crosslinker may be used to ally
crosslink gelatin beads thereby providing a reduced tibility to endogenous enzymes.
In one embodiment, the response by the biomaterial to external conditions
concerns the loss of structural integrity of the biomaterial. gh aturesensitivity
and resistance to enzymatic degradation are provided above, other mechanisms
exist by which the loss of material ity may occur in different biomaterials. These
mechanisms may include, but are not limited to thermodynamic (e.g., a phase transition
such as melting, diffusion (e.g., diffusion of an ionic crosslinker from a biomaterial into the
surrounding tissue)), chemical, enzymatic, pH (e.g., pH-sensitive liposomes), ultrasound, and
photolabile (light penetration). The exact mechanism by which the biomaterial loses
structural integrity will vary but typically the mechanism is triggered either at the time of
implantation or post-implantation.
Those of ordinary skill in the art will appreciate that other types of synthetic
or naturally-occurring materials known in the art may be used to form lds as described
herein.
Also described are constructs made from the above-referenced scaffolds or
biomaterials.
Constructs
Also described are organoids that n implantable constructs having one
or more of the cell tions described herein for the treatment of kidney disease,
anemia, or EPO deficiency in a subject in need. In one embodiment, the construct is made
up of a biocompatible material or biomaterial, scaffold or matrix composed of one or more
synthetic or naturally-occurring biocompatible materials and one or more cell tions
or admixtures of cells described herein deposited on or embedded in a e of the
scaffold by attachment and/or entrapment. In n ments, the construct is made
up of a biomaterial and one or more cell populations or admixtures of cells described herein
coated with, deposited on, deposited in, attached to, ped in, embedded in, seeded,
or combined with the biomaterial component(s). Any of the cell populations bed
herein, including enriched cell populations or admixtures thereof, may be used in
combination with a matrix to form a construct.
In another embodiment, the deposited cell population or cellular component
of the construct is a first kidney cell population enriched for oxygen-tunable EPO-producing
cells. In another embodiment, the first kidney cell population contains glomerular and
ar cells in addition to the oxygen-tunable EPO-producing cells. In one embodiment,
the first kidney cell population is a B4’ cell population. In one other embodiment, the
deposited cell population or cellular component(s) of the construct includes both the first
enriched renal cell population and a second renal cell population. In some embodiments,
the second cell population is not ed for oxygen-tunable EPO producing cells. In
another embodiment, the second cell population is enriched for renal tubular cells. In
another embodiment, the second cell population is enriched for renal r cells and
contains collecting duct epithelial cells. In other embodiments, the renal tubular cells are
characterized by the expression of one or more tubular cell markers that may include,
without limitation, megalin, cubilin, hyaluronic acid synthase 2 (HAS2), Vitamin D3 25-
Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), Aquaporin-1 (Aqp1),
Aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA g protein 3
(Gata3), FXYD -containing ion transport regulator 4 (Fxyd4), solute carrier family 9
(sodium/hydrogen exchanger), member 4 4), aldehyde dehydrogenase 3 family,
member B1 (Aldh3b1), de dehydrogenase 1 family, member A3 (Aldh1a3), and
Calpain-8 (Capn8).
In one embodiment, the cell populations deposited on or ed with
biomaterials or scaffolds to form ucts as bed herein are derived from a variety
of sources, such as autologous sources. Non-autologous sources are also suitable for use,
including without limitation, allogeneic, or syngeneic (autogeneic or isogeneic) s.
Those of ordinary skill in the art will iate there are several suitable
methods for depositing or otherwise combining cell populations with biomaterials to form a
construct.
In one embodiment, the constructs as described herein are suitable for use in
the methods of use described herein. In one embodiment, the constructs are suitable for
administration to a subject in need of treatment for a kidney disease of any etiology,
anemia, or EPO deficiency of any etiology. In other embodiments, the ucts are
suitable for administration to a subject in need of an improvement in or restoration of
erythroid homeostasis. In another embodiment, the constructs are suitable for
administration to a subject in need of ed kidney function.
] Also described is a construct for implantation into a subject in need of
improved kidney function comprising: a) a biomaterial comprising one or more
biocompatible synthetic rs or naturally-occurring proteins or peptides; and b) an
ure of mammalian renal cells derived from a subject having kidney disease
sing a first cell tion, B2, comprising an isolated, enriched tion of tubular
cells having a density between 1.045 g/mL and 1.052 g/mL and a second cell tion, B4’,
comprising erythropoietin (EPO)-producing cells and vascular cells but depleted of
glomerular cells having a density between 1.063 g/mL and 1.091 g/mL, coated with,
deposited on or in, entrapped in, suspended in, embedded in and/or otherwise ed
with the biomaterial. In certain embodiments, the admixture does not include a B1 cell
population comprising large granular cells of the collecting duct and tubular system having a
density of < 1.045 g/ml, or a B5 cell population comprising debris and small cells of low
granularity and viability with a density > 1.091 g/ml.
In one embodiment, the construct es a B4’ cell population which is
characterized by expression of a vascular marker. In some embodiments, the B4’ cell
population is not characterized by expression of a glomerular marker. In certain
embodiments, the admixture is capable of oxygen-tunable erythropoietin (EPO) expression.
In all embodiments, the admixture may be derived from mammalian kidney tissue or
ed kidney cells.
In one ment, the construct includes a biomaterial configured as a
three-dimensional (3-D) porous biomaterial suitable for entrapment and/or ment of
the admixture. In another embodiment, the construct includes a biomaterial configured as
a liquid or semi-liquid gel suitable for embedding, attaching, suspending, or g
mammalian cells. In yet another embodiment, the construct includes a biomaterial
configured comprised of a predominantly high-molecular weight species of hyaluronic acid
(HA) in hydrogel form. In another embodiment, the construct includes a biomaterial
comprised of a predominantly olecular weight species of hyaluronic acid in porous
foam form. In yet r embodiment, the construct includes a biomaterial comprised of a
poly-lactic acid-based foam having pores of between about 50 microns to about 300
microns. In still another embodiment, the construct includes one or more cell populations
that may be derived from a kidney sample that is autologous to the subject in need of
ed kidney function. In certain embodiments, the sample is a kidney biopsy. In some
embodiments, the subject has a kidney disease. In yet other embodiments, the cell
population is derived from a non-autologous kidney sample. In one embodiment, the
construct provides oid homeostasis.
Methods of use
Also described are methods for the ent of a kidney disease, anemia, or
EPO deficiency in a subject in need with SRC+ cell populations and/or organoids containing
and/or formed from the kidney cell populations and admixtures of kidney cells described
herein. In one embodiment, the method comprises administering to the t an
id(s) that includes and/or is formed from a first kidney cell tion enriched for
EPO-producing cells. In another embodiment, the first cell population is enriched for EPO-
producing cells, ular cells, and vascular cells. In another embodiment, the
oraganoid(s) may further include and/or may be formed from one or more additional kidney
cell populations. In one ment, the onal cell population is a second cell
population not enriched for EPO-producing cells. In another embodiment, the additional
cell tion is a second cell tion not enriched for EPO-producing cells, glomerular
cells, or vascular cells. In r embodiment, the organiod(s) also includes and/or is
formed from a kidney cell population or admixture of kidney cells deposited in, ted
on, embedded in, coated with, or entrapped in a biomaterial to form an implantable
construct, as described herein, for the treatment of a disease or disorder described herein.
In one embodiment, the organoids are used alone or in combination with other cells or
biomaterials, e.g., hydrogels, porous scaffolds, or native or synthetic peptides or ns, to
stimulate regeneration in acute or chronic disease states.
In another embodiment, the effective treatment of a kidney disease, anemia,
or EPO deficiency in a subject by the methods described herein can be observed through
various indicators of erythropoiesis and/or kidney function. In one embodiment, the
indicators of erythroid homeostasis include, without limitation, hematocrit (HCT),
hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC),
reticulocyte number, reticulocyte %, mean corpuscular volume (MCV), and red blood cell
distribution width (RDW). In one other embodiment, the indicators of kidney function
include, without limitation, serum albumin, albumin to globulin ratio (A/G ratio), serum
phosphorous, serum sodium, kidney size (measurable by ultrasound), serum m,
phosphorous:calcium ratio, serum potassium, proteinuria, urine creatinine, serum
creatinine, blood nitrogen urea (BUN), terol levels, triglyceride levels and glomerular
filtration rate (GFR). Furthermore, several tors of general health and well-being
include, without limitation, weight gain or loss, survival, blood pressure (mean systemic
blood pressure, diastolic blood pressure, or systolic blood pressure), and al endurance
performance.
In r embodiment, an effective treatment with SRC+ cell populations or
bioactive renal cell organoids is evidenced by stabilization of one or more indicators of
kidney function. The stabilization of kidney function is demonstrated by the observation of
a change in an indicator in a subject treated by a method as described herein as compared
to the same indicator in a subject that has not been treated by a method as described
herein. Alternatively, the stabilization of kidney function may be demonstrated by the
observation of a change in an indicator in a subject treated by a method as described herein
as ed to the same indicator in the same subject prior to treatment. The change in
the first indicator may be an increase or a decrease in value. In one embodiment, the
treatment described herein may include stabilization of blood urea nitrogen (BUN) levels in
a subject where the BUN levels observed in the t are lower as compared to a subject
with a similar disease state who has not been treated by the methods as described .
In one other embodiment, the treatment may include stabilization of serum creatinine
levels in a subject where the serum creatinine levels observed in the t are lower as
ed to a subject with a similar disease state who has not been treated by the methods
as bed herein. In r embodiment, the treatment may include stabilization of
crit (HCT) levels in a subject where the HCT levels observed in the subject are higher
as compared to a subject with a similar disease state who has not been treated by the
methods as bed herein. In another embodiment, the treatment may include
stabilization of red blood cell (RBC) levels in a t where the RBC levels observed in the
subject are higher as compared to a subject with a similar disease state who has not been
treated by the methods as described herein. Those of ordinary skill in the art will appreciate
that one or more additional tors described herein or known in the art may be
measured to determine the effective treatment of a kidney disease in the subject.
Methods and Routes of stration
The SRC+ cell populations and/or bioactive cell organoids as bed herein
can be administered alone or in combination with other bioactive components. The SRC+
cell populations and/or organoids are suitable for injection or implantation of incorporated
tissue engineering elements to the interior of solid organs to regenerate tissue.
Also described are methods of providing a bioactive cell id(s)
described herein to a subject in need. In one embodiment, the source of the bioactive cell
may be autologous or neic, syngeneic (autogeneic or isogeneic), and any ation
thereof. In ces where the source is not autologous, the methods may include the
administration of an suppressant agent. Suitable immunosuppressant drugs
include, without limitation, azathioprine, cyclophosphamide, mizoribine, ciclosporin,
tacrolimus hydrate, chlorambucil, lobenzarit disodium, auranofin, alprostadil, imus
hloride, biosynsorb, muromonab, alefacept, pentostatin, daclizumab, sirolimus,
mycophenolate mofetil, leflonomide, basiliximab, dornase α, id, cladribine,
pimecrolimus, ilodecakin, cedelizumab, efalizumab, everolimus, anisperimus, gavilimomab,
faralimomab, clofarabine, rapamycin, siplizumab, saireito, LDP-03, CD4, SR-43551, SK&F-
106615, IDEC-114, 31, FTY-720, TSK-204, LF-080299, A-86281, A-802715, GVH-313,
HMR-1279, ZD-7349, IPL-423323, 11, MT-1345, CNI-1493, CBP-2011, J-695, 0,
L-732531, ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-1g, ER-49890, ER-
38925, ISAtx-247, RDP-58, PNU-156804, LJP-1082, TMC-95A, TV-4710, PTRMG, and
AGI-1096 (see U.S. Patent No. 7,563,822). Those of ordinary skill in the art will appreciate
other suitable immunosuppressant drugs.
The treatment methods as described herein involve the delivery of SRC+ cell
populations and/or organoids described herein. In one ment, direct administration
of cells to the site of intended benefit is preferred.
A variety of means for administering organoids to subjects will, in view of this
specification, be apparent to those of skill in the art. Such methods include, but are not
limited to, intra-parenchymal injection, psular placement, trans-urethral
catheterization, and intra-renal artery catheterization.
Cells can be inserted into a delivery device or e, which facilitates
introduction by injection or implantation into the subjects. In n embodiments, the
delivery vehicle can include natural materials. In certain other embodiments, the delivery
vehicle can e synthetic materials. In one embodiment, the delivery vehicle provides a
structure to mimic or appropriately fit into the organ’s ecture. In other embodiments,
the delivery vehicle is fluid-like in nature. Such delivery devices can include tubes, e.g.,
catheters, for injecting cells and fluids into the body of a recipient subject. In a red
embodiment, the tubes additionally have a , e.g., a syringe, through which the cells of
the invention can be introduced into the t at a desired location. In some
embodiments, ian kidney-derived cell populations are formulated for
administration into a blood vessel via a catheter (where the term "catheter" is intended to
include any of the various tube-like s for delivery of substances to a blood vessel).
Alternatively, the cells can be inserted into or onto a biomaterial or scaffold, including but
not limited to textiles, such as weaves, knits, braids, meshes, and non-wovens, perforated
films, sponges and foams, and beads, such as solid or porous beads, microparticles,
nanoparticles, and the like (e.g., Cultispher-S gelatin beads - Sigma). The cells can be
prepared for delivery in a variety of different forms. For example, the cells can be
suspended in a solution or gel. Cells can be mixed with a pharmaceutically acceptable carrier
or diluent in which the cells of the invention remain viable. Pharmaceutically acceptable
carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion
media. The use of such carriers and diluents is well known in the art. The solution is
preferably sterile and fluid, and will often be isotonic. Preferably, the solution is stable
under the conditions of manufacture and e and preserved t the inating
action of microorganisms such as bacteria and fungi through the use of, for example,
parabens, butanol, phenol, ascorbic acid, thimerosal, and the like. One of skill in the
art will appreciate that the delivery vehicle used in the delivery of the cell populations and
admixtures f of the instant ion can include combinations of the abovementioned
characteristics.
Modes of administration of the organoids containing and/or formed from
isolated renal cell population(s), for example, the B2 cell population alone or admixed with
B4’ and/or B3, include, but are not limited to, intra-parenchymal injection, sub-capsular
placement, or renal artery. Additional modes of administration to be used in accordance
with the present invention include single or multiple injection(s) via direct laparotomy, via
direct laparoscopy, bdominal, or aneous. Still yet additional modes of
administration to be used in accordance with the present invention include, for example,
retrograde and opelvic infusion. Surgical means of administration include one-step
procedures such as, but not limited to, partial nephrectomy and construct implantation,
l nephrectomy, partial pyelectomy, vascularization with omentum ± peritoneum,
multifocal biopsy needle tracks, cone or pyramidal, to cylinder, and renal pole-like
replacement, as well as two-step procedures including, for example, organoid-internal
bioreactor for replanting. In one embodiment, the organoids containing and/or formed
from admixtures of cells are delivered via the same route at the same time. In another
embodiment, each of the organoids are delivered separately to specific locations or via
specific methodologies, either simultaneously or in a ally-controlled manner, by one
or more of the methods described .
The riate cell or organoid implantation dosage in humans can be
determined from existing information relating to either the activity of the ids, for
example EPO production, or extrapolated from dosing studies conducted in preclinical
studies. From in vitro culture and in vivo animal experiments, the amount of organoids can
be quantified and used in calculating an appropriate dosage of implanted al.
Additionally, the patient can be monitored to determine if additional implantation can be
made or implanted material reduced accordingly.
One or more other ents can be added to the organoids comprising
and/or formed from cell populations and admixtures thereof of the instant invention,
including selected extracellular matrix components, such as one or more types of collagen
or hyaluronic acid known in the art, growth factors, and/or cytokines, including but not
limited to VEGF, PDGF, TGFβ, FGF, IGF, platelet-rich plasma and drugs.
Those of ordinary skill in the art will appreciate the various methods of
administration suitable for the ids described herein.
Articles of Manufacture and Kits
Also described are kits comprising the polymeric matrices and scaffolds
bed herein and related als, and/or cell culture media and instructions for use.
The instructions for use may contain, for e, ctions for culture of the cells in the
formation of the SRC+ cell pouplations or organoids as described and/or administration of
the SRC+ cell pouplations or organoids. In one embodiment, described is a kit comprising a
scaffold as described herein and instructions. In yet another ment, the kit includes
an agent for detection of marker expression, reagents for use of the agent, and instructions
for use. This kit may be used for the purpose of determining the regenerative prognosis of a
native kidney in a subject following the implantation or administration of an organoid(s)
described herein. The kit may also be used to determine the biotherapeutic efficacy of an
id(s) described .
Another embodiment is an article of manufacture containing organoids
containing and/or formed from bioactive cells useful for treatment of subjects in need. The
article of cture ses a container and a label or package insert on or associated
with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The
containers may be formed from a y of materials such as glass or plastic. The container
holds a composition which is effective for treating a condition and may have a sterile access
port (for example the container may be a solution bag or a vial having a stopper pierceable
by an injection needle). The label or package insert indicates that the organoid is used for
treating the particular condition. The label or package insert will further comprise
instructions for administering the organoid to the t. Articles of manufacture and kits
comprising atorial therapies described herein are also contemplated. Package insert
refers to instructions customarily ed in commercial packages of eutic products
that n information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such therapeutic products. In one
embodiment, the package insert indicates that the organoid(s) is used for ng a disease
or disorder, such as, for example, a kidney disease or disorder. It may further include other
materials desirable from a commercial and user standpoint, including other buffers,
diluents, filters, needles, and syringes. Also described are kits which are useful for various
purposes, e.g., for assessment of regenerative outcome. Also bed are kits which
contain detection agents for urine-derived vesicles and/or their contents, e.g., c acids
(such as miRNA), es, exosomes, etc., as bed herein. Detection agents e,
without limitation, nucleic acid primers and probes, as well as antibodies for in vitro
detection of the desired target. As with the article of manufacture, the kit comprises a
container and a label or package insert on or associated with the container. The container
holds a composition comprising at least one detection agent. Additional containers may be
included that contain, e.g., diluents and buffers or control detection agents. The label or
package insert may e a description of the composition as well as instructions for the
intended in vitro, prognostic, or diagnostic use.
The following examples are offered for rative purposes only, and are not
intended to limit the scope of the present invention in any way.
EXAMPLES
e 1 - Isolation of Selected Renal Cell + (SRC+) cell populations: Isolation of
endothelial cells from renal biopsy
The following method provides an e of endothelial cell isolation
following enzymatic digestion using a previously d Collagenase/Dispase digestion
protocol 2, 3, 5. This method has been applied to diseased ZSF1 rat kidneys and to a kidney
biopsy obtained from a ensive human ESRD patient on dialysis.
Endothelial Cells (EC), vascular and lymphatic origin:
Digested kidney(s) using standard operating procedures for kidney cell
isolation, as bed infra., are filtered through a 100µm Steriflip filter (Millipore). The
remaining cell sion is neutralized with DMEM containing 5% FBS and then washed by
centrifugation at 300xg for 5 minutes. The cell pellet recovered through the 100 µm filter is
pended in fully supplemented EGM-2 growth medium (Lonza) and plated onto
fibronectin coated dishes at a cell density of 25K/cm2. The cultures are fed every 3 days.
When the EC es are 80-90% confluent, they are trypsinized and d. The
trypsinized cells are labeled with CD31 (PECAM) primary antibody and positively selected
using Miltenyi anti-CD31 microbeads. The CD31+ sorted cells are washed, counted, plated
and seeded at a density of 10K/cm2 in fully supplemented EGM-2 medium. Endothelial cell
morphology should be evident post twenty four hours and these cells can be expanded
through multiple passages. The vascular and/or lymphatic composition may be ed
using lineage-specific antibodies (e.g. VEGF3 –vascular capillary; LYVE1- lymphatic); the
specific endothelial subpopulation can be further selected/sorted using the same cell
surface phenotyping markers, using the yi micro-bead selection method.
After expansion and purification, the endothelial cell ent of the NKA
SRC can be enriched to a larger percentage (>30%) than the natural frequency as observed
through buoyant density onation (<2%). In one embodiment, for controlling the active
biological ingredients or composition of NKA, the purified EC can be combined at a selected
frequency with the B2 and/or B2-B3-B4 fractions previously described from buoyant y
gradient fractionation (e.g. 60% B2-B4 + 40% EC) prior to transplantation.
elial cell sorting using magnetic micro-beads:
Cells harvested from the fibronectin coated flasks in EGM2 medium are
stained with mouse uman CD31 primary antibody at a concentration of 0.4µg/million
cells/100µl in EGM2 medium w/o supplements for 20 minutes at 4ºC. After 20 minutes the
cells are washed and re-suspended in 12mls of EGM2 medium w/o supplements and 200 µl
of goat anti-mouse IgG1. Miltenyi eads are added at and incubated for an additional
s at 4ºC protected from light. After 20 minutes the cells are washed twice via
centrifugation (5min @ 300xg) and re-suspended in 12mls [10 million cells/ml] and purified
using a Miltenyi auto-MACS instrument (Double Positive Selection in Sensitive Mode
program).
In the e described in endothelial cells were selected (CD31+
selection) from primary ZSF1 cells. The 3.6 million cells recovered from this process were
counted and sub-cultured onto two T175 fibronectin coated flask at a concentration of 10K
per cm2. The cells were cultured for four days at 21%O2 and harvested at 85% confluency.
Following this selection and culture , the cells were d and 9.5 million cells were
recovered. The sample ted for phenotypic analysis by FACS was ~ 90% positive for
CD31 (, right FACS panel).
Human CKD Endothelial Cell Isolation, Characterization and Expansion
Human kidney cell culture:
Human kidney cells (See Table 1 below for donor information) were ed
using standard operating procedures of human kidney primary cell cnzymatic isolation and
culture, as described infra. Briefly, cells were isolated and cultured at 25K/cm2 in either T/C
treated flask with standard KGM or on fibronectin coated flask with EGM2 fully
supplemented medium. The cells were grown for three days in a 21% oxygen environment
and then the medium was changed and the cultures were moved to a 2% oxygen
environment for O/N exposure. After four days the cells were imaged to analyze culture
morphology (). They were then harvested and counted using standard methods (see
HK027 Batch Record). A sample was collected and stained to determine the percentage of
CD31+ endothelial cells (. Endothelial cells were sorted using a mouse anti-human
CD31 primary antibody (BD biosciences) and magnetic microbeads (Miltenyi). CD31+ cells
were then re-plated onto fibronectin coated flasks at a density of 10K/cm2 and cultured an
additional four days ( cell culture morphology). The cells were then counted and a
sample was collected to determine the percentage positive endothelial cells using a mouse
anti-human CD31 primary and FACS analysis (.
Table 1. Human Kidney Donor Information:
Sample ID Cause of Death BUN sCrea HCT HB Key Features
HTN for 20yrs, ESRD, on
HK027 ICH 72 11.1 42.8 14
dialysis since 07’
In conclusion, the initial culture of diseased ZSF1 cells on fibronectin yielded
a 9% CD31+ culture (<2% from ated). At p1, the 9% CD31+ fraction sub-cultured on
fibronectin yielded ~ 90% CD31+ cells that expanded nearly 3-fold from p0 to p1. Initial
culture of human CKD cells on either T/C treated or fibronectin coated flask yielded
approximately the same percentage of CD31 ve cells ( 2.1% ed to 2.7%). As
shown, it is possible to sub-culture the positively selected CD31 positive cells on fibronectin
coated flasks in fully supplemented EGM2 medium and increase the tage CD31
positive cells by 13 fold (35.5% from 2.7%) after passage 1.
ion of SRC+ cell populations: Examples of the isolation of further ive cell types:
A. Renal Epithelia: Ongoing activities are ting the entation of
the various epithelial cell compartments (parietal, proximal r, loop of Henle, distal
tubular cells) in the fractions ed h buoyant density gradients. More specifically,
the lineage tracing studies provide direct and selective confirmation of Six2+ epithelial cell
isolation; co-labeling of Six2 with a marker specific to a nephron epithelial compartment
(See Tables 2-3) confirms cell-specific detection (See Table 4). Table 4 provides a limited
panel of lial markers that characterizes p0 ZSF1 culture (combined fractions B2-B3-B4)
as a e of proximal, distal and collecting duct cell types. Using the same cell surface
detection antibodies listed in Tables 2-4, the enrichment of these lized epithelial cell
compartments through culture selection as previously bed (culture conditions along
with magnetic sorting) is contemplated.
Table 2. Marker panel for tissue specific nephron staining
Proximal Tubule Distal Tubule
n Convoluted Straight Loop Ascending Macula Convoluted Collecting
of Thick Densa Duct
Henle Limb
Cytokeratin
CK18 Pos Pos Pos Pos Pos Pos Pos
CK 7 Neg Neg Pos Pos Neg Pos Pos-ind
Other
E-CAD Neg Neg Pos Pos Pos Pos Pos
N-CAD Pos Pos Neg Neg Neg Neg Neg
rin Pos Pos Neg Neg Neg Neg Neg
Aquaporin Neg Neg Neg Neg Neg Neg Pos
THP Neg Neg Neg Pos Pos Pos Pos
Lectins
LTA Pos Pos Pos Neg Neg Med-ind
DBA Neg Neg Pos Pos Pos Pos-ind
UEA Neg Neg Neg Neg Neg Pos-ind
Pos= positive, Pos-ind = positive cell in selected population of cells, Med –ind = medulary
ting ducts stain pos
Table 3. Antibody sourcing
Antigen Isotype Manf Cat# Conc. Target
CK18 Ms IgG1 abCAM Ab668 1mg/ml Intracellular/Nephro n
CK7 Ms IgG1 abCAM Ab9021 1mg/ml Intracellular/DT
Aquaporin 1 Ms abCAM Ab9566 0.2mg/ml ne/PT
IgG2b
Aquaporin 2 rb IgG abCAM Ab6415 0.1mg/ml Membrane/CD
Tamm Horsfall Rb IgG Santa Sc- 0.2mg/ml Membrane/DT
glycoProtein Cruz 20631
Antigen Isotype Manf Cat# Conc. Target
LTA (Biotinylated) ---- Vector B-1325 2mg/ml Membrane/PT
DBA (Biotinylated) --- Vector B-1035 5mg/ml ne/DT/CD
UEA (Biotinylated) --- Vector B-1065 2mg/ml Membrane/CD
ECAD Ms BD 610182 /m
Membrane/DT/CD
IgG2a l
NCAD Ms IgG1 BD 610921 0.25mg/m Membrane/PT
Isotype ctrl Ms IgG1 BD 557273 0.5mg/ml
Isotype ctrl Ms BD 553454 0.5mg/ml
IgG2a
Isotype ctrl Ms BD 557351 ml
IgG2b
Isotype ctrl Gt IgG Invitrog 02-6202 1mg/ml
Isotype ctrl Rb IgG Invitrog 02-6102 2mg/ml
Secondary antibodies were Alexa 647 molecular probes goat anti mouse IgG , Goat anti
Rabbit IgG, and Donkey anti Goat IgG, Strep-Avidin A647. Staining following SOP’s with
1ug/ml/1x106 cells
Table 4. Preliminary ZSF1 p0 cell specific detection
Marker Compartment ZSF1 p0 % positive
AQP2 Collecting duct 42.36
DBA Distal Tubule 56.93
LTA Proximal/Loop/IMCD 39.62
CK18 Pan-epithelial 68.39
B. Glomerular Derived Cells (parietal epithelial and podocytes): Anatomical
exclusion of the pelvis improves the isolation of the ular fraction. Using standard
operating procedure for kidney digest, described infra., cells are isolated and filtered
through a 100µm Steri-flip filter (Millipore), and cell particles/clumps larger than 100µm
(contains the glomerular on) are re-digested for onal 20minutes. The digested
fraction is neutralized with DMEM medium ning 10% FBS and washed by
centrifugation (300xg for 5min). The re-suspended cell pellet is cultured in VRADD medium6
(DMEM/F12, 1uM All Trans Retinoic Acid me, Cambridge, MA), 0.1um
Dexamethasone (Sigma-Aldrich), 10-100nM Vitamin D (1,25(OH)2D3 to promote podocyte
culture, or a parietal epithelial friendly serum free medium such as (50:50 DMEM/KSFM)
fully supplemented without FBS, ed on Collagen Type 1 coated T/C plates. Cells are
seeded at a density of 25K/cm2 and subculture until cell out-growth appears. Expand and
sort outgrowth cells using primary antibody bound to Miltenyi micobeads at passage 1 using
either PEC specific markers (such as but not limited to Claudin-1) or progenitor specific
markers (e.g. CD146, CD117, SOX2, Oct 4A, CD24, CD133) or mesangial specific markers
(such as but not limited to Smooth Muscle Actin, Vimentin, Myocardin, Calbindin) or
glomerular capillary endothelial cells (e.g. VEFG3 or CD31 or LIVE1). When harvested at
optimal cell yields and ed with the B2 component at a greater than l
frequency, a higher percentage of glomerular-derived cells can be applied to patients
afflicted with glomerular disease.
C. Collecting Duct Epithelial: Anatomical exclusion of the cortex and
medullary region of the kidney improves the isolation of cells located in or near the pelvis.
Standard operating procedures for isolation and expansion and harvest of y kidney
cells for rodent, canine and human, as described infra., is followed for isolation and
expansion of collecting duct lial cells. rd operating procedures for fractionating
sub-populations of y cultured kidney cells enrich for cells of the collecting ducts in cell
fraction B1, are described herein infra. These cells contain the highest percentage of
collecting duct epithelial cells based on makers such as Aquaporin 2 and Dolichos Biflorus
Agglutinin (DBA). Alternatively, we have adapted a a/inter-medullary ting duct
(IMCD) culture protocol from Dr. Ben Humphreys (REGM media supplemented with EGF;
unpublished data). In any of these scenarios, a cell on enriched for collecting duct
epithelia can be used in combination with B2 component at higher than natural frequencies
or they can be expanded using standard KGM or equivalent and used to target ed
etiologies ated with urine concentration and may better substantiate the active
biological ingredients that could be applied to abnormalities and/or diseases of the renal
pelvis (e.g. hydro-nephrosis, reter obstruction).
e 2 - Generation and characterization of Organoids from SRC and SRC+ populations
Self-generated Organoid/Spheroid Formation
Organoids were generated following primary kidney culture, expansion and
buoyant density gradient centrifugation to isolate SRC (standard operating procedures for
generating NKA, as bed infra). Briefly, SRC were re-suspended in 100ml of renal cell
growth medium at a concentration of [1 x106 cells/ml] in spinner flasks (Corning) for 24-
48hrs on a magnetic r set at 80rpm (. Cell organoids/spheroids consist of clusters
of cells ranging in size from 50-125µM. Cell number per organoid can vary based on cell
type and size prior spinner flask e. Organoid/spheroids have been generated from
both rat and human SRC and express a tubular epithelial phenotype (.
Organoid Function and Tubulogenesis
The ability of SRC to form tubes may be applied to assess potency of NKA.
This assay was applied to the SRC organoids cultured in a 50:50 mixture of Collagen
1/Collagen IV n in 3D. Upon immuno-fluorescent staining of the cultured organoids,
the ant tubes ue to express an epithelial phenotype (.
Organoid Plus
The ability of SRC to form self-generated spheroids may prove ageous
when applying combinations of other cell types. Tubulogenesis may be enhanced with the
addition of a vascular or stem cell component. By adding a selective cell population in
culture with the SRC population during the organoid formation period, a functional unit may
be formed recapitulating key cell signaling pathways activated during regenerative
outcomes. Examples of such cell-cell interactions include but are not limited to epithelialmesenchymal
signaling events known to be pivotal during organogenesis (see Basu &
Ludlow 2012, Developmental ering the kidney-leveraging genic principles for
renal regeneration. Birth Defects Research Part C 96:30-38). While SRC are generated using
standard procedures, an endothelial cell line (HuVEC) was used as an example of an
organoid(+) combination. Eighty million SRC were labeled with a ne dye (Invitrogen
DiL red label) and added to 20 million HuVEC labeled with a different color membrane dye
(Invitrogen DiO green label) in 100ml of RCGM medium in 125ml spinner flasks at 80rpm for
48hrs (. An SRC organoid population alone was also started as control. Upon
formation of organoids, tubulogenesis assays were set-up in-vitro within Col I/IV 3D gels to
ensure the ability to form tubes with and t potential concomitant vascularization
(.
Example 3 - terization/biodistribution of SRC organoids in rodent models of kidney
disease
ZSF1 acute study evaluating bio-distribution and cell retention of SPIO labeled
organoids over a 48 hr period. Six 40+ week old ZSF1 rats were injected into the
hyma with 2.5x106 SPIO Rhodamine labeled cells (representing approximately 25000
self-generated ids in PBS) at a concentration of 50x106/ml in left caudal pole. (Figure
9). Three animals were harvested at intervals of 24 and 48 hrs post implantation. s
were evaluated by MRI and ogically using Prussian blue and H&E staining method for
cell retention and bio-distribution (Figure 10 & 11).
RESULTS
Organoids were readily labeled and traced following targeted delivery.
Organoid treatment was well tolerated with no morphological alterations observed in the
tubular or glomerular compartments. Multifocal Clusters of epitheliod cells (staining
positive by an blue) were frequently observed in the renal cortex of left kidney
(intra/inter tubular) at 24 hours post injection and to a lesser extent after 48 hours.
Example 4 - In vivo studies to trate therapeutic efficacy of SRC+ and SRC/SRC+
derived organoids: CKD (5/6 nephrectomy) immune-compromised rodent studies
Human-derived SRC+ and SRC/SRC+ organoids were evaluated for
eutic efficiency using the 5/6-nephrectomy model of chronic kidney disease in
immune-compromised rodents (NIHRNU (nude) rats; athymic rats). Establishment of the
disease state, ent ention modalities and clinical evaluation of therapeutic
response was as previously described (Genheimer et al., 2012. Molecular characterization of
the regenerative response induced by intrarenal lantation of selected renal cells in
rodent model of chronic kidney disease. Cells Tissues Organs 196: ).
Cell ion and selection: Human renal cells were isolated, expanded from
biopsy as described in Presnell (2011). Cells were sub-cultured and cryopreserved after
passage 2 in cryopreservation buffer (80% HTS/10%DMSO/10% FBS) at a concentration of
(20x106 cells/ml/vial) using a freezing rate of -1°C/min down to -80°C and then transferred
to the liquid nitrogen freezer for longer storage. Following cryopreservation, the cells were
quickly thawed at 37°C to assess recovery and ity using standard Trypan blue exclusion
method. Cells were then seeded onto tissue culture vessels at 3000 cells/cm2 and cultured
for 4 days under normoxic conditions (21%O2/5% CO2/37°C) in renal cell growth medium.
After 4 days the culture medium was changed and the cultures were incubated overnight in
a lower oxygen environment 5% CO2/37°C) prior to cell harvest and selection of SRC
by density gradient tion. Human Umbilical Vein Endothelial Cells (HUVEC) cultures
(Lonza 2389, CAT# CC2517) were expanded from early passage following eservation
until passage 3 in EGM2 fully supplemented medium. Cells were harvested and viability was
assessed using standard Trypan blue exclusion method.
Organoid Formation: Organoid cultures were established by 1) resuspending
human SRC and HUVEC’s to a concentration of 2x106 cells/ml in their tive mediums 2)
combining equal volumes of each cell preparation 25mls 6 cells/ml) into a 125ml
spinner flask ng). To the combined culture, 25mls of each medium was added to the
flasks for a final cell concentration of 1x106 cells/ml in 100mls. The flask was placed on a
magnetic stirrer in the incubator at a speed of 80RPM and cultured for 24 hrs.
Test Articles: The organoid dose was adjusted such that approximately (2- 5
x106 cells/50µl) were to be administered per rat kidney. id numbers were adjusted
to the concentration above in a larger volume of 0.5mL of DPBS to t for any loss
during shipping. An extra tube was sent as ement. The doses were sent via FEDEX to
the implantation facility in two 0.5ml microcentrifuge tubes using a CREDO cube shipper at
4-8°C.
Test Article Delivery: The remnant left kidney was ed through left flank
incision. Prior to delivery, organoids were gently resuspended by tapping or flicking the
base of the tube (no vortexing). The organoids were aspirated with an 18G blunt tip needle
into the syringe. The needle was then changed to a 23G cutting needle for ry.
ed delivery was carried out by injecting 50µL into the cortico-medullary region of the
kidney. Untreated control animals remained untreated and underwent no procedure.
RKM Model Procedures: Male NIHRNU rats 8-12 weeks old (approx. 200 g
average weight) ent 2 step nephrectomy procedures as follows: For each individual
animal the right kidney was removed and weighed, recovery allowed for 1 week, followed
by resection of tissue from caudal and cranial poles of left kidney which was also weighed.
A 2-3 week recovery and acclimation period followed prior to the beginning of the studies.
Following the ation period, blood and urine were collected for 2 utive weeks
and ed. Subsequent sampling was performed every 2 weeks thereafter. All rodents
were fed a commercially ble feed, and water was provided ad libitum.
Animals were monitored post-nephrectomy with some evidence of increasing
urine protein/urine protein creatinine (UPC) and were treated with human ids at 12
weeks post-nephrectomy. Table 5 below provides an overview of the study.
Table 5: Study Design
Nephrectomy DESCRIPTION
Batch (Treatment) Assessments
Daily
Organoid Prototype • Survival
Animals
(n=4)
(59% KMR) Bi-Weekly
• Body weights
Tx 12 weeks • Serology and
post hematology
nephrectomy • Urinalysis Panel
Term of Necropsy
Observation
Untreated • Gross pathology
204 days post
(n=8) observations
Nephrectomy • Abdomen opened
119 days post- and whole animal
Tx fixed if duled
death
Measurements: Body weights (g) were ted on a biweekly basis). Serum biochemical
and hematological evaluations were conducted for the duration of study on a bi-weekly
basis. ysis was performed biweekly At scheduled necropsy for study animals gross
observations were made, the remnant kidney excised and fixed, and animal remains fixed in
formalin. All animals and tissues were collected as needed, weighed, measured and
prepared for ogical processing. Methods used in histological evaluations of kidney
were performed. KMR is kidney mass reduction.
RESULTS:
The efficacy of human NKO was demonstrated in a renal insufficiency model
in athymic rats.
RKM Model Overview
Disease progression within the athymic KMR model was consistent with
usly described nephrectomy models and CKD-related sequelae ing disruption of
kidney protein handling, azotemia, hypercholesterolemia, anemia, and developing uremia.
The e of NIHRNU Nephrectomy model in regard to disease state ed was
sensitive to the amount of kidney tissue resected during mass reduction and the animals
utilized here demonstrated a slowly progressing early stage disease state based upon clinical
pathology monitoring and terminal ogical evaluations.
NKO Prototype Effect on In-Life Clinical Pathology
Clinical pathology measures relevant to kidney function were examined pretreatment
and compared in a paired fashion to measures taken prior to end of study. From
this comparison, as summarized in the table below, there were changes tent with
disease progression over the 4 month study in untreated control animals. Significant
ences were noted in paired BUN, Hct, Hgb, RBC, WBC, sChol, sProt, sAlb, uPro, UPC,
and spGrav. Changes in these s are typical of early stage CKD and indicative of
decreases in renal function that have yet to result in acute azotemia, end stage loss of
tubule filtration/concentration and phosphotemia. In animals treated with NKO, changes in
BUN, Hct, Hgb, RBC, WBC, and spGrav were not significant as would be ted based
upon untreated control animal outcomes (see Table 6 below).
Table 6. Treatment Group Clinical Measures Pre- and Post-Treatment.
Paired t-
Time 0 End of Study Significant
test
e Group Average SD Average SD Change p-value
sCre Control 0.48 0.07 0.51 0.21 0.598
Organoid 0.43 0.05 0.58 0.29 0.298
BUN Control 32.4 4.2 47.1 19.5 YES 0.045
Organoid 31.8 5.7 56.5 27.8 0.09
Hct Control 46.56 2.48 39.99 2.71 Yes <0.0001
Organoid 42.48 1.38 39.1 3.71 0.26
Hgb Control 14.85 0.92 12.93 0.83 Yes 0.0005
Organoid 14.18 0.25 12.63 1.05 0.0518
Paired t-
Time 0 End of Study Significant
test
Measure Group Average SD Average SD Change p-value
RBC l 8.93 0.49 7.88 0.59 Yes 0.0007
Organoid 8.51 0.17 7.76 0.68 0.124
WBC Control 6.13 0.6 8.74 2.15 Yes 0.016
Organoid 6.93 0.43 7.4 0.76 0.406
sPhos Control 7.28 0.88 6.38 1.1 0.15
Organoid 6.75 0.51 7.7 1.87 0.48
sChol Control 89.1 9.6 139 47.4 Yes 0.01
Organoid 77.8 4.1 142.5 15.4 Yes 0.005
sProt Control 5.99 0.24 5.28 0.28 Yes 0.001
id 5.83 0.26 5.38 0.1 Yes 0.04
sAlb Control 3.15 0.11 2.64 0.23 Yes 0.0003
Organoid 2.95 0.06 2.58 0.05 Yes 0.0004
uPro Control 371.8 211.5 625.6 182.7 Yes 0.007
Organoid 252 127.7 674.9 33.63 Yes 0.008
UPC Control 4.47 2.08 11.25 3.65 Yes <0.0001
Organoid 4.39 0.95 11.41 0.66 Yes 0.01
uCre Control 83.4 22.9 56.3 13.8 0.06
Organoid 55.75 17.86 58.3 3.5 0.87
uNa Control 134.38 52.63 61.13 12.88 Yes 0.005
Organoid 110.25 34.79 50.67 6.74 0.116
uK l 116 35.66 84.75 5.24 0.104
Organoid 79.5 22.75 87.67 8.56 0.549
uChlor Control 163.88 20.7 86.75 16.09 Yes 0.01
Organoid 133.25 29.28 79.33 7.02 0.24
spGrav Control 1.034 0.009 1.025 0.003 Yes 0.02
Organoid 1.027 0.009 1.026 0.005 0.73
Measure Represents Association with CKD
BUN urea handling decreased eGFR, tubular dysfunction
early indicator of decreased kidney function;
Hgb measures of anemia
EPO dysfunction
associated with increased risk for CKD
WBC* inflammation progression
decrease indicative of ed tubule
spGrav urine tration function
*the WBC here would not reflect a T-cell mediated response as these animals are athymic
Histological Overview of Kidneys from Model
Histopathology trated that control animals had progressed to mild,
developing nephropathy by terminal ice.
Injury-related findings were primarily tubular in nature with atrophy, dilation,
and proteinaceous casts usually mild in severity. Tubular basophilia and fibrosis were often
minimal and interstitial inflammation was typically minimal or absent. Glomerular changes
were minimal in ty.
Capsular fibrosis/inflammation was typically only minimal in severity and was
characterized by little inflammatory cellular content; the reaction may have been
diminished by the incomplete immune system in the athymic nude rat. Mineralization and
lymphocytic infiltration were of low incidence and severity in Batch 1 animals. Occasional
focal mineralization, common in laboratory rats, was observed.
There were no significant differences noted n untreated and NKO
treated groups.
Table 7
e Normal ted NKO Treated
Tubulo-Interstital Injury 0.0 + 0.1 1.4 ± 0.4 1.8 ± 0.4
Glomerular Injury 0.0 0.8 ± 0.3 1.2 ± 0.5
NKO Effect on Survival
All animals (59% KMR) survived until end of study (119 days post-Tx and 204
days post-nephrectomy) therefore no discernable survival benefit of NKO ent was
Engraftment of Human NKO Detected with Staining Methods at EOS
As this model allows xenogeneic application of a human product, tracking of
human cells within the rat kidney was possible through use of human cell fying
antibodies. Staining for human HLA1 within the ound of the RKM rat kidney
identified cells 4 months post-treatment, see Figure 12. Identification of staining considered
consistent with engraftment of human cells was confirmed by two independent reviewers.
lly 1 or 2 cells were identified in one of four sections stained for each animal and were
incorporated into tubules, although one r was fied within the interstitium.
CONCLUSIONS:
• Organoid (NKO) delivery mitigated significant changes associated with kidney disease
progression in control animals and included measures effected by
eic/autologous NKA deliveries to disease .
• Effected measures were consistent with those observed with syngeneic/autologous
NKA deliveries to disease models – es of anemia (Hct, Hgb, RBC),
inflammation (WBC), urine tration (spGrav) and possibly azotemia (BUN).
• Human cells in very low numbers were detected approximately 4 months post-
treatment with NKO prototype.
• Nephrectomy of nude rats (59% KMR, average) ed in a state of early stage and
chronically progressive renal insufficiency characterized by developing anemia,
proteinuria, dyslipidemia and possible tions of early stage azotemia.
• All animals survived until end of study.
• Procedures utilized to deliver human organoids to the RKM model were well
tolerated.
Example 5- Isolation & Characterization of Bioresponsive Renal Cells
A case of idiopathic progressive chronic kidney disease (CKD) with anemia in
an adult male swine (Sus scrofa) provided fresh diseased kidney tissue for the assessment of
cellular composition and characterization with direct comparison to age-matched normal
swine kidney tissue. Histological examination of the kidney tissue at the time of harvest
confirmed renal disease characterized by severe diffuse chronic interstitial fibrosis and
crescentic glomerulonephritis with ocal fibrosis. Clinical chemistry confirmed azotemia
(elevation of blood urea nitrogen and serum nine), and mild anemia (mild reduction in
hematocrit and depressed hemoglobin ). Cells were isolated, expanded, and
characterized from both diseased and normal kidney tissue. As shown in Figure 1 of Presnell
et al. WO/2010/056328 (incorporated herein by reference in its entirety), a Gomori’s
Trichrome stain highlighs the fibrosis (blue ng indicated by arrows) in the diseased
kidney tissue compared to the normal kidney tissue. Functional tubular cells, expressing
cubulin:megalin and e of receptor-mediated albumin transport, were propagated
from both normal and diseased kidney tissue. Erythropoietin (EPO)-expressing cells were
also present in the cultures and were retained through multiple passages and freeze/thaw
cycles. Furthermore, lar analyses confirmed that the EPO-expressing cells from both
normal and diseased tissue responded to hypoxic ions in vitro with HIF1α-driven
induction of EPO and other hypoxia-regulated gene targets, including vEGF. Cells were
isolated from the porcine kidney tissue via enzymatic digestion with collagenase + dispase,
and were also isolated in separate experiments by performing simple ical digestion
and explant culture. At passage two, explant-derived cell cultures containing epo-expressing
cells were subjected to both atmospheric (21%) and varying hypoxic (<5%) culture
conditions to determine whether re to hypoxia culminated in upregulation of EPO
gene expression. As noted with rodent cultures (see Example 3), the normal pig displayed
oxygen-dependent expression and regulation of the EPO gene. Surprisingly, despite the
uremic / anemic state of the CKD pig (Hematocrit <34, Creatinine >9.0) EPO expressing cells
were easily isolated and propagated from the tissue and expression of the EPO gene
remained hypoxia ted, as shown in Figure 2 of Presnell et al. WO/2010/056328
(incorporated herein by reference in its entirety). As shown in Figure 3 of Presnell et al.
WO/2010/056328 (incorporated herein by reference in its entirety), cells in the propagated
cultures trated the ability to rganize into tubule-like structures. As shown in
Figure 4 of Presnell et al. WO/2010/056328 (incorporated herein by reference in its
entirety), the ce of functional tubular cells in the culture (at passage 3) was confirmed
by observing or-mediated uptake of FITC-conjugated Albumin by the cultured cells.
The green dots (indicated by thin white ) represent endocytosed fluoresceinconjugated
n which is mediated by tubular pecific receptors, Megalin and
Cubilin, indicating protein reabosroption by functional tubular cells. The blue staining
(indicated by thick white arrows) is Hoescht-stained nuclei. Taken together, these data
suggest that functional tubular and endocrine cells can be isolated and propagated from
porcine renal tissues, even in renal tissues that have been severely compromised with CKD.
Furthermore, these findings t the advancement of autologous cell-based therapeutic
products for the treatment of CKD.
In addition, EPO-producing cells were isolated enzymatically from normal
adult human kidney (as described above in Example 1). As shown in Figure 5 of Presnell et
al. WO/2010/056328 (incorporated herein by nce in its entirety), the ion
procedure resulted in more relative EPO expression after isolation than in the initial tissue.
As shown in Figure 6 of Presnell et al. WO/2010/056328 (incorporated herein by reference
in its entirety), it is possible to maintain the human EPO producing cells in culture with
retention of EPO gene expression. Human cells were cultured/propagated on plain tissueculture
d plastic or plastic that had been coated with some extracellular matrix, such
as, for instance, fibronectin or collagen, and all were found to support EPO expression over
time.
Example 6 – Isolation & enrichment of specific bioreactive renal cells
Kidney cell isolation: Briefly, batches of 10, 2-week-old male Lewis rat kidneys
were obtained from a commercial supplier (Hilltop Lab Animals Inc.) and shipped ght
in Viaspan vation medium at a temperature around 4°C. All steps described herein
were carried out in a biological safety cabinet (BSC) to preserve sterility. The kidneys were
washed in Hank’s balanced salt solution (HBSS) 3 times to rinse out the Viaspan preservation
medium. After the third wash the remaining kidney capsules were removed as well as any
ing ltissue. The major calyx was also removed using micro dissection
techniques. The kidneys were then finely minced into a slurry using a sterile scalpel. The
slurry was then transferred into a 50ml conical centrifuge tube and weighed. A small
sample was collected for RNA and placed into an RNAse-free sterile 1.5ml micro-centrifuge
tube and snap frozen in liquid nitrogen. Once , it was then transferred to the -80
degree r until analysis. The tissue weight of 10 juvenile kidneys equaled
approximately 1 gram. Based on the weight of the batch, the digestion medium was
ed to deliver 20mls of digestion medium per 1 gram of tissue. Digestion buffer for this
procedure contained 4 Units of Dispase 1(Stem Cell Tech) in HBSS, 300Units/ml of
Collagenase type IV (Worthington) with 5mM CaCl2 (Sigma).
] The riate volume of pre-warmed digestion buffer was added to the
tube, which was then sealed and placed on a rocker in a 37ºC incubator for 20 minutes. This
first digestion step removes many red blood cells and enhances the digestion of the
remaining . After 20 minutes, the tube was removed and placed in the BSC. The
tissue was allowed to settle at the bottom of the tube and then the supernatant was
removed. The remaining tissue was then supplemented with fresh digestion buffer equaling
the starting volume. The tube was again placed on a rocker in a 37ºC incubator for an
additional 30 minutes.
After 30 minutes the digestion mixture was pipetted through a 70µm cell
strainer (BD Falcon) into an equal volume of neutralization buffer (DMEM w/ 10% FBS) to
stop the digestion reaction. The cell suspension was then washed by centrifugation at
300xg for 5 min. After centrifugation, the pellet was then re-suspended in 20mls KSFM
medium and a sample ed for cell counting and viability ment using trypan blue
exclusion. Once the cell count was calculated, 1 million cells were ted for RNA,
washed in PBS, and snap frozen in liquid nitrogen. The remaining cell suspension was
brought up to 50mls with KSFM medium and washed again by centrifugation at 300xg for 5
minutes. After washing, the cell pellet was re-suspended in a concentration of 15 million
cells per ml of KSFM.
Five milliliters of kidney cell suspension were then added to 5mls of 30%
(w/v) Optiprep® in 15ml l fuge tubes (BD Falcon) and mixed by inversion 6
times. This formed a final mixture of 15% (w/v) of Optiprep®. Post inversion, tubes were
carefully layered with 1 mL PBS. The tubes were centrifuged at 800 x g for 15 minutes
without brake. After centrifugation, the tubes were removed and a cell band was formed at
the top of the mixing nt. There was also a pellet containing red blood cells, dead cells,
and a small population of live cells that included some small less granular cells, some epoproducing
cells, some r cells, and some endothelial cells. The band was carefully
removed using a pipette and transferred to another 15ml conical tube. The gradient
medium was removed by aspiration and the pellet was collected by re-suspension in 1 ml
KSFM. The band cells and pellet cells were then recombined and re-suspended in at least 3
dilutions of the collected band volume using KSFM and washed by fugation at 300xg
for 5 minutes. Post washing, the cells were re-suspended in 20mls of KSFM and a sample for
cell ng was collected. Once the cell count was calculated using trypan blue exclusion,
1 million cells were collected for an RNA sample, washed in PBS, and snap frozen in liquid
nitrogen.
lture -up’ to enhance viability and culture performance of
Specific Bioactive Renal Cells Using Density Gradient Separation: To yield a clean, viable
population of cells for culture, a cell suspension was first generated as described above in
y Cell Isolation”. As an optional step and as a means of cleaning up the initial
preparation, up to 100 n total cells, suspended in sterile isotonic buffer were mixed
thoroughly 1:1 with an equal volume of 30% Optiprep® prepared at room temperature
from stock 60% (w/v) iodixanol (thus yielding a final 15% w/v Optiprep solution) and mixed
thoroughly by inversion six times. After mixing, 1ml PBS buffer was carefully layered on top
of the mixed cell suspension. The gradient tubes were then lly loaded into the
centrifuge, ensuring appropriate balance. The gradient tubes were centrifuged at 800 x g
for 15 minutes at 25˚C without brake. The cleaned-up cell population (containing viable and
functional collecting duct, tubular, endocrine, glomerular, and ar cells) segmented
between 6% and 8% (w/v) Optiprep®, corresponding to a density between 1.025 – 1.045
g/mL. Other cells and debris pelleted to the bottom of the tube.
Kidney Cell e: The combined cell band and pellet were then plated in
tissue culture treated triple flasks (Nunc T500) or equivalent at a cell concentration of
,000 cells per cm2 in 150mls of a 50:50 mixture of DMEM(high glucose)/KSFM containing
% (v/v)FBS, 2.5µg EGF, 25mg BPE, 1X ITS (insulin/transferrin/sodium selenite medium
supplement) with antibiotic/antimycotic. The cells were cultured in a humidified 5% CO2
tor for 2-3 days, providing a 21% atmospheric oxygen level for the cells. After two
days, the medium was changed and the cultures were placed in 2% oxygen-level
environment provided by a CO2 /Nitrogen gas multigas humidified tor (Sanyo) for
24hrs. Following the 24hr tion, the cells were washed with 60mls of 1XPBS and then
removed using 40mls 0.25% (w/v) trypsin/EDTA (Gibco). Upon removal, the cell suspension
was neutralized with an equal volume of KSFM containing 10% FBS. The cells were then
washed by centrifugation 300xg for 10 minutes. After washing, the cells were re-suspended
in 20mls of KSFM and transferred to a 50ml conical tube and a sample was collected for cell
counting. Once the viable cell count was determined using trypan blue exclusion, 1 n
cells were collected for an RNA sample, washed in PBS, and snap frozen in liquid nitrogen.
The cells were washed again in PBS and ted by centrifugation at 300xg for 5 minutes.
The washed cell pellet was re-suspended in KSFM at a concentration of 37.5 million
cells/ml.
Enriching for ic Bioactive Renal Cells Using Density Step Gradient
Separation: Cultured kidney cells, predominantly composed of renal tubular cells but
containing small subpopulations of other cell types cting duct, glomerular, vascular,
and endocrine) were separated into their component subpopulations using a density step
nt made from le concentrations w/v of nol (Optiprep). The cultures were
placed into a hypoxic environment for up to 24 hours prior to harvest and application to the
gradient. A d gradient was created by layering four different density mediums on top
of each other in a sterile 15mL conical tube, placing the solution with the highest density on
the bottom and layering to the least dense solution on the top. Cells were applied to the top
of the step gradient and centrifuged, which resulted in segregation of the population into
multiple bands based on size and granularity.
Briefly, densities of 7, 11, 13, and 16% Optiprep® (60% w/v Iodixanol) were
made using KFSM medium as diluents. For example: for 50mls of 7%(w/v) Optiprep®,
.83mls of stock 60% (w/v) Iodixanol was added to 44.17mls of KSFM medium and mixed
well by inversion. A altic pump (Master Flex L/S) loaded with sterile L/S 16 Tygon
tubing connected to sterile capillary tubes was set to a flow rate of 2 ml per minute, and 2
mL of each of the four solutions was loaded into a sterile conical 15 mL tube, beginning with
the 16% solution, followed by the 13% solution, the 11% solution, and the 7% solution.
Finally, 2 mL of cell suspension containing 75 million cultured rodent kidney cells was loaded
atop the step gradient nsions having been generated as bed above in ‘Kidney
cell Culture’). Importantly, as the pump was started to deliver the gradient solutions to the
tube, care was taken to allow the fluid to flow slowly down the side of the tube at a 45°
angle to insure that a proper interface formed n each layer of the gradient. The step
gradients, loaded with cells, were then centrifuged at 800 x g for 20 minutes without brake.
After centrifugation, the tubes were carefully removed so as not to disturb each interface.
Five distinct cell fractions resulted (4 bands and a pellet) (B1 – B4, + Pellet) (see Figure 26,
left l tube). Each on was collected using either a sterile disposable bulb pipette
or a 5ml pipette and characterized phenotypically and functionally (See Example 10 of
Presnell et al. WO/2010/056328). When rodent kidney cell sions are ted to
step-gradient fractionation immediately after isolation, the fraction enriched for tubular
cells (and containing some cells from the collecting duct) segments to a density between
1.062 – 1.088 g/mL. In contrast, when y gradient separation was performed after ex
vivo culture, the fraction enriched for tubular cells (and containing some cells from the
collecting duct) segmented to a density between 1.051 – 1.062 g/mL. Similarly, when rodent
kidney cell suspensions are subjected to step-gradient onation ately after
isolation, the fraction enriched for epo-producing cells, glomerular podocytes, and vascular
cells (“B4”) segregates at a density between 1.025 – 1.035 g/mL. In st, when density
gradient separation was med after ex vivo culture, the fraction enriched for epoproducing
cells, glomerular tes, and vascular cells (“B4”) segregated at a density
between 1.073 – 1.091 g/mL. Importantly, the post-culture distribution of cells into both the
“B2” and the “B4” fractions was enhanced by re (for a period of about 1 hour to a
period of about 24 hours) of the cultures to a hypoxic culture environment (hypoxia being
defined as <21% (atmospheric) oxygen levels prior to harvest and step-gradient procedures
(additional details ing hypoxia-effects on band distribution are provided in Example
Each band was washed by diluting with 3x the volume of KSFM, mixed well,
and centrifuged for 5 minutes at 300 x g. Pellets were re-suspended in 2mls of KSFM and
viable cells were counted using trypan blue exclusion and a hemacytometer. 1 million cells
were collected for an RNA , washed in PBS, and snap frozen in liquid nitrogen. The
cells from B2 and B4 were used for transplantation studies into uremic and anemic female
rats, generated via a two-step 5/6 nephrectomy procedure at s River Laboratories.
Characteristics of B4 were confirmed by quantitative real-time PCR, including oxygenregulated
expression of erythropoietin and vEGF, expression of glomerular markers
(nephrin, podocin), and sion of ar markers (PECAM). Phenotype of the ‘B2’
fraction was confirmed via sion of E-Cadherin, N-Cadherin, and Aquaporin-2. See
Figures 49a and 49b of Presnell et al. WO/2010/056328.
Thus, use of the step gradient strategy allows not only the enrichment for a
rare population of epo-producing cells (B4), but also a means to generate relatively enriched
fractions of functional tubular cells (B2) (see Figures 50 & 51 of Presnell et al.
WO/2010/056328). The step gradient strategy also allows EPO-producing and tubular cells
to be separated from red blood cells, cellular debris, and other potentially rable cell
types, such as large cell aggregates and certain types of immune cells.
The step gradient procedure may require tuning with regard to specific
densities employed to provide good tion of cellular components. The preferred
approach to tuning the gradient involves 1) running a continuous density gradient where
from a high density at the bottom of the gradient (16-21% Optiprep, for example) to a
relatively low density at the top of the gradient (5-10%, for example). Continuous gradients
can be prepared with any rd density gradient solution (Ficoll, Percoll, Sucrose,
iodixanol) according to standard methods (Axis Shield). Cells of interest are loaded onto the
continuous gradient and centrifuged at 800xG for 20 minutes without brake. Cells of similar
size and granularity tend to segregate together in the gradients, such that the relative
position in the gradient can be measured, and the ic gravity of the solution at that
on also measured. Thus, subsequently, a defined step gradient can be derived that
focuses isolation of particular cell populations based on their ability to transverse the
density gradient under ic conditions. Such optimization may need to be employed
when isolating cells from unhealthy vs. y tissue, or when isolating specific cells from
different species. For example, optimization was ted on both canine and human
renal cell cultures, to insure that the specific B2 and B4 subpopulations that were identified
in the rat were able from the other species. The l gradient for isolation of
rodent B2 and B4 subpopulations consists of (w/v) of 7%, 11%, 13%, and 16% Optiprep. The
optimal gradient for isolation of canine B2 and B4 subpopulations consists of (w/v) of 7%,
%, 11%, and 16% Optiprep. The optimal gradient for isolation of human B2 and B4
subpopulations consists of (w/v) 7%, 9%, 11%, 16%. Thus, the density range for localization
of B2 and B4 from ed rodent, canine, and human renal cells is provided in Table 8.
Table 8. Species Density Ranges.
Species Density Ranges g/ml
Step Gradient
Band Rodent Canine Human
B2 1.045-1.063g/ml 1.045-1.058g/ml 1.045-1.052g/ml
B4 1.073-1.091g/ml 1.091g/ml 1.063-1.091g/ml
e 7 – Low-oxygen culture prior to gradient affects band distribution, composition,
and gene expression
To determine the effect of oxygen conditions on distribution and
composition of prototypes B2 and B4, neokidney cell preparations from different species
were exposed to different oxygen conditions prior to the nt step. A rodent neokidney
tation (NKA) cell ation (RK069) was established using standard
procedures for rat cell isolation and culture initiation, as described supra. All flasks were
cultured for 2-3 days in 21% (atmospheric) oxygen conditions. Media was changed and half
of the flasks were then relocated to an oxygen-controlled incubator set to 2% oxygen, while
the remaining flasks were kept at the 21% oxygen conditions, for an onal 24 hours.
Cells were then harvested from each set of conditions using standard enzymatic ting
procedures described supra. Step gradients were prepared according to standard
procedures and the “normoxic” (21% oxygen) and “hypoxic” (2% oxygen) cultures were
ted separately and d side-by-side to identical step gradients. (Figure 27). While
4 bands and a pellet were generated in both conditions, the distribution of the cells
throughout the gradient was ent in 21% and 2% -cultured batches (Table 3).
Specifically, the yield of B2 was increased with hypoxia, with a concomitant decrease in B3.
Furthermore, the expression of B4-specific genes (such as erythropoietin) was enhanced in
the resulting gradient generated from the hypoxic-cultured cells (Figure 73 of Presnell et al.
WO/2010/056328).
A canine NKA cell preparation (DK008) was established using standard
procedures for dog cell isolation and culture (analogous to rodent isolation and e
procedures), as described supra. All flasks were cultured for 4 days in 21% (atmospheric)
oxygen conditions, then a subset of flasks were transferred to hypoxia (2%) for 24 hours
while a subset of the flasks were maintained at 21%. Subsequently, each set of flasks was
harvested and subjected to cal step gradients (Figure 28). Similar to the rat s
(Example 6), the hypoxic-cultured dog cells distributed hout the gradient differently
than the atmospheric oxygen-cultured dog cells (Table 9). Again, the yield of B2 was
increased with hypoxic exposure prior to gradient, along with a concomitant decrease in
distribution into B3.
Table 9.
Rat (RK069) Dog (DK008)
2% O2 21% O2 2% O2 21% O2
B1 0.77% 0.24% 1.20% 0.70%
B2 88.50% 79.90% 64.80% 36.70%
B3 10.50% 19.80% 29.10% 40.20%
B4 0.23% 0.17% 4.40% 21.90%
The above data show that pre-gradient exposure to hypoxia enhances
composition of B2 as well as the distribution of specific specialized cells (erythropoietinproducing
cells, vascular cells, and glomerular cells) into B4. Thus, hypoxic culture, ed
by density-gradient separation as described supra, is an effective way to generate ‘B2’ and
‘B4’ cell populations, across species.
Example 8 – Isolation of tubular/glomerular cells from human kidney
Tubular and glomerular cells were isolated and propagated from normal
human kidney tissue by the tic isolation methods described throughout. By the
gradient method described above, the tubular cell fraction was enriched ex vivo and after
culture. As shown in Figure 68 of Presnell et al. WO/2010/056328 (incorporated herein by
reference in its entirety), phenotypic attributes were maintained in isolation and
propagation. Tubular cell function, assessed via uptake of labeled albumin, was also
retained after repeated passage and cryopreservation. Figure 69 of Presnell et al.
0/056328 (incorporated herein by reference in its entirety) shows that when
tubular-enriched and tubular-depleted populations were cultured in 3D dynamic culture, a
marked se in sion of tubular , cadherin, was expressed in the tubularenriched
population. This confirms that the enrichment of tubular cells can be ined
beyond the initial enrichment when the cells are cultured in a 3D dynamic environment. The
same cultured population of kidney cells bed above in Example 7 was subjected to
flow cytometric analysis to examine d scatter and side scatter. The small, less
granular EPO-producing cell population was discernable (8.15%) and was separated via
ve selection of the small, less granular population using the g capability of a flow
ter (see Figure 70 of ll et al. WO/2010/056328 (incorporated herein by
reference in its entirety)).
Example 9 - Characterization of an unfractionated mixture of renal cells isolated from an
autoimmune glomerulonephritis patient sample
An unfractionated mixture of renal cells was isolated, as described above,
from an mune glomerulonephritis patient sample. To determine the unbiased
genotypic composition of specific subpopulations of renal cells isolated and expanded from
kidney tissue, quantitative real time PCR (qRTPCR) analysis (Brunskill et al., supra 2008) was
employed to identify differential cell-type-specific and pathway-specific gene expression
patterns among the cell subfractions. As shown in Table 6.1 of Ilagan et al.
, HK20 is an mune glomerulonephritis patient sample. As shown
in Table 6.2 of Ilagan et al. , cells generated from HK20 are lacking
glomerular cells, as determined by .
EXAMPLE 11 – Enrichment/Depletion of Viable Kidney Cell Types Using Fluorescent
Activated Cell Sorting (FACS)
One or more isolated kidney cells may be enriched, and/or one or more
specific kidney cell types may be depleted from isolated primary kidney tissue using
fluorescent ted cell g (FACS).
REAGENTS: 70% ethanol; Wash buffer (PBS ); 50:50 Kidney cell medium
(50%DMEM high glucose): 50% Keratinocyte-SFM; Trypan Blue 0.4%; Primary dies to
target kidney cell population such as CD31 for kidney endothelial cells and Nephrin for
kidney glomerular cells. Matched isotype specific fluorescent secondary antibodies; Staining
buffer ( 0.05% BSA in PBS).
PROCEDURE: Following standard procedures for cleaning the biological safety
cabinet (BSC), a single cell sion of kidney cells from either primary isolation or
cultured cells may be obtained from a T500 T/C treated flask and resuspend in kidney cell
medium and place on ice. Cell count and viability is then ined using trypan blue
exclusion method. For kidney cell enrichment/depletion of, for example, glomerular cells or
endothelial cells from a heterogeneous population, between 10 and 50 x106 live cells with a
viability of at least 70% are obtained. The heterogeneous population of kidney cells is then
stained with primary dy ic for target cell type at a starting concentration of
1µg/0.1ml of staining buffer/1 x 106 cells (titer if necessary). Target antibody can be
ated such as CD31 PE (specific for kidney endothelial cells) or un-conjugated such as
Nephrin (specific for kidney glomerular cells).
Cells are then stained for 30 minutes on ice or at 4ºC protected from light.
After 30 minutes of incubation, cells are washed by centrifugation at 300xg for 5 min. The
pellet is then resuspended in either PBS or staining buffer depending on whether a
conjugated isotype specific secondary dy is required. If cells are labeled with a
fluorochrome conjugated primary antibody, cells are ended in 2mls of PBS per 10
x107 cells and proceed to FACS aria or equivalent cell sorter. If cells are not labeled with a
fluorochrome conjugated antibody, then cells are labeled with an isotype specific
fluorochrome conjugated secondary antibody at a starting concentration of
1ug/0.1ml/1x106 cells.
Cells are then stained for 30 min. on ice or at 4ºC protected from light. After
minutes of tion, cells are washed by centrifugation at 300xg for 5 min. After
centrifugation, the pellet is resuspended in PBS at a concentration of 5x106/ml of PBS and
then 4mls per 12x75mm is transferred to a sterile tube.
FACs Aria is prepared for live cell sterile sorting per cturer’s
ctions (BD FACs Aria User Manual). The sample tube is loaded into the FACs Aria and
PMT voltages are adjusted after acquisition begins. The gates are drawn to select kidney
specific cells types using fluorescent intensity using a specific wavelength. Another gate is
drawn to select the negative population. Once the desired gates have been drawn to
encapsulate the positive target tion and the negative population, the cells are sorted
using manufacturer’s ctions.
The positive target population is collected in one 15ml conical tube and the
negative population in another 15 ml conical tube filled with 1 ml of kidney cell medium.
After collection, a sample from each tube is analyzed by flow try to determine purity.
Collected cells are washed by centrifugation at 300xg for 5 min. and the
pellet is resuspended in kidney cell medium for r analysis and experimentation.
EXAMPLE 12 – Enrichment/Depletion of Kidney Cell Types Using ic Cell Sorting
One or more ed kidney cells may be enriched and/or one or more
specific kidney cell types may be depleted from ed primary kidney tissue.
REAGENTS: 70% ethanol, Wash buffer (PBS ), 50:50 Kidney cell medium
(50%DMEM high glucose): 50% Keratinocyte-SFM, Trypan Blue 0.4%, Running Buffer(PBS,
2mM EDTA,0.5% BSA), Rinsing Buffer (PBS,2mM EDTA), Cleaning Solution (70% v/v ethanol),
Miltenyi FCR Blocking reagent, Miltenyi microbeads specific for either IgG isotype, target
antibody such as CD31(PECAM) or Nephrin, or secondary antibody.
PROCEDURE: Following standard procedures for cleaning the ical safety
cabinet (BSC), a single cell sion of kidney cells from either primary isolation or culture
is obtained and resuspended in kidney cell medium. Cell count and viability is determined
using trypan blue ion method. For kidney cell enrichment/depletion of, for example,
glomerular cells or endothelial cells from a heterogeneous population, at least 10x106 up to
4 x109 live cells with a viability of at least 70% is obtained.
The best separation for enrichment/depletion approach is determined based
on target cell of interest. For enrichment of a target frequency of less than 10%, for
e, glomerular cells using Nephrin antibody, the Miltenyi CS, or equivalent,
instrument program POSSELDS (double positive selection in sensitive mode) is used. For
depletion of a target frequency of greater than 10%, the Miltenyi autoMACS, or equivalent,
instrument program ES tion in sensitive mode) is used.
Live cells are labeled with target specific primary antibody, for example,
Nephrin rb polyclonal antibody for glomerular cells, by adding 1µg/10x106 cells/0.1ml of PBS
with 0.05% BSA in a 15ml conical centrifuge tube, followed by incubation for 15 minutes at
4ºC.
After labeling, cells are washed to remove unbound primary antibody by
adding 1-2ml of buffer per 10 x107 cells followed by centrifugation at 300xg for 5min. After
washing, isotype specific ary antibody, such as chicken anti-rabbit PE at
1ug/10x106/0.1ml of PBS with 0.05% BSA, is added, followed by incubation for 15 minutes at
4ºC.
] After incubation, cells are washed to remove unbound secondary antibody by
adding 1-2ml of buffer per 10 x107 cells ed by centrifugation at 300xg for 5 min. The
supernatant is removed, and the cell pellet is resuspended in 60µl of buffer per 10 x107 total
cells followed by addition of 20µl of FCR blocking reagent per 10 x107 total cells, which is
then mixed well.
Add 20 µl of direct MACS microbeads (such as anti-PE microbeads) and mix
and then incubate for 15 min at 4ºC.
After incubation, cells are washed by adding 10-20x the labeling volume of
buffer and centrifuging the cell sion at 300xg for 5 min. and resuspending the cell
pellet in 500µl -2mls of buffer per 10 x108 cells.
Per manufacturer’s instructions, the autoMACS system is d and primed
in preparation for magnetic cell separation using CS. New sterile collection tubes
are placed under the outlet ports. The autoMACS cell separation program is chosen. For
selection the POSSELDS program is chosen. For ion the ES program is chosen.
The labeled cells are inserted at uptake port, then beginning the program.
After cell selection or depletion, samples are collected and placed on ice until
use. Purity of the ed or selected sample is verified by flow cytometry.
EXAMPLE 13 – Cells with therapeutic potential can be isolated and propagated from
normal and chronically-diseased kidney tissue
The objective of the present study was to ine the functional
characterization of human NKA cells through high content analysis (HCA). High-content
imaging (HCI) provides simultaneous imaging of multiple sub-cellular events using two or
more fluorescent probes plexing) across a number of samples. High-content Analysis
(HCA) provides simultaneous quantitative measurement of le cellular ters
captured in High-Content . In brief, unfractionated (UNFX) cultures were generated
hwareb et al., supra 2008) and maintained independently from core biopsies taken
from five human kidneys with advanced chronic kidney disease (CKD) and three non-CKD
human kidneys using standard biopsy procedures. After (2) passages of UNFX ex vivo, cells
were harvested and subjected to density gradient methods (as in Example 2) to generate
subfractions, ing subfractions B2, B3, and/or B4.
Human kidney tissues were procured from non-CKD and CKD human donors
as summarized in Table 10.1 of Ilagan et al. . Figure 4 of Ilagan et al.
shows histopathologic features of the HK17 and HK19 samples. Ex vivo
cultures were established from all non-CKD (3/3) and CKD (5/5) kidneys. High content
analysis (HCA) of albumin transport in human NKA cells defining s of interest (ROI) is
shown in Figure 5 (HCA of albumin transport in human NKA cells) of Ilagan et al.
. Quantitative comparison of albumin transport in NKA cells derived
from non-CKD and CKD kidney is shown in Figure 6 of Ilagan et al. .
As shown in Figure 6 of Ilagan et al. , albumin transport
is not mised in CKD-derived NKA cultures. Comparative analysis of marker
expression between tubular-enriched B2 and tubular cell-depleted B4 subfractions is shown
in Figure 7 8/19) of Ilagan et al. .
] Comparative functional analysis of albumin transport between tubularenriched
B2 and tubular cell-depleted B4 subfractions is shown in Figure 8 of Ilagan et al.
. Subfraction B2 is ed in proximal tubule cells and thus exhibits
increased albumin-transport function.
Albumin uptake: Culture media of cells grown to ency in 24-well,
collagen IV plates (BD Biocoat ™) was replaced for 18-24 hours with phenol red-free, serumfree
, low-glucose DMEM (pr-/s-/lg DMEM) ning 1X antimycotic/antibiotic and 2mM
glutamine. Immediately prior to assay, cells were washed and incubated for 30 s with
pr-/s-/lg DMEM + 10mM HEPES, 2mM glutamine,1.8mM CaCl2, and 1mM MgCl2. Cells were
exposed to 25μg/mL rhodamine-conjugated bovine albumin (Invitrogen) for 30 min, washed
with ice cold PBS to stop endocytosis and fixed immediately with 2% paraformaldehyde
containing 25 μg/mL Hoechst nuclear dye. For inhibition experiments, 1μM receptorassociated
protein (RAP) (Ray Biotech, Inc., Norcross GA) was added 10 minutes prior to
albumin addition. Microscopic imaging and analysis was performed with a BD Pathway™ 855
High-Content BioImager (Becton Dickinson) (see Kelley et al. Am J Physiol Renal Physiol.
2010 Nov; 299(5):F1026-39. Epub Sep 8, 2010).
] In conclusion, HCA yields cellular level data and can reveal populations
dynamics that are undetectable by other assays, i.e., gene or protein sion. A
quantifiable ex-vivo HCA assay for ing n transport (HCA-AT) function can be
utilized to characterize human renal tubular cells as components of human NKA prototypes.
HCA-AT enabled comparative evaluation of ar function, showing that albumin
transport-competent cells were retained in NKA cultures derived from human CKD kidneys.
It was also shown that specific ctions of NKA cultures, B2 and B4, were distinct in
phenotype and function, with B2 representing a tubular nriched fraction with
enhanced albumin transport ty. The B2 cell subpopulation from human CKD are
phenotypically and functionally analogous to rodent B2 cells that demonstrated efficacy in
vivo (as shown above).
ture References
1. Kelley, R. et al. Intra-renal Transplantation of Bioactive Renal Cells Preserves Renal
Functions and Extends Survival in the ZSFI model of Progressive Diabetic Nephropathy. 2011
American Diabetes Association (ADA) Conference. (2011).
2. Kelley, R. et al. r cell-enriched subpopulation of primary renal cells improves
survival and augments kidney function in rodent model of chronic kidney disease. Am J
Physiol Renal Physiol 299, F1026-1039.
3. Presnell, S.C. et al. (2011) Isolation, Characterization, and Expansion Methods for
Defined y Renal Cell Populations from Rodent, Canine, and Human Normal and
Diseased Kidneys. Tissue Eng Part C Methods 17:261-273.
4. Humphreys, B.D. et al. Repair of injured proximal tubule does not involve specialized
itors. Proc Natl Acad Sci U S A 108, 9226-9231.
. Aboushwareb, T. et al. opoietin producing cells for potential cell therapy.
World J Urol 26, 295-300 (2008).
6. Takano, Y. et al. Recovery and maintenance of nephrin expression in cultured
podocytes and identification of HGF as a repressor of nephrin. Am J l Renal Physiol
292, F1573-1582 (2007).
7. Basu, J. et al. Expansion of the human adipose-derived stromal vascular cell fraction
yields a population of smooth muscle-like cells with markedly distinct phenotypic and
functional properties relative to mesenchymal stem cells. Tissue Eng Part C Methods 17,
843-860.
8. Basu, J. et al. Organ specific regenerative markers in peri-organ adipose: kidney.
Lipids Health Dis 10, 171.
9. Xinaris C, et al. In vivo maturation of functional renal organoids formed from
embryonic cell sions. J Am Soc Nephrol 23,1-12. (2012).
. Buzhor et al. Kidney Spheroids Recapitulate Tubular Organoids Leading to Enhanced
Tubulogenic Potency of Human Kideny-Derived Cells Tissue Engineering Part A, (2011).
11. Brown, SA et al., Impaired renal autoregulatory y in dogs with reduced renal
mass. J Am Soc Nephrol. 5:1768-74 (1995).
12. Robertson, J.L. et al., erm renal ses to high dietary protein in dogs with
75% nephrectomy. Kidney Int. 29:511-9 .
13. Urie, B.K. et al., Evaluation of clinical status, renal on, and hematopoietic
variables after unilateral ctomy in canine kidney donors. J Am Vet Med Assoc.
230:1653-6 (2007).
What is claimed is:
1. A method of forming a spheroid comprising:
suspension culturing a heterogenerous renal cell population and a non-renal
bioactive cell population in a three-dimensional (3D) culture system
wherein the 3D culture system comprises a spinner and lacks an ous
ld to which cells of the heterogeneous renal cell population and the bioactive
cell population attach;
wherein the heterogenous renal cell population is ed for renal
erythropoietin (EPO)-producing cells, and
n the non-renal bioactive cell population is a non-renal endothelial cell
population, a non-renal endothelial itor population, a non-renal mesenchymal
stem cell population, or a non-renal adipose-derived progenitor population.
2. The method of claim 1 wherein the EPO-producing cells increase EPO expression in
response to hypoxic culture conditions.
3. The method of claim 2 wherein the heterogeneous renal cell population is further
enriched for ular cells and vascular cells.
4. The method of claim 1 wherein the non-renal bioactive cell population is the non-renal
endothelial cell population.
. The method of claim 4 wherein the non-renal endothelial cell population is a cell
line.
6. The method of claim 4 wherein the nal endothelial cell population comprises
human umbilical vein endothelial cells.
7. The method of claim 1 wherein the heterogeneous renal cell population and the nonrenal
ive cell population are xenogeneic, eic, allogeneic, or autologous.
8. The method of claim 1 wherein the heterogeneous renal cell tion and the nonrenal
ive cell population are cultured separately for a first time period, then combined
and cultured for a second time period.
9. The method of claim 8 wherein the second time period is at least 24 hours.
. The method of claim 9 wherein the second time period is 24 hours to 72 hours.
11. The method of claim 1 wherein the renal cell population and bioactive cell population
are at a ratio of 1:1.
12. A spheroid formed according to the method of any one of claims 1-11.
13. An isolated cluster of cells comprising a heterogeneous renal cell population and a nonrenal
bioactive cell tion,
wherein the heterogeneous renal cell population is enriched for renal EPO-producing
cells,
wherein the non-renal bioactive cell population is a non-renal endothelial cell
population, a non-renal endothelial progenitor cell population, a non-renal
mesenchymal stem cell tion, or a non-renal adipose-derived progenitor
population; and
wherein the cluster of cells is cultured in media in suspension without attachment to
a scaffold.
14. The isolated cluster of cells of claim 13 n the non-renal bioactive cell population
is a non-renal endothelial cell population.
. The isolated cluster of cells of claim 13 wherein the heterogeneous renal cell
population is further enriched for ular and vascular cells.
16. The isolated cluster of cells of claim 14 wherein the non-renal endothelial cell
population is a cell line.
17. The isolated cluster of cells of claim 14 n the non-renal endothelial cell
population comprises human umbilical vein endothelial cells.
18. The isolated cluster of cells of claim 13, wherein the non-renal bioactive cell population
is the non-renal mesenchymal stem cell tion.
19. The isolated cluster of cells of claim 13, wherein the non-renal bioactive cell population
is the adipose-derived progenitor cell population.
. The ed cluster of cells of any one of claims 13-19, wherein the EPO-producing
cells increase EPO expression in response to hypoxic e conditions.
21. The isolated cluster of cells of claim 20, wherein the hypoxic conditions comprise
culturing at less than 5% oxygen.
22. The isolated cluster of cells of claim 21, n the hypoxic conditions comprise
culturing at less than 3% .
23. An injectable formulation comprising at least one cluster of cells as claimed in any one
of claims 13-19 and a liquid medium.
24. The formulation of claim 23, wherein the liquid medium is ed from a cell growth
medium, Dulbecco’s phosphate buffered saline and combinations thereof.
. The formulation of claim 24 wherein the liquid medium is Dulbecco’s phosphate
ed saline.
26. The formulation of claim 23, wherein the cluster of cells is ded in the liquid
medium.
27. An injectable formulation comprising at least one cluster of cells as claimed in any of
claims 13-19 and a hydrogel.
28. The ation of claim 27, wherein the hydrogel comprises gelatin.
29. The formulation of claim 28, n the gelatin is present in the formulation at about
0.5% to about 1% (w/v).
. The formulation of claim 28, wherein the gelatin is present in the formulation at about
0.75% (w/v).
31. Use of at least one cluster of cells as claimed in any one of claims 13-19 for the
manufacture of a medicament for treating kidney disease in a subject.
32. Use of at least one cluster of cells as claimed in any one of claims 13-19 and a liquid
medium in the manufacture of a medicament for treating kidney disease in a subject.
33. Use of at least one r of cells as claimed in any one of claims 13-19 and a hydrogel
in the manufacture of a medicament for treating kidney disease in a subject.
34. The use of any one of claims 31 to 33, wherein the medicament is formulated for
administration by ion.
. The method according to any one of claims 1 to 11, substantially as herein described
with reference to any example thereof.
36. The spheroid according to claim 12, substantially as herein described with reference to
any example thereof.
37. The isolated cluster of cells according to any one of claims 13 to 22, substantially as
herein described with reference to any example thereof.
38. The formulation ing to any one of claims 23 to 30, substantially as herein
described with reference to any example thereof.
39. The use according to any one of claims 31 to 34, substantially as herein described with
reference to any e thereof.
1/12
104 104
Alexa Fluor 488-A 103 103 CD31+ CD31+
9.32% 0.24%
102 102
101 Alexa Fluor 488-A 101
100 100
100 101 102 103 104 100 101 102 103 104
SSC-A SSC-A
ZSF1 Rat UNFX t ZSF1 post sort Neg flow thru
2/12
Alexa Fluor 488-A 103 CD31+
89.94%
100 101 102 103 104
SSC-A
ZSF1 post sort CD31 + ion
3/12
104 104
2.15% (+) 0.5% (+)
103 103
PE-A 102 PE-A 102
101 101
100 100
100 101 102 103 104 100 101 102 103 104
SSC-A SSC-A
FX Norm p0 HK27 Norm Band 2
104 104
0.78% (+) 2.7% (+)
103 103
PE-A 102 PE-A 102
101 101
100 100
100 101 102 103 104 100 101 102 103 104
SSC-A SSC-A
HK27 2%O2 Band 2 HK27 UNFXp0 in EGM-2 on
fibronectin
4/12
104 104
0% (+) 35.5% (+)
103 103
PE-A 102 PE-A 102
101 101
100 100
100 101 102 103 104 100 101 102 103 104
SSC-A SSC-A
HK27p0 CD31 neg sort HK27 CD31 + p1 in EGM-2 on
ectin
1A
/12
104 104
Alexa Fluor 594-A 103 Alexa Fluor 594-A 103
0.19% 91.23%
102 102
101 101
100 100
100 101 102 103 104 100 101 102 103 104
SSC-A SSC-A
Unlabeled l BWH001p1 2%O2 UNFX
1B
1C
6/12
7/12
8/12
9/12
/12
A B
11/12
12/12
C F
B E
A D
Claims (39)
1. A method of forming a spheroid comprising: suspension culturing a heterogenerous renal cell population and a non-renal bioactive cell population in a three-dimensional (3D) culture system wherein the 3D culture system comprises a spinner and lacks an ous ld to which cells of the heterogeneous renal cell population and the bioactive cell population attach; wherein the heterogenous renal cell population is ed for renal erythropoietin (EPO)-producing cells, and n the non-renal bioactive cell population is a non-renal endothelial cell population, a non-renal endothelial itor population, a non-renal mesenchymal stem cell population, or a non-renal adipose-derived progenitor population.
2. The method of claim 1 wherein the EPO-producing cells increase EPO expression in response to hypoxic culture conditions.
3. The method of claim 2 wherein the heterogeneous renal cell population is further enriched for ular cells and vascular cells.
4. The method of claim 1 wherein the non-renal bioactive cell population is the non-renal endothelial cell population.
5. The method of claim 4 wherein the non-renal endothelial cell population is a cell line.
6. The method of claim 4 wherein the nal endothelial cell population comprises human umbilical vein endothelial cells.
7. The method of claim 1 wherein the heterogeneous renal cell population and the nonrenal ive cell population are xenogeneic, eic, allogeneic, or autologous.
8. The method of claim 1 wherein the heterogeneous renal cell tion and the nonrenal ive cell population are cultured separately for a first time period, then combined and cultured for a second time period.
9. The method of claim 8 wherein the second time period is at least 24 hours.
10. The method of claim 9 wherein the second time period is 24 hours to 72 hours.
11. The method of claim 1 wherein the renal cell population and bioactive cell population are at a ratio of 1:1.
12. A spheroid formed according to the method of any one of claims 1-11.
13. An isolated cluster of cells comprising a heterogeneous renal cell population and a nonrenal bioactive cell tion, wherein the heterogeneous renal cell population is enriched for renal EPO-producing cells, wherein the non-renal bioactive cell population is a non-renal endothelial cell population, a non-renal endothelial progenitor cell population, a non-renal mesenchymal stem cell tion, or a non-renal adipose-derived progenitor population; and wherein the cluster of cells is cultured in media in suspension without attachment to a scaffold.
14. The isolated cluster of cells of claim 13 n the non-renal bioactive cell population is a non-renal endothelial cell population.
15. The isolated cluster of cells of claim 13 wherein the heterogeneous renal cell population is further enriched for ular and vascular cells.
16. The isolated cluster of cells of claim 14 wherein the non-renal endothelial cell population is a cell line.
17. The isolated cluster of cells of claim 14 n the non-renal endothelial cell population comprises human umbilical vein endothelial cells.
18. The isolated cluster of cells of claim 13, wherein the non-renal bioactive cell population is the non-renal mesenchymal stem cell tion.
19. The isolated cluster of cells of claim 13, wherein the non-renal bioactive cell population is the adipose-derived progenitor cell population.
20. The ed cluster of cells of any one of claims 13-19, wherein the EPO-producing cells increase EPO expression in response to hypoxic e conditions.
21. The isolated cluster of cells of claim 20, wherein the hypoxic conditions comprise culturing at less than 5% oxygen.
22. The isolated cluster of cells of claim 21, n the hypoxic conditions comprise culturing at less than 3% .
23. An injectable formulation comprising at least one cluster of cells as claimed in any one of claims 13-19 and a liquid medium.
24. The formulation of claim 23, wherein the liquid medium is ed from a cell growth medium, Dulbecco’s phosphate buffered saline and combinations thereof.
25. The formulation of claim 24 wherein the liquid medium is Dulbecco’s phosphate ed saline.
26. The formulation of claim 23, wherein the cluster of cells is ded in the liquid medium.
27. An injectable formulation comprising at least one cluster of cells as claimed in any of claims 13-19 and a hydrogel.
28. The ation of claim 27, wherein the hydrogel comprises gelatin.
29. The formulation of claim 28, n the gelatin is present in the formulation at about 0.5% to about 1% (w/v).
30. The formulation of claim 28, wherein the gelatin is present in the formulation at about 0.75% (w/v).
31. Use of at least one cluster of cells as claimed in any one of claims 13-19 for the manufacture of a medicament for treating kidney disease in a subject.
32. Use of at least one cluster of cells as claimed in any one of claims 13-19 and a liquid medium in the manufacture of a medicament for treating kidney disease in a subject.
33. Use of at least one r of cells as claimed in any one of claims 13-19 and a hydrogel in the manufacture of a medicament for treating kidney disease in a subject.
34. The use of any one of claims 31 to 33, wherein the medicament is formulated for administration by ion.
35. The method according to any one of claims 1 to 11, substantially as herein described with reference to any example thereof.
36. The spheroid according to claim 12, substantially as herein described with reference to any example thereof.
37. The isolated cluster of cells according to any one of claims 13 to 22, substantially as herein described with reference to any example thereof.
38. The formulation ing to any one of claims 23 to 30, substantially as herein described with reference to any example thereof.
39. The use according to any one of claims 31 to 34, substantially as herein described with reference to any e thereof.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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NZ752702A NZ752702B2 (en) | 2013-05-08 | 2014-05-08 | Organoids comprising isolated renal cells and uses thereof |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361855146P | 2013-05-08 | 2013-05-08 | |
US61/855,146 | 2013-05-08 | ||
US201361855152P | 2013-05-09 | 2013-05-09 | |
US61/855,152 | 2013-05-09 | ||
PCT/US2014/037275 WO2014182885A2 (en) | 2013-05-08 | 2014-05-08 | Organoids comprising isolated renal cells and uses thereof |
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NZ713875A NZ713875A (en) | 2021-11-26 |
NZ713875B2 true NZ713875B2 (en) | 2022-03-01 |
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