NZ752702B2 - Organoids comprising isolated renal cells and uses thereof - Google Patents
Organoids comprising isolated renal cells and uses thereof Download PDFInfo
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- NZ752702B2 NZ752702B2 NZ752702A NZ75270214A NZ752702B2 NZ 752702 B2 NZ752702 B2 NZ 752702B2 NZ 752702 A NZ752702 A NZ 752702A NZ 75270214 A NZ75270214 A NZ 75270214A NZ 752702 B2 NZ752702 B2 NZ 752702B2
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- cells
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- kidney
- cell population
- renal
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- 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
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/25—Urinary tract cells, renal cells
- C12N2502/256—Renal cells
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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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- C12N5/0697—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
This application is a divisional of New Zealand patent application 713875, which is
the national phase entry in New Zealand of PCT international ation
(published as ) filed 8 May 2014, the contents of
which are all incorporated herein by reference.
Field of the Invention
The present sure is directed to admixtures of selected bioactive primary renal
cells and further bioactive cell populations, and methods of treating a subject in need
thereof. The present disclosure is further directed to organoids comprising isolated renal
cells, including tubular and erythropoietin (EPO)-producing kidney cell populations, and
methods of ng a subject in need with the organoids.
Background of the Invention
Chronic Kidney Disease (CKD) affects over 19M people in the United States and is
frequently a consequence of metabolic disorders involving obesity, diabetes, and
hypertension. Examination of the data reveals that the rate of increase is due to the
development of renal e secondary to ension and non-insulin ent diabetes
mellitus (NIDDM) (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda,
MD, al Institutes of Health, National Institute of Diabetes and Digestive and Kidney
Diseases, 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 proteinuria 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: 6,
1998). CKD patients in stages 1-3 of progression are managed by lifestyle changes and
pharmacological entions aimed at controlling the underlying disease state(s), while
patients in stages 4-5 are managed by dialysis and a drug regimen that lly es
anti-hypertensive agents, erythropoiesis stimulating agents (ESAs), iron and vitamin D
mentation. According 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 is ($65,405), the healthcare
cost for maintenance 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 ural costs and does not include treatment of other
complications, emergency procedures, or ancillary procedures such as the placement of
ar grafts for dialysis access. Combined medicare costs for CKD and ESRD in 2005
totaled $62B – representing 19% of all medicare spending for that year d States Renal
Data System: Costs of CKD and ESRD. ed. Bethesda, MD, National utes of Health,
National Institute of Diabetes and Digestive and Kidney Diseases, 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 benefit from whole kidney
transplant far s 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 ency on dialysis and to fill the void left by
the shortage of donor kidneys.
Progressive renal e s from a combination of the initial disease injury (e.g,
hypertension), followed by a maladaptive renal response to that injury. Such a se
includes the production of pro-inflammatory and pro-fibrotic cytokines and growth factors.
Therefore, one gy to slow CKD progression is to ameliorate the inflammatory and
fibrotic response as well as mitigate 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 animals.
Patients 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 erythropoiesis. Erythroid homeostasis is dependent on both the
production of erythropoietin (EPO) by lized 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 negative s of uremic factors on the actions 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
ic delivery of recombinant EPO or EPO s to restore erythroid mass. Dialysis
offers survival benefit to patients in mid-to-late stage renal failure, but causes significant
quality-of-life issues. Kidney transplant is a highly desired (and often the only) option for
ts in the later stages of renal failure, but the supply of high-quality donor kidneys 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 oid 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 transiently stimulate erythropoiesis and RBC number by in vivo delivery of epo-producing
cells. However, to date, none of these approaches have d regulated erythroid
homeostasis or long-term in vivo functionality. Consequently, HCT and RBC number are
often increased beyond normal values, leading to themia vera and other
complications. ry of EPO-producing cells that are therapeutically-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 isms
intact. It is important to note that EPO-deficient anemias, while prevalent in ts with
kidney e, can also develop as a result of other disease states, including heart failure,
organ system failure, and other chronic diseases.
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 bioactive renal cells, including tubular and
erythropoietin (EPO)-producing kidney cell populations, and methods of isolating and
ing 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
In a first aspect the invention provides a method of forming a spheroid comprising:
suspension culturing a heterogeneous renal cell population and a bioactive cell population
in cell culture media in a 3-dimensional (D) culture system,
wherein the 3D culture system comprises a spinner and lacks an exogenous scaffold to
which cells of the heterogenerous renal cell population attach;
wherein the heterogeneous renal cell population is enriched for renal tubular cells, and
wherein the bioactive cell population is a non-renal endothelial cell population, a al
endothelial progenitor cell population, a non-renal mesenchymal stem cell tion, or a
non-renal adipose-derived progenitor cell tion.
[008a] In another aspect the invention provides a spheroid made according the method of
the invention.
[008b] In a r aspect the invention provides an isolated cluster of cells comprising
aheterogeneous renal cell tion and a bioactive cell population, wherein the
heterogeneous renal cell population is enriched for renal tubular cells,
wherein the bioactive cell population is a nal endothelial cell tion, a non-renal
endothelial progenitor cell population, a nal mesenchymal stem cell population, or a
non-renal adipose-derived progenitor cell population; and
wherein the cluster of cells is cultured in media in suspension without ment to a
[008c] In another aspect the invention provides an injectable formulation comprising at
least one cluster of cells according to the invention and a liquid medium.
[008d] In a further aspect the invention provides an injectable formulation comprising at
least one cluster of cells according to the invention and a hydrogel.
[008e] In another aspect the invention relates to use of at least one cluster of cells
according to the invention and a liquid medium in the cture of a ment to
treat kidney e in a subject in need thereof.
[008f] In a further aspect the invention relates to the use of at least one cluster of cells
according to the invention and a el in the manufacture of a medicament to treat
kidney disease in a subject in need thereof.
[008g] 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 ce. The
reader is directed to the accompanying claim set which defines the scope of the invention.
Described herein are organoids, methods 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.
Described is a method of forming an organoid comprising a heterogeneous renal cell
tion and a bioactive cell population. In one emodiment, the d comprises
culturing the heterogenerous renal cell population and a bioactive cell population in a
e system ed from the group consisting of i) 2D culture; ii) 3D e: COL(I) gel;
iii) 3D culture: Matrigel; iv) 3D culture: spinners, followed by COL(I)/Matrigel; and v) 3D
culture: COL(IV) gel. In some embodiments, the heterogeneous renal cell population
comprises a bioactive renal cell population. In certain embodiments, the heterogeneous
renal cell population comprises a B2 cell population comprising an enriched population of
tubular cells, and wherein the geneous 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 tion 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
embodiments, the endothelial cell population is derived from human umbilical cord. In
some embodiments, the bioactive cell population comprise endothelial progenitor cells. In
some embodiments, 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 tion and the bioactive
cell population 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
population are combined 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 described is is an organoid. In some embodiments, the organoids are made
according to the s 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 n embodiments, the elial cell
population comprises HUVEC cells.
In some ments, the heterogeneous renal cell population comprises a B2 cell
population comprising an enriched population of tubular cells, and n the
geneous renal cell population is depleted of a B1 cell population. In certain
ments, the heterogeneous renal cell population is further depleted of a B5 cell
population. In select embodiments, the heterogeneous renal cell population comprises a
cell population ed from B2, B2/B3, B2/B4, and B2/B3/B4. In some embodiments, the
heterogeneous renal cell population comprises erythropoetin (EPO)-producing cells.
Described is an injectable formulation comprising at least one id and a liquid
medium. In one embodiment, the the liquid medium is selected from a cell growth medium,
DPBS and combinations thereof. In another embodiment, the ids are ded 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
throughout 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 embodiment, the cell-stabilizing biomaterial comprises a hydrogel. In one other
embodiment, the hydrogel comprises gelatin. In other embodiments, the gelatin is present
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).
Described is a method of treating kidney e in a t in need comprising
administering at least one organoid comprising a heterogeneous renal cell population and a
bioactive cell population. In some embodiments, the bioactive cell population is an
endothelial cell population. In certain embodiments, the endothelial cell population is a cell
line. In one embodiment, the endothelial cell population comprises HUVEC cells.
In most ments, the heterogeneous renal cell population ses a B2 cell
population comprising an enriched population of tubular cells, and wherein the
heterogeneous renal cell tion is depleted of a B1 cell population. In select
embodiments, the heterogeneous renal cell population is further depleted of a B5 cell
population. In some embodiments, the heterogeneous renal cell population ses a
cell population selected from B2, B2/B3, B2/B4, and B4. In most embodiments, the
heterogeneous renal cell population comprises opoetin (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 subject is a mammal selected from dogs, cats, , 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 ed.
Also bed is the use of an organoid in the ation of a medicament for the
treatment of kidney disease.
BRIEF DESCRIPTION OF THE DRAWINGS
depicts cell culture morphology of y 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. ve flow through (CD31-) from
Miltenyi micro-beads selection;and E. 90% CD31+ cells selected at the end of passage 1
grown on fibronectin coated flasks in EGM2 fully supplemented medium.
shows human cell culture morphology viewed at 5x. A. Unfractionated kidney
cells at the end of p0 that were grown in 21%O2 on TC-treated 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 endothelial cells in
ed samples during culture period. The unfractionated (UNFX) endothelial composition
was <3% at p0 and was ed ~ 15 fold at p1 when plated on fibronectin and cultured in
EGM-2 media.
1 shows Bigeneic xP reporters for e 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 ion.
depicts phase imaging (10x) of A. rat and B. human SRC showing mity 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 organoid tube formation (ringed in white). B. Phase image at
higher magnification (ringed in white) along with the remnant organoids. C. GGT-1
phenotypic sion (green), nuclear (blue) D. CK18 expression (green), nuclear ( blue) at
20x magnification.
depicts membrane 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 ce of both SRC population (red) and endothelial
cells (green). When , populations appear . Nuclear staining (blue) at 20x
magnification.
depicts SPIO ine d id (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 showing 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 showing the HLA1 staining of human cells in a rat
kidney that had been administered the organoids described herein. A. Normal human
; 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 staining is present in end of study non-treated ed 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 typically lighter in color but is sometimes dark and
of size r than cells. Panels D, E and F demonstrate lower magnification utilized for
ing to identify dark staining cells, then higher magnification confirmation of HLA1
ng 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 s and humans has been recently ished 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 density gradient centrifugation.
Described herein is the isolation, identification and expansion of selected individual
cell populations of the various n compartments or niches, such that novel, le
ed 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 le, individual cell types that make up the nephron and
combined as ed admixtures enables improved targeted treatment of specific renal
disease s; and/or at least provide the public with a useful choice.
Cell types such as the vascular endothelium, tubular and collecting duct epithelium,
interstitial cells, glomerular cells, mesenchymal stem cells, etc., can be isolated, fied
and ed ex-vivo. While each cell type may require a unique method and media
ation for sub-culture, they can be added back in selective combinations or admixtures
as organoid rs to provide enhanced delivery and improved treatment for an
underlying renal tissue/ cell ency associated with a specific acute and/or chronic
kidney disease patient syndrome/cohort 8.
Described herein are 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 ability to isolate, characterize and expand
resident renal endothelial cells using selective culture systems and magnetic sorting allows
for enrichment of a previously characterized selective regenerative renal epithelial cell (SRC)
population 2, 4 with a specific percentage of purified renal endothelial cells (SRC+). The SRC+
cell populations described herein are comprised of selected renal cells (SRCs or BRCs), as
previously described and also ed herein, and further bioactive cell populations,
including but not limted to, endothelial cells, elial progenitors, mesenchymal stem
cells, and/or adipose-derived progenitors. In one embodiment, the further bioactive cell
populations are sourced from renal s. In other embodiments, the further bioactive
cell populations are sourced from non-renal sources.
Described herein are organoids comprising and/or formed from heterogenous
mixtures or ons of selected or bioactive renal cells (SRCs or BRCs) or SRC+ cell
poulations, methods of isolating and culturing the same, as well as s of treatment
using the organoids as described herein. Directed delivery of SRC or SRC+ populations to
the kidney as organoids is expected to e cell retention, thereby leading to improved
overall therapeutic es. The present ption is also directed to methods of
treatment using SRC+ cell populations.
Definitions
Unless defined otherwise, 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. (Edited 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 described herein, which could be used in the practice of the
present invention. Indeed, the present invention is in no way limited to the methods and
materials bed.
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
tion may be subsequently cultured in vitro. Those of ordinary skill in the art will
appreciate that various methods for isolating and ing cell populations for use with the
present invention 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, heterogeneous cell
population derived from the kidney. For example, a geneous 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 cultures of ian cells, established from
kidney biopsies or whole kidney tissue. An unfractionated heterogeneous cell population
may also be referred to as a non-enriched cell population.
The term “native ” shall mean the kidney of a living subject. The subject may
be healthy or un-healthy. An unhealthy subject may have a kidney disease.
The term “regenerative effect” shall mean an effect which provides a benefit to a
native kidney. 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, ular 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 ive cell ations and/or
admixtures described herein to e a regenerative effect.
The term ture” as used herein refers to a combination of two or more
ed, enriched cell populations derived from an unfractionated, heterogeneous cell
population. According to certain embodiments, the cell populations of the present
invention are renal cell populations.
An “enriched” cell population or preparation refers to a cell population derived from
a starting kidney cell population (e.g., an unfractionated, heterogeneous cell population)
that ns a greater percentage of a specific cell type than the percentage 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 suspension from a kidney biopsy or
cultured mammalian kidney cells) that contains a percentage of cells capable of producing
EPO that is greater than the percentage of cells capable of producing EPO in the starting
population. For example, 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 invention may be enriched for one or more cell types and depleted of one or more
other cell types. For example, an enriched oducing cell tion may be ed
for interstitial fibroblasts and depleted of tubular cells and collecting duct epithelial cells
ve to the interstitial lasts and tubular cells in a non-enriched cell population, i.e.
the starting cell population from which the ed 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 regulated
manner, as demonstrated by oxygen-tunable EPO expression from the
endogenous native EPO gene.
In another embodiment, an enriched cell population, which ns a greater
tage 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 ine 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
starting kidney cell population that lacks or is deficient in one or more cell types, e.g.,
vascular, glomerular or ine, 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 embodiment, the B4’ cell population is
derived from a subject having focal segmental ulosclerosis (FSGS). In another
embodiment, the B4’ cell population is derived from a subject having autoimmune
glomerulonephritis. In another embodiment, B4’ is a cell population d from a starting
cell population including all cell types, e.g., vascular, glomerular, or ine cells, which is
later depleted of or made ent in one or more cell types, e.g., vascular, glomerular, or
ine cells. In yet another embodiment, B4’ is a cell population derived from a starting
cell population including all cell types, e.g., vascular, glomerular, or endocrine 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 tion may be enriched for glomerular cells but depleted of vascular and/or
endocrine cells. In another 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 ar and endocrine cells but depleted of
glomerular 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 bed 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 sed above that contains a
percentage of cells expressing one or more vascular, glomerular and proximal tubular
markers with some EPO-producing cells that is greater than the tage of cells
expressing one or more vascular, glomerular 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 r percentage
of proximal tubular cells as well as vascular and glomerular cells as compared to the starting
population. In one embodiment, the B3 cell population contains a greater percentage of
proximal tubular cells as compared to the starting tion but a lesser percentage of
proximal tubular cells as compared to the B2 cell population. In another embodiment, the
B3 cell population ns a greater percentage of vascular and glomerular cells markers
with some EPO-producing cells as compared to the ng 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 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 tubular cell markers that is r than the
percentage of cells expressing one or more tubular cell markers in the starting tion.
For example, the term “B2” refers to a cell population derived from a starting kidney cell
population that contains a greater percentage of r 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 ed of EPO-producing cells, glomerular cells, and ar 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 population is depleted of one or more cell types such that the depleted
cell population contains a lesser proportion of the cell type(s) 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 ed include cells with large granularity of the
collecting duct and tubular system having a density of < about 1.045 g/ml, referred to as
“B1”. In certain other embodiments, the cell types that may be depleted e 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 relatively
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
relative to standard e conditions in which cells are cultured at atmospheric oxygen
levels (about 21%). Non-hypoxic ions are referred to herein as normal or normoxic
culture conditions.
The term “oxygen-tunable” as used herein refers to the ability of cells to modulate
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 tension (regardless of the pre-induction or starting oxygen tension).
The term oid” 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 “spheroid” does not
imply that the aggregate is a geometric sphere. The aggregate may be highly organized with
a well defined logy or it may be an unorganized mass; it may e 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. Included in this tion are ids and organotypic
cultures.
The term oid” as used herein refers to a heterogeneous 3D agglomeration of
cells that recapitulates aspects of cellular rganization, architecture and signaling
interactions present in the native organ. The term “organoid” es spheroids or cell
clusters formed from suspension cell cultures.
The term “biomaterial” as used here refers to a natural or synthetic biocompatible
material that is suitable for introduction 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,
ric matrices and scaffolds. Those of ordinary skill in the art will appreciate that the
biomaterial(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 scaffold 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, ed to, seeded, or entrapped in a biomaterial made up of one or
more synthetic or lly-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 patible materials used to form the
scaffold/biomaterial is selected to direct, facilitate, or permit the formation of multicellular,
three-dimensional, organization of at least one of the cell populations deposited n.
The one or more biomaterials used to generate the construct may also be selected to direct,
facilitate, or permit dispersion and/or integration of the construct or cellular components of
the construct with the endogenous host tissue, or to direct, facilitate, or permit the survival,
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 lar marker, the expression or presence of which in
a cultured cell population can be detected by standard methods (or methods disclosed
) and is consistent with one or more cells in the cultured cell population being a
particular type of cell. The marker may be a ptide sed by the cell or an
identifiable physical location on a chromosome, such as a gene, a ction endonuclease
recognition 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 ”, or some segment of DNA with no known coding function. The
biomarkers may be cell-derived, e.g., secreted, products.
The terms “differentially 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 include 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 ted at the nucleic acid level or protein level, or may be t to alternative ng
to result in a different polypeptide product. Such differences may be ced by a change
in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for
example. Differential gene expression may include a comparison of expression between
two or more genes or their gene ts, 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 ative, differences in
the temporal or cellular expression pattern in a gene or its sion products among, for
example, the first cell and the second cell. For the purpose of this disclosure, “differential
gene expression” is considered to be present when there is a difference between the
expression of a given gene in the first cell and the second cell. The differential expression of
a marker may be in cells from a patient before administration of a cell tion,
admixture, or construct (the first cell) relative to expression in cells from the patient after
administration (the second cell).
The terms it”, “down-regulate”, “under-express” and “reduce” are used
interchangeably and 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 reduced relative to one or more ls, such
as, for e, one or more positive and/or negative controls. The under-expression may
be in cells from a patient before administration of a cell population, ure, or construct
relative to cells from the patient after administration.
The term “up-regulate” or “over-express” is used to mean that the sion 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
negative 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 production of functional EPO protein by the EPO-
producing cells of a t, 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 general,
anemia can occur with a decline or loss of kidney function (e.g., chronic renal failure),
anemia ated with relative EPO deficiency, anemia associated with congestive heart
failure, 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 ions such as HIV, and anemia of c diseases such as autoimmune
diseases (e.g., toid arthritis), liver disease, and multi-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 e” as used herein refers to disorders associated with
any stage or degree of acute or chronic organ failure that results in a loss of the organ’s
ability to perform its function.
The term “kidney disease” as used herein refers to disorders associated with any
stage or degree of acute or chronic renal failure 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 ary to a variety of conditions, including (but
not limited to) heart failure, hypertension, diabetes, autoimmune disease, or liver disease.
Kidney e may be a ion of chronic renal failure that develops after an acute
injury to the kidney. For example, injury to the kidney by ia and/or exposure to
toxicants may cause acute renal failure; lete recovery 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 es for kidney e, anemia, EPO deficiency, tubular transport
deficiency, or glomerular tion deficiency wherein the object is to reverse, prevent or
slow down (lessen) the targeted disorder. Those in need of treatment include those already
having a kidney disease, anemia, EPO deficiency, tubular transport ency, or glomerular
filtration deficiency as well as those prone to having a kidney disease, anemia, EPO
deficiency, tubular transport deficiency, or glomerular filtration ency or those in whom
the kidney disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular
filtration deficiency is to be prevented. The term ment” as used herein includes the
stabilization and/or improvement of kidney function.
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, , or EPO deficiency. Such subjects e
t 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, , or EPO
deficiency, 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 nt” refers to any single animal, more preferably a mammal (including
such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows,
pigs, sheep, and non-human primates) for which treatment is desired. Most preferably, the
patient herein is a human.
The term “sample” or “patient sample” or “biological sample” shall generally mean
any biological sample obtained from a subject or patient, body fluid, body tissue, cell line,
tissue culture, or other source. The term es tissue biopsies such as, for example,
kidney biopsies. The term includes cultured cells such as, for example, cultured mammalian
kidney cells. Methods 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 bed herein. The test sample may originate from various sources in the
mammalian subject including, without tion, blood, semen, serum, urine, bone marrow,
, tissue, etc.
The term “control” or “control sample” 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 description 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, without limitation,
samples derived from subjects that have been treated with cological 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 described
herein. 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 e. 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 prognosis”, “regenerative prognosis”, or “prognostic for
regeneration” generally refers to a forecast or prediction of the le regenerative
course or outcome of the administration or implantation of a cell population, admixture or
construct described . For a regeneration prognosis, the forecast or prediction may be
informed by one or more of the following: improvement of a functional organ (e.g., the
) after implantation or administration, development of a functional kidney after
implantation or administration, development of improved kidney function or ty after
implantation or administration, and expression of certain markers by the native kidney
ing implantation or administration.
“Regenerated organ” refers to a native organ after implantation or administration of
a cell tion, admixture, or construct as described . The regenerated organ is
characterized by various indicators including, without limitation, development of function or
capacity in the native organ, improvement of function or capacity in the native organ, and
the expression of certain markers in the native organ. Those of ordinary skill in the art will
iate that other indicators may be le for characterizing a regenerated organ.
“Regenerated kidney” 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 s indicators including, without limitation, development of function or
ty in the native kidney, ement of function or capacity in the native kidney, and
the sion of certain markers in the native kidney. Those of ordinary skill in the art will
appreciate that other indicators may be suitable for characterizing a regenerated .
The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When reting ents in this specification and claims which include
the term “comprising”, 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.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of ation, this is generally for the purpose of
ing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission
that such documents, or such sources of information, in any jurisdiction, are prior art, or
form part of the common general knowledge in the art.
SRC+ cell populations
Described herein 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 further bioactive cell
tions, including but not limted to, endothelial cells, endothelial progenitors,
mesenchymal stem cells, adipose-derived progenitors, for use in the treatment of acute or
chronic kidney disease. 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 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, e-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 red components or
cell types. bed herein, are methods for preparing the SRC+ cell populations, as
described herein. The further bioactive components, e.g., endothelial cells, endothelial
progenitors, mesenchymal stem cells, adipose-derived progenitors, comprising the cell
population, may be present in any percentage ient to improve a cell or tissue
deficiency.
The cell populations of the invention may comprise one or more further bioactive
components, for example but not limited to, endothelial cells, endothelial progenitors,
mesenchymal stem cells, adipose-derived progenitors, sing the cell population. In
certain embodiments, the cell population comprises two further bioactive components. In
certain embodiments, the cell population comprises three further bioactive components. In
certain ments, the cell tion comprises four r bioactive components. In
certain embodiments, the cell population comprises five further bioactive components. In
certain embodiments, the cell population comprises six further bioactive components. In
certain embodiments, the cell population comprises seven further bioactive components. In
n embodiments, the cell population comprises eight further bioactive components. In
n embodiments, the cell tion comprises nine further bioactive components. In
certain embodiments, the cell population comprises ten further bioactive components.
In one embodiment, a further bioactive component is an epithelial cell. In one
embodiment, the epithelial cell is a proximal tubular epithelial cell. In another embodiment,
the epithelial cell is a distal tubular epithelial cell. In another embodiment, the epithelial cell
is a parietal lial cell.
In another embodiment, a further ive component is an epithelial cell. In one
embodiment, the epithelial cell is a venous elial cell. In one embodiment, the
epithelial cell is an arterial endothelial cell. In one embodiment, the lial cell is a
capillary endothelial cell. In one embodiment, the epithelial cell is a tic endothelial
cell.
In another ment, 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 component is a mesenchymal stem cell. In
another embodiment, a further ive component is a progenitor of endothelial,
mesenchymal, epithelial or hematopoietic lineage. In another embodiment, a further
bioactive component is a itor 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 bioactive component is a derivative of a stem cell of
any origin or derivation, including but not limited to nic (ES) and induced pluripotent
(iPS) stem cell, wherein the derivative may be ted 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 ment, a further bioactive component is a derivative of
a progenitor of endothelial, mesenchymal, epithelial or hematopoietic e 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 ment, a further bioactive ent 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 les 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 component is an intersitial
cell. In one embodiment, the interstitial cell is a supportive last. In r
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 neic to the subject. In certain
embodiments, a further bioactive ent is derived from a source that is autologous to
the subject, which further bioactive component is a combined admixture with a still further
ive components which are derived from a source that is allogeneic to the subject.
Described herein are methods for the targeted ration of renal mass and
functionality by directed delivery of the cell populations and/or organoids and/or
biomaterials described herein. Also described herein are methods for the rescue and/or
ry of renal functionality in patients having acute or chronic renal disease by
administration of cell populations and/or organoids and/or biomaterials described herein.
Therapeutic ids
Described herein are organoids comprising and/or formed from the bioactive
components described herein, e.g., B2, B4, and B3, which are depleted of inactive or
undesired components, e.g., B1 and B5, alone or d for use in the treatment of acute
and/or chronic kidney disease. Described herein are organiods 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 red ment, 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 specific cellular
ents of B3 and/or B4. In all embodiments, the organoids described herein are
formed and cultured ex vivo. In all embodiments, the organoids may further comprise
and/or be formed from further bioactive cell populations, including but not limted to,
endothelial cells, endothelial progenitors, hymal stem cells, adipose-derived
progenitors. In one embodiment, the further bioactive cell populations, including but not
limted to, elial cells, endothelial progenitors, mesenchymal stem cells, adiposederived
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 described herein 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
comprises a B4 cell population. In yet another embodiment, the heterogeneous renal cell
population r comprises a B3 population. In still another embodiment, the
geneous renal cell population further ses a B5 population.
In n embodiments, the cell tion 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.
Described herein are methods of forming organoids comprising and/or formed from
bioactive components described herein, e.g., B2, B4, and B3, which are ed of inactive
or undesired components, e.g., B1 and B5, alone or admixed. In one embodiment,
described are organiods 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 therapeutic properties, e.g., stabilization and/or improvement and/or regeneration
of kidney function, alone or when admixed with other ive subfractions, e.g., B2 and/or
B3. In a preferred embodiment, the ive 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 ents of B3 and/or B4.
In one embodiment, the organoids described 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 embodiment, the heterogenous renal cell tion 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 comprises a B5 population.
In certain embodiments, the cell population ses a B2 cell population, wherein
the B2 cell population comprises an enriched population of r cells, and is depleted of
a B1 cell population, and/or a B5 cell population.
Described herein are methods of forming organoids using the bio active cell
ations and/or admixtures described herein. General metho ds for generating s
from primary renal cell populations using 3D COL(I) gel culture are known in the art, for
example, as in Joraku et al., Methods. 2009 Feb;47(2):129-33. In all embodiments, the
organoids described herein are formed and cultured ex vivo.
In some ments, ion 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 culture: 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 ive cell ations and/or
admixtures bed herein may be induced in 2D culture. In one embodiment, the
bioactive cell preparations and/or admixtures bed herein are seeded on standard 2D
plastic-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 general, cell populations may be grown past ence 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 r embodinment, organoids formed from the bioactive cell preparations
and/or admixtures described herein may be induced in 3D culture. In one embodiment, the
organoid is generated with the cell populations described herein together with a biomaterial
scaffold of natural or synthetic origin. In one embodiment, formulated the bioactive cell
ations and/or admixtures described herein may be orated into a collagen (I)
gel, collagen (IV) gel, el or a mixture of any of these as previously described (see
Guimaraes-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 t 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 ted 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
networks of tubular structures organize as lattices and rings form throughout the gel matrix
by the ive 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 spinner flasks or low-bind
plasticware. In one embodiment, cells may be cultured in media in spinner 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 g of tubular structures from cultured
spheroids.
Cell populations
The SRC+ cell populations and/or organoids described herein may contain and/or be
formed from isolated, heterogeneous populations of kidney cells, and admixtures f,
enriched for specific bioactive components or cell types and/or depleted of ic inactive
or undesired components or cell types for use in the treatment of kidney disease, i.e.,
providing stabilization and/or improvement and/or regeneration 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
fractions, and/or admixtures of cells described herein may be derived from y
individuals, individuals with a kidney disease, or subjects as described herein.
Bioactive cell populations
Described herein are 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 population potentially having
therapeutic properties upon administration to a subject. For e, upon administration
to a subject in need, a id comprising a bioactive renal cell tion can provide
stabilization and/or improvement and/or regeneration of kidney function in the t.
The eutic properties may include a regenerative effect.
Bioactive cell populations include, t limitation, stem cells (e.g., pluripotent,
multipotent, oligopotent, or unipotent) such as embryonic stem cells, amniotic stem cells,
adult stem cells (e.g., hematopoietic, y, intestinal, mesenchymal, placental, lung,
bone marrow, blood, umbilical cord, endothelial, dental pulp, adipose, neural, olfactory,
neural crest, testicular), induced 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 heterogeneous in nature.
Those of ry skill in the art will appreciate other bioactive cell populations that are
suitable for use in generating the organoids 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 e to generate an organoid to be administered to the kidney.
Described herein areorganoids comprising certain ctions of a
heterogeneous population of renal cells, enriched for bioactive components and depleted of
inactive or undesired ents provide superior therapeutic and regenerative outcomes
than the starting population. For e, bioactive 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 on.
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 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 tion 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.
The B2 cell population is characterized by expression of a tubular cell marker
selected from the group ting of one or more of the following: megalin, cubilin,
hyaluronic acid synthase 2 (HAS2), Vitamin D3 25-Hydroxylase (CYP2D25), N-cadherin
(Ncad), E-cadherin , Aquaporin-1 (Aqp1), rin-2 (Aqp2), RAB17, member RAS
oncogene family (Rab17), GATA binding 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), 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 t
density between about 1.063 g/ml and about 1.073 g/ml (rodent), n 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
e). 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 ),
solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), alanyl
(membrane) aminopeptidase (Anpep), transmembrane protein 27 (Tmem27), acyl-CoA
synthetase medium-chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3),
fructose-1,6- biphosphatase 1 (Fbp1), and alanine-glyoxylate aminotransferase 2 (Agxt2).
B3 is also characterized by the vascular expression marker Platelet endothelial cell adhesion
molecule ) and the glomerular expression 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
ular marker set containing one or more of the following: n (Podn), and
Nephrin (Neph); and an oxygen-tunable EPO enriched population compared to
unfractionated (UNFX), B2 and B3. B4 is also characterized by the expression of one or
more of the following markers: ine (C-X-C motif) or 4 (Cxcr4), endothelin
receptor type B (Ednrb), collagen, type V, alpha 2 (Col5a2), Cadherin 5 (Cdh5), plasminogen
activator, tissue (Plat), angiopoietin 2 (Angpt2), kinase insert domain n or (Kdr),
secreted protein, acidic, cysteine-rich (osteonectin) (Sparc), serglycin (Srgn), TIMP
metallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless-type MMTV
integration site family, member 4 , regulator of G-protein signaling 4 (Rgs4), Platelet
elial cell on 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 (rodent), between about 1.063
g/ml and about 1.091 g/mL (human and canine).
The B4’ cell population is defined 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 endocrine marker EPO. In
one embodiment, the B4’ cell tion 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 population is characterized by the sion of a vascular
marker set containing one or more of the following: PECAM, vEGF, KDR, HIF1a and by the
sion of an endocrine marker EPO. In another embodiment, 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 (rodent), between about 1.063 g/ml and
about 1.091 g/mL (human and canine).
In one embodiment, described are organoids containing an isolated, enriched
B4’ population of human renal cells comprising at least one of erythropoietin (EPO)-
producing cells, ar 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 expression
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 oxygen-tunable erythropoietin (EPO) expression.
In one embodiment, the organoid as described herein contains the B4’ cell
tion but does not e 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 id does not include a B1 cell population sing 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 tion organoid does not include a B5 cell
population comprising debris and small cells of low granularity and ity 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 n 1.045 g/mL
and 1.052 g/mL; a B1 cell population comprising large granular cells of the collecting duct
and tubular 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.
Described herein areorganoids ning admixtures of human renal cells
comprising a first cell population, B2, comprising an isolated, enriched population of r
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
ting 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 certain ment, 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 ments, B2 r comprises collecting duct epithelial
cells. In one embodiment, the id ns or is formed from an admixture of cells
that 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 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 ment, the organoid contains an admixture that is capable of
providing a regenerative stimulus upon in vivo delivery. In other embodiments, the
admixture is e 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 tion is derived from a subject having
kidney e.
Described herein areorganoids containing an isolated, enriched B4’
population of human renal cells comprising at least one of erythropoietin 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 expression of a
vascular marker. In certain embodiments, the B4’ cell population is not characterized by
expression of a glomerular marker. 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 tion-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 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 comprising large granular cells of the collecting duct
and tubular 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.
Described herein areorganoids containing an admixture of human renal cells
sing a first cell tion, B2, comprising 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 opoietin producing cells and vascular cells but depleted of
glomerular cells having a y 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
sing debris and small cells of low granularity and viability with a density > 1.091 g/ml.
In certain ment, the B4’ cell population is characterized by sion of a vascular
marker. In one embodiment, the B4’ cell population is not terized by sion of a
ular marker. In certain embodiments, B2 further comprises collecting duct epithelial
cells. In one embodiment, the ure of cells is capable of receptor-mediated albumin
uptake. In another ment, 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 populations may be derived from kidney tissue or cultured kidney cells.
Described herein are organiods containing a heterogeneous renal cell
population comprising a combination of cell ons or enriched cell populations (e.g., B1,
B2, B3, B4 (or B4’), and B5). In one embodiment, the combination has a buoyant density
between about 1.045 g/ml and about 1.091 g/ml. In one other embodiment, the
ation 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 ment, the combination has a buoyant density
as ined by separation on a density gradient, e.g., by centrifugation. In yet another
embodiment, the combination of cell fractions 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 ation of an organoid comprising the
combination of B2, B3, and B4 (or B4’) cell fractions.
The inventors of the present invention 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 e of
providing a regenerative stimulus upon in vivo delivery. In other embodiments, the
admixture is e of reducing the decline of, izing, 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 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 preferred
embodiment, the admixture comprises B2 in combination with B3 and/or B4’. In other
red embodiments, the admixture consists of or consists essentially of (i) B2 in
combination with B3 and/or B4; or (ii) B2 in combination 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 subject
may be the same subject from which the B4’ fraction was obtained. In contrast 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 lar weight species of
hyaluronic acid (HA) both in vitro and in vivo, through the s 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 expected tion of high-molecular-weight
HA within the treated . y, the 5/6 Nx model left ted resulted in fibrosis
with limited detection of HAS-2 and little tion of high-molecular-weight HA. Without
wishing to be bound by , it is hypothesized that this anti-inflammatory high-molecular
weight species of HA produced 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, bed herein are organoids containing the bioactive
renal cells described herein along with a biomaterial comprising hyaluronic acid. Also
described herein is the provision of a biomaterial component of the regenerative stimulus
via direct production or ation of production by the implanted cells.
Described herein areorganoids containing and/or generated from isolated,
heterogeneous populations 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 embodiment, the cell
tions are d from a kidney biopsy. In r embodiment, the cell populations
are derived from whole kidney . 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 unfractionated cell
populations, also referred to herein as non-enriched cell populations.
Described herein are organoids containing and/or generated from isolated
populations of erythropoietin (EPO)-producing kidney cells that are further ed such
that the proportion of EPO-producing cells in the ed subpopulation is greater ve
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 tion of
interstitial fibroblasts and a lesser tion of tubular cells relative to the interstitial
fibroblasts and tubular cells contained in the unenriched l 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 tion. In such embodiments, these populations are referred to herein as the
“B4” cell population.
Described herein are organoids containing and/or generated 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 EPO-producing cells, e.g., B4. In another embodiment, the EPO-producing cell
population is a first cell population that is not enriched for EPO-producing 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 another
embodiment, the tubular cell phenotype may be indicated by the presence of one tubular
cell marker. In another embodiment, the r 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), aldehyde dehydrogenase 3 family,
member B1 b1), aldehyde dehydrogenase 1 , 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 vasculature.
The organoids described herein may include or be formed from EPO-
producing kidney cell populations containing B4 or B4’ in the form of an admixture 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 n 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 e
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 embodiment, the
EPO expression is d when the cell population is cultured under conditions where the
cells are subjected to a reduction in available oxygen levels in the culture system as
ed to a cell population ed at normal atmospheric (~21%) levels of available
oxygen. In one embodiment, EPO-producing cells ed in lower oxygen conditions
express greater levels of EPO ve to oducing 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 reduced oxygen is reduced
relative to the culturing of cells at normal heric 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%
oxygen, 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 culturing
cells at about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about
% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen,
about 20% , or about 21% oxygen.
In one other embodiment, induction or increased expression of EPO is
ed and can be observed by culturing cells at about less than 5% ble oxygen and
comparing EPO expression levels to cells cultured at atmospheric (about 21%) oxygen. In
another embodiment, the ion of EPO is obtained in a culture of cells capable of
expressing EPO by a method that includes a first culture phase in which the culture 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 cultured at
about less than 5% available oxygen. In another embodiment, the EPO expression that is
responsive 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 bed 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 ion 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, intermittent, or continuous fluid flow are ed in such a manner that they are
present as 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 ted 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 directionally through or across the lding. 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
finding that organoids comprising and/or formed from n subfractions of a
heterogeneous population of renal cells, enriched for bioactive components and ed of
inactive or red components, provide superior therapeutic and regenerative outcomes
than the starting population. In red embodiments, the organoids described herein
contain cellular tions 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 population comprises large, granular cells of the ting duct
and tubular , 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 buoyant density greater than about 1.091 g/ml.
Methods of isolating and culturing cell populations
The SRC+ cell populations and/or organoids described herein contain and/or
are formed from cell populations that have been isolated and/or cultured from kidney
tissue. s are provided herein for separating and isolating the renal cellular
components, e.g., enriched cell populations that are contained in the organoids for
therapeutic use, including the treatment of kidney disease, anemia, EPO deficiency, tubular
ort deficiency, and glomerular filtration deficiency. In one embodiment, the cell
populations are ed 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 es
of renal cells that have been cultured in hypoxic culture conditions prior to separation on a
density gradient provides for enhanced distribution and ition 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 specific cellular components
during the hypoxic culture period; 2) alterations in cell granularity and/or size in se to
the hypoxic culture, thereby effecting alterations in t density and uent
localization during density gradient separation; and 3) alterations in cell gene / protein
expression in se to the hypoxic culture period, y 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 isolating the cell populations of the
invention include separation on a density gradient based on the differential specific y
of different cell types ned within the population of interest. The specific gravity of any
given cell type can be nced by the degree of granularity within the cells, the
intracellular volume of water, and other factors. Described herein are optimal gradient
conditions for isolation 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 on, i.e., B4 cell population, derived from a
heterogeneous population of renal cells. In other embodiments, a density gradient is used
to obtain enriched subpopulations of tubular cells, ular cells, and endothelial 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 ar 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
debris. In one other ment, the endocrine, glomerular, and/or vascular cells are
ted 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 ar debris.
In one embodiment, the the organoids of the present ption contain
and/or are formed from cell populations generated by using, in part, the OPTIPREP® (Axis-
Shield) density gradient medium, comprising 60% nonionic ted 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 e markers known in the art, comprising necessary features for isolating the cell
populations of the t invention may be used in accordance with the invention. It
should also be recognized 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 separate cellular subpopulations via flow cytometry
(forward scatter = a reflection of size via flow cytometry, and side scatter = a reflection of
arity). Importantly, the density gradient medium should have low toxicity towards
the ic cells of interest. While the y gradient medium should have low toxicity
toward the specific cells of interest, the instant description contemplates the use of nt
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 populations of the t invention
recovered by the gradient comprising iodixanol are iodixanol-resistant, as there is an
appreciable loss of cells between the loading 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 iodixanol 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 tions for the ids described herein is also contemplated. In
addition, the density gradient medium should also not bind to proteins in human plasma or
adversely affect key functions of the cells of st.
Described herein 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 . 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
populations that have been enriched and/or depleted of kidney cell types using magnetic
cell sorting. In one embodiment, kidney cell types may be enriched and/or depleted using
the Miltenyi CS® 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 ion. In
one embodiment, the cell populations ed via three-dimensional culturing and
continuous ion demonstrate greater cellularity and onnectivity when compared
to cell populations cultured statically. In another embodiment, the cell populations cultured
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 demonstrate greater levels of glucose
and glutamine consumption when compared to cell tions ed statically.
As described herein, low or hypoxic oxygen conditions may be used in the
methods to prepare the cell tions 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 described herein.
Biomaterials
As described in Bertram et al. U.S. Published Application 20070276507,
polymeric matrices or lds 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 t invention may be threedimensional
and shaped to conform to the dimensions and shapes of an organ or tissue
structure. For example, in the use of the ric scaffold for treating kidney disease,
anemia, EPO ency, tubular transport deficiency, or glomerular filtration ency, 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 special needs of the patient. In another embodiment, the
ric matrix or scaffold may be a patible, porous ric 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,
osic ester, fluorinated polyethylene, phenolic, polymethylpentene, polyacrylonitrile,
polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate,
polycyanoarylether, polyester, polyestercarbonate, polyether, polyetheretherketone,
polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin,
polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, enylene sulfide,
polypropylene, yrene, polysulfide, polysulfone, trafluoroethylene,
polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated
cellulose, silicone, urea-formaldehyde, collagens, ns, ectin, glycosaminoglycans,
silk, elastin, alginate, hyaluronic acid, agarose, or copolymers or physical blends thereof.
Scaffolding configurations may range from liquid hydrogel suspensions to soft porous
scaffolds to rigid, shape-holding porous scaffolds.
The scaffold may be ed of any material form of biomaterial including
but not limited to diluents, cell carriers, micro-beads, material fragments, scaffolds of
synthetic composition including but not limited to PGA, PLGA, PLLA, OPLA, electrospun
bers and foams of synthetic composition including but not limited to PGA, PLGA,
PLLA, OPLA, ospun nanofibers.
Hydrogels may be formed from a variety of ric materials and are
useful in a variety of biomedical applications. Hydrogels can be described physically as
three-dimensional ks 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 ce of hydrophilic residues. Hydrogels swell extensively without
changing their nous structure. The basic physical features of hydrogel can be
ically modified, 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 synthetically derived material or combinations thereof, that is biologically inert and
physiologically compatible with mammalian tissues. The hydrogel material preferably does
not induce an matory response. Examples of other materials which can be used to
form a hydrogel e (a) modified alginates, (b) polysaccharides (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 thixotropic 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
rs, that are suitable for making hydrogels for use in accordance with the t
invention.
Scaffolding or biomaterial teristics may enable cells to attach and
interact with the scaffolding or biomaterial material, and/or may provide porous spaces into
which cells can be entrapped. In one embodiment, the porous scaffolds or biomaterials
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
. In r embodiment, the scaffolds or biomaterials allow or promote for cell:cell
and/or cell:biomaterial interactions within the scaffold to form constructs as described
herein.
] In one embodiment, the erial used in accordance with the present
invention is comprised of onic acid (HA) in hydrogel form, containing HA molecules
ranging in size from 5.1 kDA to >2 x 106 kDa. In another ment, the biomaterial used
in accordance with the present invention is comprised of hyaluronic acid in porous foam
form, also containing HA molecules g in size from 5.1 kDA to >2 x 106 kDa . In yet
another embodiment, the biomaterial used in ance with the present invention is
comprised of of a poly-lactic acid (PLA)-based foam, having an open-cell 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 ),
especially after intra-renal implantation.
The biomaterials bed herein may also be designed or adapted to
respond to n external ions, 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
described can be varied by ing the percentage of a biomaterial in the organoid.
Alternatively, erials may be chemically cross-linked to provide greater resistance to
enzymatic degradation. For instance, a carbodiimide crosslinker may be used to chemically
crosslink gelatin beads thereby providing a reduced susceptibility to endogenous enzymes.
In one embodiment, the response by the biomaterial to external conditions
concerns the loss of structural integrity of the biomaterial. Although aturesensitivity
and resistance to enzymatic degradation are ed above, other mechanisms
exist by which the loss of material integrity may occur in different biomaterials. These
isms may include, but are not limited to thermodynamic (e.g., a phase transition
such as g, ion (e.g., diffusion of an ionic crosslinker from a biomaterial into the
surrounding tissue)), chemical, enzymatic, pH (e.g., pH-sensitive mes), 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 scaffolds as described
herein.
Also described are constructs made from the referenced scaffolds or
biomaterials.
Constructs
Described herein are organoids that n implantable constructs having
one or more of the cell populations 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 populations
or admixtures of cells described herein deposited on or embedded in a surface of the
scaffold by attachment and/or entrapment. In certain embodiments, the uct is made
up of a biomaterial and one or more cell populations or admixtures of cells described herein
coated with, ted on, deposited in, attached to, entrapped in, embedded in, seeded,
or combined with the biomaterial component(s). Any of the cell populations bed
, 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
vascular 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 ment, the
deposited cell population or cellular component(s) of the construct includes both the first
enriched renal cell tion and a second renal cell population. In some embodiments,
the second cell population is not enriched for oxygen-tunable EPO producing cells. In
another embodiment, the second cell population is enriched for renal tubular cells. In
another ment, the second cell population is enriched for renal tubular cells and
contains collecting duct epithelial cells. In other embodiments, the renal r cells are
characterized by the expression of one or more tubular cell markers that may include,
without tion, 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 ), GATA binding n 3
(Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute carrier family 9
(sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family,
member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and
Calpain-8 (Capn8).
In one embodiment, the cell populations deposited on or combined with
biomaterials or lds to form constructs as bed herein are derived from a variety
of sources, such as autologous sources. tologous sources are also suitable for use,
including t limitation, allogeneic, or syngeneic (autogeneic or isogeneic) sources.
Those of ordinary skill in the art will appreciate there are several suitable
methods for depositing or otherwise combining cell populations with erials to form a
construct.
In one embodiment, the constructs useful in the present invention 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 ent for a kidney disease of any
etiology, anemia, or EPO deficiency of any etiology. In other embodiments, the constructs
are suitable for administration to a t 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 improved kidney function.
] In yet another embodiment, described is a construct for implantation into a
subject in need of improved kidney function comprising: a) a erial comprising one or
more biocompatible synthetic polymers or lly-occurring ns or peptides; and b)
an admixture of mammalian renal cells derived from a subject having kidney disease
comprising a first cell population, B2, comprising 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 ar 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, ed in and/or otherwise combined
with the erial. 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 includes a B4’ cell population which is
characterized by sion of a vascular marker. In some embodiments, the B4’ cell
population is not characterized by expression of a ular marker. In certain
embodiments, the ure is capable of oxygen-tunable erythropoietin (EPO) expression.
In all embodiments, the admixture may be derived from mammalian kidney tissue or
cultured kidney cells.
In one embodiment, the construct includes a biomaterial configured as a
three-dimensional (3-D) porous biomaterial suitable for ment and/or attachment of
the admixture. In another embodiment, the construct includes a biomaterial configured as
a liquid or semi-liquid gel suitable for embedding, attaching, ding, or coating
ian cells. In yet another embodiment, the construct includes a biomaterial
configured comprised of a predominantly olecular weight species of hyaluronic acid
(HA) in el form. In another embodiment, the construct includes a biomaterial
comprised of a predominantly high-molecular weight species of hyaluronic acid in porous
foam form. In yet another ment, 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 tions
that may be derived from a kidney sample that is autologous to the t in need of
improved 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 erythroid homeostasis.
Methods of use
Described herein are methods for the ent of a kidney disease, anemia,
or EPO deficiency in a t in need with SRC+ cell populations and/or organoids
containing and/or formed from the kidney cell tions and admixtures of kidney cells
described herein. In one embodiment, the method comprises administering to the subject
an organoid(s) that includes and/or is formed from a first kidney cell tion enriched for
EPO-producing cells. In r embodiment, the first cell population is enriched for EPO-
producing cells, glomerular cells, and vascular cells. In another embodiment, the
oraganoid(s) may further include and/or may be formed from one or more onal kidney
cell populations. In one embodiment, the additional cell population is a second cell
population not enriched for EPO-producing cells. In another embodiment, the additional
cell population is a second cell population not enriched for oducing cells, glomerular
cells, or vascular cells. In another embodiment, the organiod(s) also includes and/or is
formed from a kidney cell population or admixture of kidney cells deposited in, deposited
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 es or proteins, to
ate regeneration in acute or chronic disease states.
The effective treatment of a kidney disease, anemia, or EPO deficiency in a
subject by the methods as described herein can be observed h 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 calcium, orous:calcium ratio,
serum potassium, proteinuria, urine creatinine, serum nine, blood nitrogen urea
(BUN), cholesterol levels, triglyceride levels and glomerular filtration rate (GFR).
Furthermore, several indicators of general health and well-being include, without limitation,
weight gain or loss, survival, blood pressure (mean systemic blood re, diastolic blood
pressure, or systolic blood pressure), and physical endurance performance.
In another embodiment, an effective treatment with SRC+ cell populations or
bioactive renal cell organoids is evidenced by ization of one or more tors of
kidney function. The stabilization of kidney function is demonstrated by the ation of
a change in an indicator in a subject treated by a method as described herein as compared
to the same indicator in a t that has not been treated by a method as described
herein. Alternatively, the ization 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 compared 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 ment, the
treatment provided by the present description may include stabilization of blood urea
nitrogen (BUN) levels in a subject where the BUN levels observed in the subject are lower as
ed to a subject with a similar disease state who has not been treated by the methods
as bed herein. In one other embodiment, the treatment may include stabilization of
serum creatinine levels in a t where the serum creatinine levels observed in the
subject are lower as compared to a subject with a similar disease state who has not been
treated by the methods as described herein. In another embodiment, the treatment may
include stabilization of hematocrit (HCT) levels in a subject where the HCT levels observed in
the subject are higher as compared to a subject with a similar e state who has not
been treated by the methods as described 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.
s and Routes of Administration
The SRC+ cell populations and/or bioactive cell as described 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.
] Described herein aremethods of providing a ive cell organoid(s)
described herein to a subject in need. In one embodiment, the source of the bioactive cell
may be autologous or allogeneic, syngeneic eneic or isogeneic), and any combination
thereof. In instances where the source is not autologous, the methods may include the
administration of an suppressant agent. Suitable suppressant drugs
include, without limitation, azathioprine, cyclophosphamide, mizoribine, ciclosporin,
tacrolimus hydrate, chlorambucil, lobenzarit disodium, auranofin, alprostadil, gusperimus
hydrochloride, biosynsorb, muromonab, alefacept, pentostatin, daclizumab, sirolimus,
mycophenolate mofetil, leflonomide, basiliximab, dornase α, bindarid, cladribine,
pimecrolimus, ilodecakin, cedelizumab, efalizumab, everolimus, anisperimus, gavilimomab,
faralimomab, clofarabine, rapamycin, siplizumab, saireito, LDP-03, CD4, SR-43551, SK&F-
106615, IDEC-114, IDEC-131, 0, TSK-204, LF-080299, A-86281, A-802715, GVH-313,
HMR-1279, 9, IPL-423323, CBP-1011, 5, CNI-1493, CBP-2011, J-695, LJP-920,
L-732531, ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-1g, 90, ER-
38925, ISAtx-247, RDP-58, PNU-156804, LJP-1082, TMC-95A, TV-4710, PTRMG, and
96 (see U.S. Patent No. 7,563,822). Those of ordinary skill in the art will iate
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, sub-capsular placement, trans-urethral
catheterization, and intra-renal artery catheterization.
Cells can be inserted into a delivery device or vehicle, which facilitates
introduction by injection or implantation into the subjects. In certain embodiments, the
delivery vehicle can include l materials. In certain other embodiments, the delivery
vehicle can include synthetic materials. In one ment, 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 t. In a preferred
embodiment, the tubes additionally have a , e.g., a syringe, through which the cells of
the invention can be introduced into the subject at a desired location. In some
embodiments, mammalian kidney-derived cell tions 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 systems 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 ent forms. For example, the cells can be
suspended in a solution or gel. Cells can be mixed with a pharmaceutically able carrier
or diluent in which the cells of the ion remain viable. Pharmaceutically acceptable
carriers and diluents include saline, aqueous buffer solutions, solvents and/or sion
media. The use of such carriers and ts is well known in the art. The solution is
ably sterile and fluid, and will often be isotonic. ably, the solution is stable
under the conditions of cture and storage and preserved against the contaminating
action of microorganisms such as bacteria and fungi through the use of, for example,
parabens, chlorobutanol, 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 thereof of the instant invention can include combinations of the abovementioned
characteristics.
Modes of administration of the organoids containing and/or formed from
ed renal cell tion(s), for example, the B2 cell population alone or d with
B4’ and/or B3, include, but are not limited to, intra-parenchymal injection, sub-capsular
placement, or renal artery. Additional modes of administration useful accordance with the
present invention include single or multiple injection(s) via direct laparotomy, via direct
laparoscopy, transabdominal, or percutaneous. Still yet additional modes of administration
useful in accordance with the present invention include, for example, rade and
opelvic infusion. Surgical means of administration include one-step procedures such
as, but not d to, partial ctomy and construct implantation, partial nephrectomy,
partial pyelectomy, vascularization with omentum ± peritoneum, multifocal biopsy needle
tracks, cone or pyramidal, to cylinder, and renal pole-like replacement, as well as ep
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 r embodiment, each of the
organoids are delivered separately to ic ons or via specific methodologies, either
simultaneously or in a temporally-controlled manner, by one or more of the methods
described herein.
The appropriate cell or organoid implantation dosage in humans can be
determined from existing information relating to either the activity of the organoids, 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 ating 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 components can be added to the organoids comprising
and/or formed from cell populations and admixtures thereof of the instant invention,
including selected extracellular matrix ents, 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
Described herein are kits comprising the polymeric matrices and scaffolds as
described and related materials, and/or cell culture media and instructions for use. The
instructions for use may contain, for example, instructions for e of the cells in the
formation of the SRC+ cell pouplations or ids as described herein and/or
administration of the SRC+ cell pouplations or organoids. In one embodiment, described is a
kit comprising a scaffold as described herein and ctions. In yet another embodiment,
the kit es an agent for detection of marker sion, 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 organoid(s) described herein.
Another embodiment is an article of manufacture containing ids
containing and/or formed from bioactive cells useful for treatment of subjects in need. The
article of manufacture comprises a container and a label or package insert on or associated
with the container. Suitable containers include, for e, bottles, vials, syringes, etc. The
containers may be formed from a variety 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 ion. The label or package insert will further comprise
instructions for stering the organoid to the t. Articles of manufacture and kits
sing combinatorial ies described herein are also contemplated. Package insert
refers to instructions arily included in commercial packages of therapeutic products
that contain information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such eutic products. In one
embodiment, the package insert indicates that the organoid(s) is used for treating a disease
or disorder, such as, for example, a kidney disease or disorder. It may further include other
materials desirable from a cial and user standpoint, including other buffers,
diluents, filters, needles, and syringes. Kits are also described that are useful for various
purposes, e.g., for assessment of rative outcome. Kits can be provided which n
detection agents for derived vesicles and/or their contents, e.g., nucleic acids (such as
miRNA), vesicles, es, etc., as described herein. ion agents include, without
limitation, nucleic acid primers and probes, as well as antibodies for in vitro ion 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
provide a description of the composition as well as instructions for the ed in vitro,
prognostic, or diagnostic use.
The following examples are offered for illustrative es only, and are not
intended to limit the scope of the present invention in any way.
EXAMPLES
Example 1 - Isolation of Selected Renal Cell + (SRC+) cell populations: ion of
endothelial cells from renal biopsy
The following method provides an example of endothelial cell isolation
following enzymatic digestion using a previously adapted 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 hypertensive 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
ing cell suspension 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
ectin coated dishes at a cell density of 25K/cm2. The cultures are fed every 3 days.
When the EC cultures are 80-90% confluent, they are nized and d. The
trypsinized cells are labeled with CD31 ) primary antibody and positively ed
using Miltenyi anti-CD31 microbeads. The CD31+ sorted cells are washed, counted, plated
and seeded at a density of 10K/cm2 in fully mented EGM-2 medium. Endothelial cell
logy should be t post twenty four hours and these cells can be expanded
through multiple es. The vascular and/or lymphatic composition may be analyzed
using lineage-specific antibodies (e.g. VEGF3 –vascular capillary; LYVE1- lymphatic); the
specific elial subpopulation can be further selected/sorted using the same cell
surface phenotyping markers, using the Miltenyi micro-bead selection method.
After expansion and purification, the endothelial cell component of the NKA
SRC can be enriched to a larger percentage (>30%) than the natural frequency as observed
through buoyant density fractionation (<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 density
gradient fractionation (e.g. 60% B2-B4 + 40% EC) prior to transplantation.
Endothelial cell sorting using magnetic beads:
Cells harvested from the fibronectin coated flasks in EGM2 medium are
stained with mouse anti-human CD31 primary antibody at a concentration of 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 microbeads are added at and incubated for an additional
minutes 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 example 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 ed for four days at 21%O2 and harvested at 85% confluency.
Following this selection and culture period, the cells were counted and 9.5 million cells were
recovered. The sample collected for phenotypic analysis by FACS was ~ 90% positive for
CD31 (, right FACS panel).
Human CKD Endothelial Cell Isolation, terization and ion
Human kidney cell culture:
] Human kidney cells (See Table 1 below for donor information) were isolated
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 es 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 d to determine the tage 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 ted to ine 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 TC-treated). 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 positive cells ( 2.1% compared 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 percentage CD31
positive cells by 13 fold (35.5% from 2.7%) after passage 1.
Isolation of SRC+ cell populations: Examples of the isolation of r ive cell types:
A. Renal Epithelia: g activities are evaluating the entation of
the various epithelial cell compartments (parietal, proximal tubular, loop of Henle, distal
tubular cells) in the fractions isolated through buoyant y gradients. More specifically,
the lineage tracing studies e direct and ive 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 epithelial s that characterizes p0 ZSF1 culture (combined fractions B2-B3-B4)
as a mixture of proximal, distal and collecting duct cell types. Using the same cell surface
ion antibodies listed in Tables 2-4, the enrichment of these specialized epithelial cell
compartments through culture selection as previously described (culture conditions along
with magnetic sorting) is contemplated.
Table 2. Marker panel for tissue ic nephron staining
Proximal Tubule Distal Tubule
Antigen 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
Aquaporin 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 = ve cell in selected population of cells, Med –ind = medulary
collecting 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 ml Membrane/PT
IgG2b
Aquaporin 2 rb IgG abCAM Ab6415 0.1mg/ml Membrane/CD
Tamm ll Rb IgG Santa Sc- 0.2mg/ml Membrane/DT
glycoProtein Cruz 20631
LTA (Biotinylated) ---- Vector B-1325 2mg/ml Membrane/PT
DBA (Biotinylated) --- Vector B-1035 5mg/ml Membrane/DT/CD
UEA (Biotinylated) --- Vector B-1065 2mg/ml Membrane/CD
ECAD Ms BD 610182 0.25mg/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 ml
IgG2a
Isotype ctrl Ms BD 557351 0.5mg/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 tment 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 lial and podocytes): Anatomical
exclusion of the pelvis improves the isolation of the glomerular fraction. Using standard
operating procedure for kidney digest, bed infra., cells are isolated and filtered
through a 100µm Steri-flip filter (Millipore), and cell particles/clumps larger than 100µm
(contains the glomerular fraction) are re-digested for additional 20minutes. The digested
fraction is neutralized with DMEM medium ning 10% FBS and washed by
fugation (300xg for 5min). The re-suspended cell pellet is cultured in VRADD medium6
F12, 1uM All Trans Retinoic Acid (Genzyme, Cambridge, MA), 0.1um
Dexamethasone -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, cultured 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 yi ads at passage 1 using
either PEC ic markers (such as but not limited to Claudin-1) or progenitor ic
s (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
ular capillary endothelial cells (e.g. VEFG3 or CD31 or LIVE1). When harvested at
optimal cell yields and combined with the B2 component at a greater than natural
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 ing procedures for isolation and expansion and harvest of primary kidney
cells for rodent, canine and human, as described infra., is followed for isolation and
expansion of collecting duct epithelial cells. Standard operating procedures for onating
sub-populations of primary 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 papilla/inter-medullary collecting duct
(IMCD) culture protocol from Dr. Ben Humphreys (REGM media supplemented with EGF;
unpublished data). In any of these scenarios, a cell fraction enriched for collecting duct
lia 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 diseased
etiologies ated with urine tration 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, vaso-ureter obstruction).
Example 2 - Generation and characterization of ids 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 described 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 stirrer set at 80rpm (. Cell organoids/spheroids consist of clusters
of cells g in size from 50-125µM. Cell number per organoid can vary based on cell
type and size prior spinner flask culture. Organoid/spheroids have been generated from
both rat and human SRC and express a r 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 ids cultured in a 50:50 mixture of Collagen
1/Collagen IV gelatin in 3D. Upon immuno-fluorescent ng of the cultured organoids,
the ant tubes ue to express an epithelial phenotype (.
Organoid Plus
The ability of SRC to form self-generated ids may prove advantageous
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 tion during the organoid formation period, a functional unit may
be formed recapitulating key cell signaling pathways activated during regenerative
outcomes. es of such cell-cell interactions include but are not limited to epithelial-
mesenchymal signaling events known to be pivotal during organogenesis (see Basu &
Ludlow 2012, Developmental engineering the kidney-leveraging genic ples for
renal regeneration. Birth Defects ch 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 membrane 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 without potential concomitant vascularization
(.
Example 3 - Characterization/biodistribution of SRC ids 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
parenchyma with 2.5x106 SPIO Rhodamine labeled cells (representing approximately 25000
self-generated organoids 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. Kidneys
were evaluated by MRI and histologically using Prussian blue and H&E ng 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 rs of epitheliod cells (staining
ve by Prussian 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 s to demonstrate therapeutic efficacy of SRC+ and SRC/SRC+
derived ids: CKD (5/6 ctomy) immune-compromised rodent studies
Human-derived SRC+ and SRC/SRC+ organoids were evaluated for
therapeutic 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, treatment intervention modalities and clinical evaluation of therapeutic
response was as previously described (Genheimer et al., 2012. Molecular characterization of
the regenerative response d by intrarenal transplantation of selected renal cells in
rodent model of chronic kidney disease. Cells s Organs 196: 374-84).
Cell isolation 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 en freezer for longer storage. Following cryopreservation, the cells were
quickly thawed at 37°C to assess ry and viability using standard Trypan blue exclusion
method. Cells were then seeded onto tissue culture vessels at 3000 cm2 and cultured
for 4 days under normoxic ions (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 (2%O2/5% CO2/37°C) prior to cell harvest and selection of SRC
by density nt separation. Human Umbilical Vein Endothelial Cells (HUVEC) es
(Lonza 2389, CAT# CC2517) were expanded from early e ing cryopreservation
until passage 3 in EGM2 fully supplemented medium. Cells were harvested and viability was
assessed using standard Trypan blue ion method.
Organoid ion: Organoid cultures were established by 1) resuspending
human SRC and HUVEC’s to a concentration of 2x106 cells/ml in their respective mediums 2)
combining equal volumes of each cell preparation 25mls (50x106 ml) into a 125ml
spinner flask (Corning). To the combined culture, 25mls of each medium was added to the
flasks for a final cell concentration of 1x106 cells/ml in . 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. Organoid numbers were adjusted
to the concentration above in a larger volume of 0.5mL of DPBS to account for any loss
during shipping. An extra tube was sent as replacement. 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 accessed 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 delivery.
Targeted 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 ) underwent 2 step nephrectomy procedures as follows: For each individual
animal the right kidney was removed and weighed, recovery d for 1 week, followed
by resection of tissue from caudal and l poles of left kidney which was also weighed.
A 2-3 week recovery and acclimation period followed prior to the beginning of the studies.
ing the acclimation period, blood and urine were collected for 2 consecutive weeks
and analyzed. Subsequent sampling was med every 2 weeks thereafter. All rodents
were fed a commercially available feed, and water was provided ad libitum.
Animals were monitored post-nephrectomy with some evidence of increasing
urine n/urine protein creatinine (UPC) and were treated with human organoids at 12
weeks ephrectomy. Table 5 below es 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
Untreated
Term of
(n=8) Necropsy
Observation
• Gross ogy
204 days post
observations
Nephrectomy
119 days post- • Abdomen opened
Tx and whole animal
fixed if unscheduled
death
Measurements: Body weights (g) were collected on a biweekly . Serum biochemical
and hematological evaluations were conducted for the on of study on a bi-weekly
basis. Urinalysis 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 s and tissues were ted as , weighed, measured and
prepared for histological processing. Methods used in histological evaluations of kidney
were performed. KMR is kidney mass ion.
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
previously described nephrectomy models and CKD-related sequelae including disruption of
kidney protein handling, azotemia, hypercholesterolemia, anemia, and ping uremia.
The outcome of NIHRNU Nephrectomy model in regard to disease state achieved 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 histological evaluations.
NKO Prototype Effect on In-Life Clinical Pathology
Clinical ogy measures relevant to kidney function were examined pretreatment
and compared in a paired n to measures taken prior to end of study. From
this comparison, as summarized in the table below, there were changes consistent with
disease progression over the 4 month study in untreated control animals. Significant
differences were noted in paired BUN, Hct, Hgb, RBC, WBC, sChol, sProt, sAlb, uPro, UPC,
and . s in these markers 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 otemia. In animals treated with NKO, changes in
BUN, Hct, Hgb, RBC, WBC, and spGrav were not significant as would be predicted 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
Measure 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
RBC Control 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 l 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
id 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
id 110.25 34.79 50.67 6.74 0.116
uK Control 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
Paired t-
Time 0 End of Study Significant
test
Measure Group Average SD Average SD Change p-value
Organoid 133.25 29.28 79.33 7.02 0.24
spGrav l 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 sed 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 impaired tubule
spGrav urine concentration function
*the WBC here would not reflect a T-cell mediated response as these animals are c
Histological Overview of Kidneys from Model
Histopathology demonstrated that control animals had progressed to mild,
developing pathy by terminal ice.
Injury-related gs were primarily tubular in nature with y, 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 severity.
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 lete 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 tory rats, was observed.
There were no significant differences noted between ted and NKO
treated groups.
Table 7
Measure Normal Untreated 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) ore no nable survival benefit of NKO treatment was
detected.
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 background 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.
Typically 1 or 2 cells were identified in one of four sections d for each animal and were
incorporated into tubules, although one cluster was identified within the interstitium.
CONCLUSIONS:
• Organoid (NKO) delivery mitigated significant changes associated with kidney disease
ssion in l animals and included measures effected by
syngeneic/autologous NKA deliveries to disease models.
• Effected measures were consistent with those observed with syngeneic/autologous
NKA deliveries to disease models – measures of anemia (Hct, Hgb, RBC),
inflammation (WBC), urine concentration (spGrav) and ly 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) resulted in a state of early stage and
chronically progressive renal insufficiency characterized by developing anemia,
proteinuria, dyslipidemia and le indications of early stage azotemia.
• All animals survived until end of study.
• Procedures utilized to r 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 terized by severe diffuse chronic interstitial fibrosis and
crescentic glomerulonephritis with multifocal fibrosis. Clinical try confirmed azotemia
tion of blood urea nitrogen and serum creatinine), and mild anemia (mild reduction in
hematocrit and depressed hemoglobin levels). Cells were ed, expanded, and
characterized from both ed 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 ghs the fibrosis (blue staining indicated by ) in the diseased
kidney tissue compared to the normal kidney tissue. Functional r cells, sing
cubulin:megalin and capable 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, molecular analyses confirmed that the EPO-expressing cells from both
normal and diseased tissue responded to hypoxic conditions 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 mechanical digestion
and explant culture. At passage two, explant-derived cell cultures containing pressing
cells were subjected to both atmospheric (21%) and varying hypoxic (<5%) e
conditions to ine whether exposure to a culminated in upregulation of EPO
gene expression. As noted with rodent cultures (see Example 3), the normal pig displayed
oxygen-dependent sion 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 regulated, 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 demonstrated the y to self-organize into tubule-like structures. As shown in
Figure 4 of Presnell et al. WO/2010/056328 (incorporated herein by reference in its
entirety), the presence of functional tubular cells in the e (at passage 3) was confirmed
by observing receptor-mediated uptake of FITC-conjugated n by the cultured cells.
The green dots (indicated by thin white arrows) represent endocytosed sceinconjugated
albumin which is mediated by r cell-specific receptors, Megalin and
Cubilin, indicating n 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 s, even in renal tissues that have been severely compromised with CKD.
Furthermore, these findings support 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. 0/056328 (incorporated herein by reference in its entirety), the isolation
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 ty), it is possible to maintain the human EPO ing cells in culture with
retention of EPO gene expression. Human cells were cultured/propagated on plain tissueculture
treated 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 ion: Briefly, batches of 10, 2-week-old male Lewis rat kidneys
were obtained from a commercial supplier (Hilltop Lab Animals Inc.) and shipped overnight
in Viaspan preservation medium at a temperature around 4°C. All steps described herein
were d 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 ing kidney capsules were removed as well as any
remaining stromaltissue. 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 fuge 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 frozen, it was then transferred to the -80
degree freezer until analysis. The tissue weight of 10 juvenile kidneys d
approximately 1 gram. Based on the weight of the batch, the digestion medium was
adjusted 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 appropriate 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 s. This
first digestion step removes many red blood cells and enhances the digestion of the
remaining tissue. 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
er (BD Falcon) into an equal volume of neutralization buffer (DMEM w/ 10% FBS) to
stop the ion on. 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 acquired for cell counting and ity assessment using trypan blue
exclusion. Once the cell count was calculated, 1 million cells were collected for RNA,
washed in PBS, and snap frozen in liquid en. 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 conical centrifuge tubes (BD Falcon) and mixed by inversion 6
times. This formed a final mixture of 15% (w/v) of Optiprep®. Post ion, tubes were
carefully layered with 1 mL PBS. The tubes were centrifuged at 800 x g for 15 minutes
without brake. After fugation, the tubes were removed and a cell band was formed at
the top of the mixing gradient. There was also a pellet ning red blood cells, dead cells,
and a small population of live cells that included some small less granular cells, some epoproducing
cells, some tubular 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 centrifugation at 300xg
for 5 minutes. Post washing, the cells were re-suspended in 20mls of KSFM and a sample for
cell counting was collected. Once the cell count was calculated using trypan blue exclusion,
1 million cells were collected for an RNA , washed in PBS, and snap frozen in liquid
nitrogen.
Pre-Culture ‘Clean-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
“Kidney Cell Isolation”. As an optional step and as a means of ng up the initial
ation, up to 100 million total cells, suspended in e 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 sion. The gradient tubes were then carefully loaded into the
centrifuge, ensuring riate balance. The nt tubes were centrifuged at 800 x g
for 15 s at 25˚C without brake. The cleaned-up cell population (containing viable and
functional collecting duct, tubular, endocrine, glomerular, and vascular cells) segmented
between 6% and 8% (w/v) Optiprep®, corresponding to a y between 1.025 – 1.045
g/mL. Other cells and debris pelleted to the bottom of the tube.
Kidney Cell Culture: 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 e 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
incubator 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% -level
environment provided by a CO2 gen gas multigas humidified incubator (Sanyo) for
24hrs. Following the 24hr incubation, the cells were washed with 60mls of 1XPBS and then
removed using 40mls 0.25% (w/v) trypsin/EDTA (Gibco). Upon removal, the cell sion
was neutralized with an equal volume of KSFM containing 10% FBS. The cells were then
washed by fugation 300xg for 10 minutes. After washing, the cells were re-suspended
in 20mls of KSFM and transferred to a 50ml l tube and a sample was collected for cell
counting. Once the viable cell count was determined using trypan blue exclusion, 1 million
cells were collected for an RNA sample, washed in PBS, and snap frozen in liquid nitrogen.
The cells were washed again in PBS and collected by centrifugation at 300xg for 5 minutes.
The washed cell pellet was re-suspended in KSFM at a tration of 37.5 million
cells/ml.
Enriching for Specific Bioactive Renal Cells Using Density Step Gradient
tion: Cultured kidney cells, predominantly composed of renal tubular cells but
containing small subpopulations of other cell types (collecting duct, glomerular, vascular,
and endocrine) were separated into their component subpopulations using a density step
gradient made from multiple concentrations w/v of iodixanol (Optiprep). The cultures were
placed into a hypoxic environment for up to 24 hours prior to harvest and application to the
gradient. A stepped 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 nt and centrifuged, which resulted in segregation of the population into
multiple bands based on size and granularity.
y, densities of 7, 11, 13, and 16% Optiprep® (60% w/v Iodixanol) were
made using KFSM medium as ts. 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 peristaltic pump (Master Flex L/S) loaded with sterile L/S 16 Tygon
tubing connected to sterile ary 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.
y, 2 mL of cell suspension containing 75 million cultured rodent kidney cells was loaded
atop the step gradient (suspensions having been ted as described 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 nt. The step
gradients, loaded with cells, were then centrifuged at 800 x g for 20 minutes t 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 conical tube). Each fraction 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 suspensions are subjected 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 r 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 fractionation immediately after
isolation, the fraction ed for epo-producing cells, glomerular podocytes, and vascular
cells (“B4”) segregates at a density between 1.025 – 1.035 g/mL. In contrast, when density
nt separation was performed after ex vivo culture, the fraction enriched for epoproducing
cells, glomerular podocytes, 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” ons was enhanced by exposure (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 radient 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 sample, 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 ctomy procedure at Charles River Laboratories.
Characteristics of B4 were med by tative real-time PCR, including oxygenregulated
expression of erythropoietin and vEGF, expression of glomerular markers
(nephrin, podocin), and expression of vascular markers ). Phenotype of the ‘B2’
fraction was confirmed via expression 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 oducing 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 oducing and tubular cells
to be separated from red blood cells, cellular debris, and other potentially undesirable cell
types, such as large cell aggregates and certain types of immune cells.
The step gradient procedure may e 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 nt (5-10%, for example). Continuous gradients
can be prepared with any standard y gradient solution (Ficoll, Percoll, Sucrose,
iodixanol) according to standard methods (Axis Shield). Cells of interest are loaded onto the
continuous nt and centrifuged at 800xG for 20 minutes t 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 specific gravity of the solution at that
position also measured. Thus, subsequently, a defined step gradient can be derived that
focuses isolation of particular cell tions based on their ability to transverse the
density gradient under specific conditions. Such optimization may need to be employed
when isolating cells from unhealthy vs. healthy tissue, or when isolating specific cells from
different species. For e, optimization was conducted on both canine and human
renal cell cultures, to insure that the specific B2 and B4 subpopulations that were identified
in the rat were isolatable from the other s. 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 l gradient for isolation of human B2 and B4
subpopulations consists of (w/v) 7%, 9%, 11%, 16%. Thus, the y range for localization
of B2 and B4 from cultured 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 1.063-1.091g/ml 1.063-1.091g/ml
Example 7 – ygen 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, ney cell preparations from different species
were exposed to different oxygen conditions prior to the gradient step. A rodent neokidney
augmentation (NKA) cell preparation (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 tor set to 2% oxygen, while
the remaining flasks were kept at the 21% oxygen conditions, for an additional 24 hours.
Cells were then harvested from each set of conditions using standard tic harvesting
procedures described supra. Step gradients were ed according to rd
procedures and the “normoxic” (21% oxygen) and “hypoxic” (2% oxygen) cultures were
ted separately and applied 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
hout the gradient was different in 21% and 2% oxygen-cultured batches (Table 3).
Specifically, the yield of B2 was increased with hypoxia, with a concomitant decrease in B3.
Furthermore, the expression of cific genes (such as erythropoietin) was enhanced in
the resulting nt 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 ion and culture
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 ined at 21%. Subsequently, each set of flasks was
harvested and subjected to identical step gradients (Figure 28). Similar to the rat results
(Example 6), the hypoxic-cultured dog cells distributed throughout the nt 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 bution of specific lized cells (erythropoietinproducing
cells, vascular cells, and glomerular cells) into B4. Thus, hypoxic culture, followed
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 ular cells were isolated and propagated from normal
human kidney tissue by the enzymatic isolation methods described throughout. By the
gradient method described above, the r cell fraction was ed 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 utes 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.
WO/2010/056328 (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 expression of tubular , cadherin, was expressed in the renriched
population. This confirms that the enrichment of tubular cells can be maintained
beyond the initial enrichment when the cells are cultured in a 3D dynamic environment. The
same cultured population of kidney cells described above in Example 7 was subjected to
flow cytometric analysis to examine forward scatter and side scatter. The small, less
granular EPO-producing cell population was discernable (8.15%) and was separated via
positive selection of the small, less ar population using the sorting capability of a flow
cytometer (see Figure 70 of Presnell et al. WO/2010/056328 (incorporated herein by
reference in its ty)).
e 9 - Characterization of an unfractionated mixture of renal cells isolated from an
mune glomerulonephritis patient sample
An unfractionated mixture of renal cells was isolated, as described above,
from an autoimmune 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 ential ype-specific and pathway-specific gene expression
patterns among the cell subfractions. As shown in Table 6.1 of Ilagan et al.
, HK20 is an autoimmune glomerulonephritis patient sample. As shown
in Table 6.2 of Ilagan et al. , cells generated from HK20 are lacking
glomerular cells, as ined by qRTPCR.
EXAMPLE 11 – Enrichment/Depletion of Viable Kidney Cell Types Using Fluorescent
ted 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 activated cell sorting (FACS).
REAGENTS: 70% ethanol; Wash buffer (PBS ); 50:50 Kidney cell medium
(50%DMEM high glucose): 50% Keratinocyte-SFM; Trypan Blue 0.4%; Primary antibodies to
target kidney cell population such as CD31 for kidney elial 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 ng the biological safety
cabinet (BSC), a single cell suspension of kidney cells from either primary ion 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 ity 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 tion of kidney cells is then
d with primary dy specific 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
conjugated such as CD31 PE (specific for kidney endothelial cells) or un-conjugated such as
n (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 antibody is ed. If cells are labeled with a
fluorochrome conjugated primary antibody, cells are resuspended 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 incubation, 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 manufacturer’s
instructions (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 ic wavelength. Another gate is
drawn to select the ve tion. Once the desired gates have been drawn to
encapsulate the positive target population and the negative tion, the cells are sorted
using manufacturer’s instructions.
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 cytometry to determine purity.
Collected cells are washed by centrifugation at 300xg for 5 min. and the
pellet is resuspended in kidney cell medium for further analysis and experimentation.
EXAMPLE 12 – Enrichment/Depletion of Kidney Cell Types Using Magnetic Cell Sorting
One or more isolated kidney cells may be enriched and/or one or more
specific kidney cell types may be depleted from isolated y kidney tissue.
TS: 70% ethanol, Wash buffer (PBS ), 50:50 Kidney cell medium
EM high glucose): 50% Keratinocyte-SFM, Trypan Blue 0.4%, Running Buffer(PBS,
2mM EDTA,0.5% BSA), Rinsing Buffer (PBS,2mM EDTA), ng on (70% v/v ethanol),
Miltenyi FCR Blocking reagent, Miltenyi microbeads ic for either IgG isotype, target
antibody such as CD31(PECAM) or Nephrin, or secondary antibody.
PROCEDURE: Following standard procedures for cleaning the biological safety
cabinet (BSC), a single cell suspension of kidney cells from either y isolation or culture
is obtained and resuspended in kidney cell medium. Cell count and viability is determined
using trypan blue exclusion method. For kidney cell enrichment/depletion of, for e,
glomerular cells or elial 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
example, glomerular cells using Nephrin antibody, the Miltenyi autoMACS, or equivalent,
instrument program POSSELDS (double positive selection in sensitive mode) is used. For
ion of a target frequency of r than 10%, the yi autoMACS, or equivalent,
instrument program DEPLETES (depletion 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 fuge tube, followed by incubation for 15 minutes at
4ºC.
After labeling, cells are washed to remove d primary dy by
adding 1-2ml of buffer per 10 x107 cells followed by centrifugation at 300xg for 5min. After
washing, isotype specific secondary antibody, such as n 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 d ary antibody by
adding 1-2ml of buffer per 10 x107 cells followed 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 t 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 te for 15 min at 4ºC.
After incubation, cells are washed by adding 10-20x the labeling volume of
buffer and centrifuging the cell suspension 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 cleaned and primed
in preparation for magnetic cell separation using autoMACS. 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 depletion the DEPLETES 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 depleted 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 determine the functional
characterization of human NKA cells h high content analysis (HCA). ontent
imaging (HCI) provides aneous imaging of multiple sub-cellular events using two or
more fluorescent probes (multiplexing) across a number of samples. High-content Analysis
(HCA) provides simultaneous quantitative ement of multiple cellular parameters
captured in High-Content Images. In brief, unfractionated (UNFX) cultures were generated
(Aboushwareb et al., supra 2008) and maintained ndently 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, including 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 s. Ex vivo
cultures were established from all non-CKD (3/3) and CKD (5/5) kidneys. High t
analysis (HCA) of albumin transport in human NKA cells defining regions of interest (ROI) is
shown in Figure 5 (HCA of albumin transport in human NKA cells) of Ilagan et al.
. Quantitative comparison of albumin ort in NKA cells d
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
sion between tubular-enriched B2 and tubular cell-depleted B4 subfractions is shown
in Figure 7 (CK8/18/19) 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 enriched in proximal tubule cells and thus ts
increased albumin-transport on.
Albumin uptake: Culture media of cells grown to confluency 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) containing 1X antimycotic/antibiotic and 2mM
glutamine. Immediately prior to assay, cells were washed and incubated for 30 minutes with
/lg DMEM + 10mM HEPES, 2mM glutamine,1.8mM CaCl2, and 1mM MgCl2. Cells were
d 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 receptor-
associated 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 son) (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 expression. A
quantifiable ex-vivo HCA assay for measuring albumin 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 cellular function, g that n
transport-competent cells were retained in NKA cultures derived from human CKD kidneys.
It was also shown that specific subfractions of NKA cultures, B2 and B4, were distinct in
ype and on, with B2 representing a tubular cell-enriched fraction with
enhanced albumin transport activity. The B2 cell subpopulation from human CKD are
phenotypically and functionally analogous to rodent B2 cells that demonstrated efficacy in
vivo (as shown above).
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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 suspensions. J Am Soc Nephrol 23,1-12. (2012).
. Buzhor et al. Kidney ids Recapitulate Tubular ids Leading to Enhanced
Tubulogenic Potency of Human Kideny-Derived Cells Tissue Engineering Part A, .
11. Brown, SA et al., Impaired renal gulatory ability in dogs with reduced renal
mass. J Am Soc Nephrol. 5:1768-74 (1995).
12. Robertson, J.L. et al., Long-term renal responses to high dietary protein in dogs with
75% nephrectomy. Kidney Int. 29:511-9 (1986).
13. Urie, B.K. et al., Evaluation of clinical status, renal function, and hematopoietic
variables after unilateral nephrectomy 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 heterogeneous renal cell population and a bioactive cell
population in cell culture media in a 3-dimensional (D) culture ,
wherein the 3D culture system comprises a spinner and lacks an exogenous scaffold
to which cells of the heterogenerous renal cell population attach;
wherein the heterogeneous renal cell population is ed for renal r
cells, and
wherein the 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 cell
population.
2. The method of claim 1 wherein the heterogeneous renal cell population further
comprises epithelial cells from the collecting duct.
3. The method of claim 1 wherein the bioactive cell population is the non-renal
elial cell population.
4. The method of claim 3 wherein the non-renal endothelial cell population is a cell
line.
. The method of claim 4 wherein the cell line comprises human umbilical vascular
endothelial cells.
6. The method of claim 1 wherein the heterogeneous renal cell tion and the
ive cell population are xenogeneic, syngeneic, allogeneic, or gous.
7. The method of claim 1 wherein the heterogeneous renal cell population and the
bioactive cell population are cultured tely for a first time period, combined
and cultured for a second time period.
8. The method of claim 7 n the second time period is at least 24 hours.
9. The method of claim 8 wherein the second time period is 24 hours to 72 hours.
. The method of claim 1 wherein cells of the heterogeneous renal cell population and
the bioactive cell population are at a ratio of 1:1.
11. A id made according the method of any one of claims 1 to 10.
12. An ed cluster of cells comprising a heterogeneous renal cell population and a
ive cell population, wherein the heterogeneous renal cell population is
enriched for renal r cells,
n the 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 cell
population; and
wherein the cluster of cells is cultured in media in suspension without
attachment to a scaffold.
13. The isolated cluster of cells of claim 12 wherein the bioactive cell population is the
non-renal endothelial cell tion.
14. The isolated cluster of cells of claim 13 wherein the heterogeneous renal cell
population further comprises epithelial cells from the collecting duct.
. The isolated cluster of cells of claim 13 wherein the non-renal endothelial cell
population is a cell line.
16. The isolated cluster of cells of claim 15 wherein the cell line ses human
umbilical vascular endothelial HUVEC cells.
17. An injectable formulation comprising at least one cluster of cells as claimed in any of
claims 12-16 and a liquid medium.
18. The formulation of claim 17, wherein the liquid medium is selected from a cell
growth medium, Dulbecco’s ate buffered saline DPBS and combinations
thereof.
19. The formulation of claim 18 wherein the liquid medium is DPBS.
. The formulation of claim 17, n the at least cluster of cells is suspended in the
liquid medium.
21. An injectable formulation comprising at least one cluster of cells as claimed in any of
claims 12 to 16 and a hydrogel.
22. The formulation of claim 21, wherein the hydrogel comprises gelatin.
23. The ation of claim 22, wherein the gelatin is present in the formulation at
about 0.5% to about 1% (w/v).
24. The formulation of claim 22, wherein the gelatin is present in the formulation at
about 0.75% (w/v).
. Use of at least one cluster of cells as claimed in any one of claims 12-16 and a liquid
medium in the manufacture of a medicament to treat kidney e in a subject in
need thereof.
26. Use of at least one cluster of cells as claimed in any one of claims 12-16 and a
hydrogel in the cture of a medicament to treat kidney disease in a subject in
need thereof.
27. The use according to claim 25 or claim 26 wherein to treat kidney disease in the
subject comprises an improvement in any one of the following measures of anemia
(Hct, Hgb, RBC), mation (WBC), urine tration (spGrav) and azotemia
(BUN).
28. The use of any one of claims 25 to 27, wherein the ment is ated for
administration by injection.
29. The method according to any one of claims 1 to 10, substantially as herein described
with reference to any example thereof.
. The spheroid according to claim 11, substantially as herein described with reference
to any example thereof.
31. The isolated cluster of cells according to any one of claims 12 to 16, substantially as
herein described with reference to any example thereof.
32. The formulation according to any one of claims 17 to 24, substantially as herein
described with reference to any example thereof.
33. The use according to any one of claims 24 to 28, substantially as herein bed
with reference to any example 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
led control 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
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 | ||
NZ713875A NZ713875B2 (en) | 2013-05-08 | 2014-05-08 | Organoids comprising isolated renal cells and uses thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ752702A NZ752702A (en) | 2021-11-26 |
NZ752702B2 true NZ752702B2 (en) | 2022-03-01 |
Family
ID=
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