Isolated renal cells and uses thereof

Abstract

To provide an isolated renal cell including a renal tubule and a cell mass of a kidney that produces erythropoietin (EPO), and a method for isolating and culturing the cell, and a method for treating a subject who needs it by using the cell mass.SOLUTION: In one embodiment, a mixture of human renal cells includes a first cell mass including isolated and condensed mass of renal tubule cells having the density of 1.045 g / mL to 1.052 g / mL, and a second cell mass including a B2 cell mass, cells producing erythropoietin (EPO), glomerular cells, and vascular cells, where the mixture does not include the B1 cell mass including a collecting duct having the density of less than 1.045 g / mL and renal tubule based large-sized granular cells, or a B5 cell mass including debris and small cells of low particle size and survival rate having the density exceeding 1.091 g / mL.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application was filed on November 12, 2008 under 35 U.S.C. § 119(e). U.S. Provisional Application No. 61 / 114,025, filed November 12, 2008; No. 114,030, filed December 5, 2008, No. 61 / 201,056, 20 No. 61 / 201,305 filed December 8, 2008, and No. 61 / 201,305 filed December 10, 2008 This application claims priority to application Ser. No. 61 / 121,311, filed on 2006 / 01 / 121, which is hereby incorporated by reference. The entire contents of which are incorporated herein by reference. The subject matter of this application is filed on November 12, 2009. No. 61 / 260,833 filed on Dec. 1, 2002, the disclosure of which is incorporated herein by reference. be absorbed.

[0002] Technical Field The present invention relates to a renal tubular cell population and a cell population that produces erythropoietin (EPO). Isolated renal cells comprising a population of cells, methods for isolating and culturing the same, and the cell population The present invention is directed to a method of treating a subject in need thereof using the method. [Background technology]

[0003] Background technology Chronic kidney disease (CKD) affects more than 19 million people in the United States and is associated with obesity, diabetes, and This is often the result of metabolic diseases related to diabetes and hypertension. , which are themselves two diseases that are on the rise worldwide: hypertension and insulin-independent diabetes. It has been shown that this condition is caused by renal failure secondary to non-inferiority diabetes mellitus (NIDDM). (United States Renal Data System:Costs o f CKD and ESRD. ed. Bethesda, MD, Nation al Institutes of Health, National Institute Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238). Obesity, hypertension, and blood sugar Poor management has been shown to be an independent risk factor for kidney damage, It causes lesions of the kidneys and tubules, leading to proteinuria and other systemic effects on the filtration function of the kidney. (Aboushwareb, et al., World J Urol, 26: 295-300, 2008, Amann, K. et. al., Nephrol Dial Transplant, 13: 1958-6 6, 1998). CKD patients in stages 1-3 of progression are at increased risk for lifestyle changes and underlying are managed by pharmacological interventions aimed at controlling the underlying condition(s), while Stage 5 patients receive dialysis and usually antihypertensive drugs, erythropoietin-stimulating agents (ESAs), iron and vitamin D supplementation. According to the USRDS, the average patient with end-stage renal disease (ESRD) receives injectable erythropoiesis stimulating Over $600 per month for ESAs, vitamin D supplements, and iron supplements (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, MD , National Institutes of Health, Nationa l Institute of Diabetes and Digestive an d Kidney Diseases, 2007 pp 223-238). Years of Dialysis Combined with the average annual cost ($65,405), the medical costs to maintain one patient are The amount rose to over $72,000 during that time (United States Renal Da ta System: Costs of CKD and ESRD. ed. Be thesda, MD, National Institutes of Healt h, National Institute of Diabetes and Diabetes Gestive and Kidney Diseases, 2007 pp 223 -238), this figure reflects only the cost of standard procedures and does not include other complications. This does not include treatment, emergency care, or ancillary procedures such as placement of vascular grafts for dialysis access. The combined medical costs for CKD and ESRD in 2005 totaled $62 billion accounted for 19% of total medical expenses that year (United States Rena l Data System: Costs of CKD and ESRD. Bethesda, MD, National Institutes of H. health, National Institute of Diabetes an d Digestive and Kidney Diseases, 2007 pp. 223-238) For stage 4-5 patients, it is a preemptive measure to avoid dialysis, Kidney transplantation is a viable option when dialysis is no longer sufficient to control the condition. However, the number of stage 5 CKD patients in the United States who could benefit from whole kidney transplantation (400,000) 0 or more) is the number of suitable donor kidneys available in any given year (approximately 16,000). (Powe, NR et al., Am J Kidney Dis, 53: S37-45, 2009). Therefore, it is important to delay the dependence on dialysis. New treatment paradigms are being developed to fill the gaps caused by the shortage of donor kidneys by reducing or eliminating the need for donor kidneys. It is needed.

[0004] Progressive kidney disease is characterized by an initial disorder (e.g., hypertension) and subsequent inadequate treatment for that disorder. This is caused by a combination of aggressive renal responses, including pro-inflammatory and pro-fibrotic responses. It also promotes the production of cytokines and growth factors, thus slowing the progression of CKD. One strategy is to ameliorate the inflammatory and fibrotic response and promote repair and / or regeneration of kidney tissue. The goal is to reduce or reverse kidney degeneration by

[0005] Chronic renal failure is widespread not only in humans but also in some domestic animals. , not only experience loss of kidney function (uremia), but also have difficulty producing sufficient numbers of red blood cells through erythropoiesis. Anemia also develops due to a decrease in the bone marrow's ability to produce red blood cells (RBCs). The production of erythropoietin (EPO) by specialized interstitial fibroblasts present in the kidney, and The ability of targeted erythroid progenitor cells in the bone marrow to generate more RBCs in response to EPO Anemia in renal failure is caused by a decrease in the amount of EPO produced in the kidney and uremia. This is due to both the adverse effects of steroids on the action of EPO in the bone marrow.

[0006] The clinical approach to treating chronic renal failure to date has been to restore renal filtration and urinary Dialysis and kidney transplantation for production and recombinant EPO to restore red blood cell mass or systemic delivery of EPO analogs. Dialysis is used for patients with mid- to late-stage renal failure. Although it improves survival rates, it also causes serious quality of life problems. It is a highly desirable (and often the only) option for patients with renal failure. However, the supply of high-quality donor kidneys is insufficient to meet the demands of the kidney failure population. Bolus administration of recombinant EPO for steroid use is now associated with serious downstream health risks. This has led to a black box warning from the FDA for the drug, and Alternative therapies to restore hemoglobin homeostasis should be further explored. The in vivo efficacy and safety of the EPO-producing cells generated by the therapy will be investigated. These studies demonstrated that in vivo delivery of EPO-producing cells can stimulate erythropoiesis. It has been found that it can transiently stimulate RBC counts. Among these approaches, the regulation of erythrocyte homeostasis or long-term in vivo Therefore, the number of HCT and RBCs has not increased beyond normal limits. It often increases, causing polycythemia vera and other complications. Delivery of EPO-producing cells offers advantages over delivery of recombinant EPO, increasing HCT Not only does it promote erythrocyte homeostasis, but it also restores erythroid homeostasis without impairing both positive and negative regulatory mechanisms. Anemia due to EPO deficiency is common in patients with kidney disease. However, it can also occur as a result of other conditions, including heart failure, multiple organ system failure, and other chronic diseases. It is important to note that

[0007] The kidney is a unique organ composed of many different (more than 10) specialized cell types. All of these cell types develop from the intermediate mesoderm, but upon maturation, they undergo morphological and and functionally distinct compartments, as well as blood filtration, urine production, acid-base regulation, and electrolyte erythropoietin (Epo), vitamin D, renin, and angiotensin II They form an anatomical unit that acts in concert to provide regulation of endocrine functions such as the production of hormones. As will be demonstrated by the following examples, the intracellular compartments of the kidney are involved in homeostatic functions. The cells of the afferent arteriole are highly interdependent for the production of blood. It acts in concert with tubule cells (macula densa) to regulate blood flow through the glomerulus (Castrop, H. Acta Physiol (Oxf),189:3-14,2007). Kidney Protein processing by the endocytosis pathway involves receptor-mediated endocytosis and specialized proximal tubule cells. In conjunction with protein reabsorption from the glomerular filtrate by the fenestrated endothelial cells of the glomerulus, podocytes , and regulated by the basement membrane (Jarad, G & Miner, JH. C urr Opin Nephrol Hypertens,18:226-32,200 9) The production of active vitamin D by renal tubular cells is mediated by the deposition of extracellular matrix and the production of vitamin D by interstitial cells. Direct and indirect mechanisms regulating epithelial-to-myofibroblast conversion and epithelial-to-mesenchymal transition Thus, it regulates the homeostasis of stromal cells (Tan, X, et al. J Steroid Biochem Mol Biol, 103:491-6, 2007). Regardless of the specific example, all cell-cell interactions in the kidney involve spatial and structural relationships. At the cellular level, the progression of CKD depends, at least in part, on cellular dysfunction or homeostasis. loss of a specific cell type or one or more cells due to loss of cell-cell interactions that maintain Therefore, a successful regenerative approach to the treatment of CKD may involve the loss of vascular function. To some extent, it restores homeostasis through the restoration of cellular organization and intercellular communication. It must be established.

[0008] The aim of this study is to investigate whether renal tubular transport or Epo The enhancement of specific kidney functions, such as the production of ATP, is contemplated. The majority of these studies have focused on therapeutic intervention of acute renal failure (ARF) using stem or progenitor cell types. (Hopkins, C, et al. J Pathol, 217: 265-81, 2009). MSC(Humphreys BD & Bonventr e JV, Annu Rev Med 2008,59:311-325), endothelial progenitor Cells (EPC) (Chade AR, et al., Circulation 20 09,119:547-557,Patschan D, et al.,Curr O pin Pharmacol 2006,6:176-183), and fetal cells or Tissue primordium (Hammerman MR, Curr Opin Nephrol Hy pertens 2001, 10:13-17, Kim SS, et al, St em Cells 2007, 25:1393-1401, Marshall D. et al., Exp Physiol 2007, 92:263-271, Yok oo T, et al., J Am Soc Nephrol 2006, 17: Various cell types, including erythropoietin (ER) and erythropoietin (ER), were delivered intrarenally or intrarenally just before or immediately after ARF induction. Many preclinical studies have been conducted involving the administration of steroids or systemic delivery of steroids to treat ARF in humans. As an adjunct to conventional dialysis for the treatment of urinary tract infections, extracorporeal hollow fiber filtration devices containing renal tubular cells are used. The vise was tested (Ding, F & Humes, HD. Nephron E xp Nephrol, 109:e118-22, 2008, Humes, HD , et al.Kidney Int, 66: 1578-88, 2004, H Nat Biotechnol, 17: 451 -5, 1999). Transplantation of mesenchymal stem cells via the renal artery has also been reported following cardiovascular surgery. It has been clinically tested in patient populations at high risk for subsequent ARF episodes (W estenfelder, C. Experimental Biology.New Orleans, LA, 2009). Limited availability of cell-based therapeutic interventions for CKD. Quantitative preclinical studies are being conducted (Chade, AR, et al. Circula tion,119:547-57,2009,Eliopoulos,N,et al. .J Am Soc Nephrol,17:1576-84,2006,Kucic, T,et al..Am J Physiol Renal Physiol,295: F488-96, 2008). The combination of fetal kidney primordium + / - mesenchymal stem cells was It has been investigated in rodents (Yokoo, T, et al. Transpla ntation, 85: 1654-8, 2008, Yokoo, T. et. al. J Am Soc Nephrol, 17: 1026-34, 2006) Whole fetal kidney tissue transplanted into a suitable environment, such as the omentum, develops into a renal structure with limited function. However, as a component of the embryonic tissue primordium, The therapeutic role of MSCs is unclear, and there is currently no provision of human fetal kidney tissue for therapeutic purposes. Other studies have shown that glomerulonephritis, protein loss, and , and irradiated COL4A3(- / - When cells derived from normal donor bone marrow were transplanted into mice, leaky glomerular podocytes were produced. -By replacing the cells with normal cells that do not have the gene mutation, the progression in the model was partially delayed (Prodromidi, EI, et al. Stem Cells, 24: 2448-55, 2006, Sugimoto, H, e. t al. Proc Natl Acad Sci USA, 103: 732 1-6, 2006). Cell transplantation significantly improved sCREAT, BUN, and sodium levels. Although the stabilization was highly evaluated, the untreated / renally impaired controls for comparison in Study 24 Chade et al. used a porcine model of unilateral renal artery stenosis to examine the effects of injury on the renal artery. The effects of intrarenally delivered autologous EPCs were examined 6 weeks after transplantation (Chade AR, et al., Circulation 2009, 119:547-557). EPCs are , some improvement in tubulointerstitial fibrosis, significant improvement in glomerular sclerosis, and improved renal blood flow. Although the treatment improved, no change in blood pressure was observed (Chade AR, et al. al., Circulation 2009, 119:547-557). Studies examining the in vivo efficacy of cell-based CKD treatments have shown transient and / or has had partial effects but has gathered both systemic and histological evidence of function. There is little evidence of clinically meaningful benefit following intervention in models of CKD progression. The limited number of studies providing evidence suggests that cell-based therapies may fully restore kidney function. However, there are concerns that it may stabilize renal function and slow progression. Regenerative therapies that address this unmet medical need within this patient population. can be done.

[0009] Reproducible in vivo model(s) of progressive CKD allow for the evaluation of the therapeutic potential of candidate therapies. There are numerous models of ARF, including chemically induced and ischemic / Reperfusion-induced tubular damage, but without significant intervention, progressive and Few models of early and end-stage CKD exist. The procedure reproducibly generates end-stage and progressive renal failure, and can improve blood pressure, glomerular filtration rate ( Decreased GFR, elevated serum creatinine (sCREAT) and BUN, mesangial and several of the symptoms of CKD, including tubulointerstitial fibrosis, hyperlipidemia, hyperphosphatemia, and anemia. It results in systemic and histologically detectable disease with several key features (Kaufm an,JM,et al.. Kidney Int, 6: 10-7, 19742 2, Platt, R, et al. Clin Sci (Lond), 11: 2 17-31,1952,Ormrod,D&Miller,T.Nephron,26: 249-54, 1980, Brenner, BM. Am J Physiol, 249: F324-37, 1985). The presence of these clinically significant features is due to the technical reproducibility This, combined with its availability and commercial availability, led to the selection of this model for the studies described herein. provided a basis for the selection. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, there is a need to slow progression in this patient population, improve quality of life, and save the healthcare system money. New treatments that provide substantial and durable augmentation of kidney function to reduce long-term costs A new treatment paradigm is needed. Regenerative medicine technologies offer next-generation treatment options for CKD. It is possible. [Means for solving the problem]

[0011] In one aspect, the present invention provides a human erythrocyte colony comprising a first cell population, B2, and a second cell population. B1 provides a mixture of renal cells, B2 contains an isolated, enriched population of renal tubular cells, and B3 contains a second cell The population includes erythropoietin (EPO)-producing cells, glomerular cells, and vascular cells. In some embodiments, the mixture further comprises a third cell population. In one embodiment, the B2 cell population further comprises collecting duct epithelial cells. In one embodiment, the B2 cell population is hypoxia-tolerant. Hyaluronic acid (H) synthase-2 is expressed both in vitro and in vivo. A) can produce and / or stimulate the production of high molecular weight species. In certain embodiments, the B2 cell population is iodixanol-resistant. The B2 cell population has a density of about 1.045 g / mL to about 1.052 g / mL. In one embodiment, the second cell population is a B4 cell population. The 4 cell population has a density of about 1.063 g / mL to about 1.091 g / mL. In one embodiment, the second cell population is a B3 cell population. The cell population has a density of about 1.052 g / mL to about 1.063 g / mL. In some embodiments, the mixture includes both B2 and B3 cell populations.

[0012] In a further embodiment, the mixture is purified of inert or undesirable components. In one embodiment, the mixture does not contain or has been depleted of the B1 cell population. In one embodiment, the B1 cell population is a population having a density of less than about 1.045 g / mL. In another specific embodiment, the mixture comprises B5 In one embodiment, the mixture is free of or has had the cell population removed. Contains low granularity and viability debris and small cells with a density greater than about 1.091 g / mL. It does not contain the B5 cell population, which includes In certain embodiments, the mixture of cells is used to stabilize and / or improve kidney function and / or In one embodiment, the mixture provides a method for the treatment of albumin receptor-mediated uptake. In another embodiment, the mixture of cells is capable of incorporating oxygen-tunable E. coli. In yet another embodiment, the mixture is capable of expressing estrogen (EPO). Produces high molecular weight species of hyaluronic acid (HA) both in vitro and in vivo, and and / or cells expressing HAS-2 that can stimulate its production. In embodiments, the mixture can provide a regenerative stimulus upon delivery in vivo. In some embodiments, the mixture, upon delivery in vivo, induces glomerular filtration, tubular reabsorption, urinary production, Reduce, stabilize, or improve metabolic and / or endocrine dysfunction It is possible.

[0013] In all embodiments, the first and second cell populations are derived from kidney tissue or cultured In one embodiment, B2 is derived from a kidney cell. Vitamin D3 25-hydroxylase (CYP2D25) ), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (A qp1), aquaporin-2 (Aqp2), RAB17, a member of the RAS oncogene family Rab17, GATA binding protein 3 (Gata3), and FXYD domain-containing Ion transport regulator 4 (Fxyd4), solute transporter family 9 (sodium / Hydrogen exchanger), member-4 (Slc9a4), aldehyde dehydrogenase 3 family, member Aldh3b1, aldehyde dehydrogenase 1 family, member A3 (Aldh3b1) tubule cell endothelial cell membrane protein (TMP) selected from the group consisting of dh1a3), and calpain-8 (Capn8). In another embodiment, B2 is characterized by expression of the collecting duct marker Aqua. They are further characterized by expression of the porin-4 (Aqp4) marker.

[0014] In all embodiments, B4 comprises PECAM, VEGF, KDR, HIF1a. The tumor may be characterized by expression of a vascular marker selected from the group consisting of: In this study, B4 inhibits the expression of the glomerular markers podocin (Podn) and nephrin (Neph). In another embodiment, B4 is unfractionated (UNFX), B 2, and characterizing the oxygen-tunable EPO-enriched population compared with B3 cell populations. In another embodiment, B4 is a chemokine (CXC motif) receptor 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 protein Protein receptor (Kdr), secreted protein, acidic, cysteine-rich (osteone) Cutin (Sparc), Serglycin (Srgn), TIMP metallopeptidase inhibitor Timp3, Wilms' tumor 1 (Wt1), wingless type MMTV integration site family Wnt4, regulator of G-protein signaling 4 (Rgs4) , platelet endothelial cell adhesion molecule (Pecam), and erythropoietin (Epo) The cells are characterized by the expression of a marker selected from the group.

[0015] In all embodiments, B3 comprises aquaporin 7 (Aqp7), FXYD domain. Ion transport regulator 2 (Fxyd2), solute transporter family 17 (phosphate phosphate), Thorium), member-3 (Slc17a3), solute transporter family-3, member-1 (S lc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase (Napsa), solute transporter family 2 (facilitative glucose transporter), member 2 (S lc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 2 7 (Tmem27), acyl-CoA synthetase medium-chain family member 2 (Acsm2), Glutathione peroxidase 3 (Gpx3), fructose-1,6-bisphosphatase -ase 1 (Fbp1), and alanine aminotransferase 2 (Agxt2) In another specific embodiment, B 3 is a vascular-expressed marker, platelet endothelial cell adhesion molecule (Pecam), and a glomerular-expressed marker. -Features podocin (Podn).

[0016] In one aspect, the present invention provides a human kidney comprising a first cell population, B2, and a second cell population. A second cell collection B1 provides a mixture of cells, B2 containing an isolated, enriched population of renal tubular cells. The group identified the vascular-expressed marker platelet endothelial cell adhesion molecule (Pecam) and the glomerular-expressed marker - comprising one or more cell populations that express podocin (Podn).

[0017] In another aspect, the present invention provides an isolated, enriched population of human renal cells comprising a B2 cell population. wherein B2 comprises an isolated, enriched population of renal tubular cells. The cell populations are expressed through the expression of HAS-2 (hyaluronan synthase-2) in vitro and in vivo. and / or produce high molecular weight species of hyaluronic acid (HA) both in vitro and in vivo. In certain embodiments, the B2 cell population can stimulate the production of about 1.04 B1 cell population, which includes large granular cells of the collecting duct and tubular system with a density of less than 5 g / mL In another embodiment, the B2 cell population comprises a B2 cell population of about 1.052 g / mL to about 1.0 Glomerular cells, which produce erythropoietin (EPO), have a density of 63 g / mL. and does not include a B3 cell population, which includes vascular cells. The population contained a B4 cell population having a density of about 1.063 g / mL to about 1.091 g / mL. In yet another embodiment, the B2 cell population has a density greater than about 1.091 g / mL. The B5 cell population contains debris and small cells of low granularity and viability.

[0018] In another aspect, the present invention provides a cell suspension comprising: a) a non-enriched heterogeneous kidney cell population; b) exposing the culture medium to hypoxic culture conditions; and c) extracting a first cell fraction containing a B2 cell population. In one embodiment, the present invention provides a method for preparing a human B2 cell population comprising: The B2 cell population obtained by this method represents a greater proportion of the urine when compared to the non-enriched cell population. Tubular cells and a smaller proportion of EPO-producing cells, glomerular cells, and vascular cells In another embodiment, the method further comprises the step of: In order to separate the above cell fractions, a step including contacting the cell suspension with a density gradient is carried out. and the first cell fraction further comprises a pH between about 1.045 g / mL and about 1.052 g / mL. present in the gradient after centrifugation at a specific density of 1000 kJ / ml.

[0019] In yet another aspect, the present invention provides a method for producing a cell line comprising: a) a non-enriched heterogeneous kidney cell population; b) subjecting the suspension to hypoxic culture conditions; and c) extracting a first cell fraction containing a B4 cell population. In one embodiment, the present invention provides a method for preparing a human B4 cell population, the method comprising: The B4 cell population obtained by the method of the invention has a greater percentage of B4 cells than non-enriched cell populations. EPO-producing cells, vascular cells, and glomerular cells, as well as a smaller proportion of EP In another embodiment, the cells include cells that do not produce O, non-vascular cells, and non-glomerular cells. The method may further comprise, between step a) and step b), separating one or more cell fractions: The method further includes contacting the cell suspension with a density gradient, wherein the first cell fraction comprises , in the gradient after centrifugation at a specific density between about 1.063 g / mL and about 1.091 g / mL exists in.

[0020] In yet another aspect, the present invention provides a method for producing a cell line comprising: a) a non-enriched heterogeneous kidney cell population; b) exposing the suspension to hypoxic culture conditions; and c) extracting a first cell fraction containing a B3 cell population. In another embodiment, a method for preparing a human B3 cell population is provided, comprising: The method may further comprise, between step a) and step b), separating one or more cell fractions: The method further includes contacting the cell suspension with a density gradient, wherein the first cell fraction comprises in the gradient after centrifugation at a specific density between 1.052 g / mL and approximately 1.063 g / mL. exist.

[0021] In another aspect, the present invention provides a cell suspension comprising a non-enriched heterogeneous kidney cell population. b) subjecting the cell suspension to hypoxic culture conditions; and c) isolating the cell suspension from one or more individual cells within the cell population. Suitable for flow cytometry instruments that allow simultaneous measurement of forward and side scatter in cells. c) selecting a subpopulation of cells from the cell population; and d) selecting a subpopulation of cells from the cell population. and e) isolating a subpopulation of B4 cells from the cell population. Also provided is a method for producing a B2 cell preparation comprising a subpopulation of B2 cells, the subpopulation of B2 cells being more abundant than the majority of the population. It is characterized by high forward scatter and high side scatter.

[0022] In another aspect, the present invention provides a cell suspension comprising: a) a non-enriched heterogeneous kidney cell population; b) subjecting the cell suspension to hypoxic culture conditions; and c) isolating the cell suspension from one or more individual cells within the cell population. Suitable for flow cytometry instruments that allow simultaneous measurement of forward and side scatter in cells. c) selecting a subpopulation of cells from the cell population; and d) selecting a subpopulation of cells from the cell population. and e) isolating a subpopulation of B4 cells from the cell population. The present invention provides a method for producing a B4 cell preparation comprising a subpopulation of B4 cells, the subpopulation of B4 cells being more abundant than the majority of the population. In all embodiments, the forward scatter is Corresponds to cell size. In all embodiments, side scatter corresponds to the granularity of the cells.

[0023] In yet another embodiment, the present invention provides a method for administering a stable and / or improved and (c) providing an implantable structure for providing improved renal function, the structure comprising: a) one or more biocompatible synthetic a) a biomaterial comprising a synthetic polymer or a naturally occurring protein or peptide; and b) said biomaterial. coated on, deposited on or in, entrapped in, suspended in a material a first cell population formed on, embedded within, and / or otherwise combined therewith; and a mixture of mammalian kidney cells comprising B1, B2, and a second cell population. coated on, deposited on or in, or entrapped in a biomaterial, suspended therein, embedded therein, and / or otherwise combined therewith In another embodiment, the second cell population is B4. or deposited in, trapped in, suspended in, embedded in, and / or the second cell population otherwise combined therewith is B3. In the form of coated on, deposited on or in, or entrapped within a biomaterial suspended therein, embedded therein, and / or otherwise combined therewith The mixture further comprises a third cell population. deposited on or in, trapped in, suspended in, The mixture embedded in and / or otherwise combined with B3 and B4 In all embodiments, the mixture comprises mammalian kidney tissue or cultured kidney cells. In one embodiment, the structure is suitable for capturing and / or attaching a compound. In another embodiment, the biomaterial is configured as a porous, three-dimensional (3-D) biomaterial. In the method, the structure embeds, attaches, suspends, or coats mammalian cells. The biomaterial may be configured as a liquid or semi-liquid gel suitable for use in a surgical procedure. In terms of morphology, the structure is composed of primarily high molecular weight species of hyaluronic acid (HA) in hydrogel form. In another embodiment, the structure comprises a hyaluronic acid biomaterial in the form of a porous foam. In another embodiment, the structure comprises a biomaterial comprising predominantly high molecular weight species of uronate. , including biomaterials containing HA molecules ranging in size from 5.1 kDa to over 2 x 106 kDa. In yet another embodiment, the structure has pores of about 50 microns to about 300 microns. In yet another embodiment, the structure comprises a biomaterial comprising a polylactic acid-based foam. In one embodiment, the kidney sample comprises one or more cell populations derived from an autologous kidney sample. In a further embodiment, the structure is one or more biopsies derived from a non-autologous kidney sample. In one embodiment, the structure provides red blood cell homeostasis.

[0024] In yet another aspect, the present invention provides a method for treating kidney disease in a subject in need thereof. a) a mixture of mammalian kidney cells comprising a first cell population, B2, and a second cell population; administering to the subject a composition comprising the compound of formula (I) or (II), and b) determining whether the level of an indicator of renal function is increased relative to the level of the indicator in a control. and determining in a test sample from the subject that the difference in the indicator level differs from the difference in the indicator level. indicates a reduction, stabilization, or amelioration of decline in one or more kidney functions in a subject. In one embodiment, the method includes a mixture of cells comprising a third cell population. In another embodiment, the second cell population is B4 or B3. In certain embodiments, the kidney disease treated by the methods of the present invention is erythrocyte sequestration (ERS) or B3. In certain embodiments, the EPO deficiency is anemia. In some embodiments, the EPO deficiency or anemia is secondary to renal failure in the subject. In some other embodiments, the EPO deficiency or anemia is caused by chronic renal failure, primary renal failure, or chronic EPO deficiency, chemotherapy or antiviral treatment, non-myeloid cancer, HIV infection, liver disease , heart failure, rheumatoid arthritis, or multiple organ system failure. In certain embodiments, the composition used in the method comprises one or more biocompatible Biomaterials including synthetic polymers and / or naturally occurring proteins or peptides are also included. The mixture may be coated on, deposited on or in a biomaterial or entrapped therein. suspended therein, embedded therein, and / or otherwise combined therewith. do.

[0025] In yet another aspect, the present invention provides a cell preparation and mixture thereof, or a method for treating a cell in need thereof. The transfer of the present invention to the preparation of a medicament useful for treating kidney disease, anemia, or EPO deficiency in elephants. The use of implantable structures is provided.

[0026] In one embodiment, the present invention provides a method for treating a cancer cell that has been exposed to an oxygen level of about 1% to about 5% for about 12 to about 24 hours. After that, a density gradient containing a density range of about 1.045 g / mL to about 1.052 g / mL was used. providing a selected population of kidney cells that are separable by centrifugation through a density gradient, The population was (i) in the gradient after centrifugation at a density between 1.045 g / mL and approximately 1.052 g / mL and (ii) kidney urinary markers characterized by the expression of at least one renal tubule cell marker. (iii) renal tubule cells capable of receptor-mediated albumin transport; (iv) delivering to a subject at risk for or having kidney disease. Sometimes, one or more kidney functions can be regulated.

[0027] In yet another aspect, the present invention provides a method for treating a cancer cell at an oxygen level of about 1% to about 5% for about 12 hours to about 24 hours. After exposure to the bell, the gradient contained a portion with a density of about 1.063 g / mL to about 1.091 g / mL. A selected population of kidney cells that can be separated by density gradient centrifugation using The cell population is (i) centrifuged at a density of 1.063 g / mL to about 1.091 g / mL. (ii) express oxygen-tunable erythropoietin (EPO) (iii) cells at risk of or associated with kidney disease, including cells that cause kidney disease, glomerular cells, and vascular cells; (iv) capable of modulating one or more renal functions when delivered to a subject having the same 66. The method of claim 65, wherein the population of renal cells enhances regulation of one or more renal functions upon simultaneous administration. It is possible.

[0028] In yet another aspect, the present invention provides a population of kidney cells, the cells being: i) cultured for 24 to 72 hours; The medium was cultured in high glucose DMEM with 5% fetal bovine serum at 37°C and 21% oxygen for a period of 10 min. 25,000 cells / cm in a medium consisting of a 1:1 mixture with fully supplemented KSFM. Plated in adherent culture on standard tissue-culture-treated plastic dishes at an initial density of 2; ii) exposed to 50–100% medium changes with the same medium and incubated for 18 h at 37°C and 2% oxygen; iii) harvested via trypsinization, resuspended, and serum-free Washed with KSFM medium or PBS; iv) loaded onto a prepared density gradient, The gradient contains layers of defined density from 1.045 g / mL to about 1.052 g / mL, with larger at least one layer of higher density and at least one layer of lower density, Prepare gradients in 15 mL conical tubes with a total volume of at least 5 mL and at most 14 mL of liquid. The number of cells loaded onto the gradient is at least 50 million but not more than 100 million, ) Gradient was run by centrifugation at 800xG for 20-30 minutes without braking; 1.045g divided at a density of 1.052 g / mL to 1.052 g / mL; and / or megalin, cubilin, Hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP 2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin -1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member-RAS oncogene Rab17 family, GATA binding protein 3 (Gata3), FXYD domain Ion transport regulator 4 (Fxyd4), solute transporter family 9 (NaTc), Sodium / hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family -, member-B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member-A 3 (Aldh1a3), calpain-8 (Capn8), and aquaporin-4 (Aq p4) characterized by a marker selected from the group consisting of markers; and / or kidney disease stabilize or reduce the decline in one or more renal functions in immunocompetent subjects with , or can be improved.

[0029] In yet another aspect, the present invention provides a population of kidney cells, the cells being: i) cultured for 24 to 72 hours; The medium was cultured in high glucose DMEM with 5% fetal bovine serum at 37°C and 21% oxygen for a period of 10 min. 25,000 cells / cm in a medium consisting of a 1:1 mixture with fully supplemented KSFM. Plated in adherent culture on standard tissue-culture-treated plastic dishes at an initial density of 2; ii) exposed to 50–100% medium changes with the same medium and incubated for 18 h at 37°C and 2% oxygen; iii) harvested via trypsinization, resuspended, and serum-free Washed with KSFM medium or PBS; iv) loaded onto a prepared density gradient, The gradient contains layers of defined density from 1.063 g / mL to about 1.091 g / mL, with larger at least one layer of higher density and at least one layer of lower density, Prepare gradients in 15 mL conical tubes with a total volume of at least 5 mL and at most 14 mL of liquid. the number of cells loaded onto the gradient is at least 50 million but not more than 100 million; v) Gradient run by centrifugation at 800xG for 20-30 minutes without braking; 1.063 divided into densities of 1.091 g / mL to approximately 1.091 g / mL; and / or VEGF, KDR, HIF1a, podocin (Podn) or nephrin (Neph), chemokine (CX -C motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), Collagen, type V, alpha 2 (Col5a2), cadherin 5 (Cdh5), pla Platinum activator, tissue (Plat), angiopoietin 2 (Angpt2), Kdr receptor, secreted protein, acidic, cytoplasmic Inosin-rich (osteonectin) (Sparc), serglycin (Srgn), TIMP Metallopeptidase inhibitor 3 (Timp3), Wilms' tumor 1 (Wt1), wingless type MMTV integration site family, member 4 (Wnt4), G-protein signaling 4 Regulator of platelet endothelial cell adhesion molecule (Pecam), a regulator of erythropoietin (Rgs4), and / or kidney disease; stabilize or reduce the decline in one or more renal functions in immunocompetent subjects with , or can be improved.

[0030] In one aspect, the present invention provides an isolated method for producing erythropoietin (EPO)-producing kidney cells. In one embodiment, the population is a single cell population of mammalian cells that produce EPO. In another embodiment, the population is an isolated, enriched population of erythropoietin (EPO). a greater proportion of EPO-producing mammalian cells than a non-enriched population containing EPO-producing mammalian cells. isolated and enriched mammalian cells that produce erythropoietin (EPO), including cells that It is a group.

[0031] The population may be derived from kidney tissue or cultured kidney cells. The sample may be derived from a kidney tissue sample obtained from a subject or a cultured kidney tissue sample. In another embodiment, the cell population is a mammalian cell that produces EPO. A population containing a greater proportion of EPO-producing cells than a non-enriched population containing mammalian cells. In certain other embodiments, the cell population is a mammalian cell line that produces erythropoietin (EPO). It contains a smaller proportion of kidney tubular cells than the non-enriched population containing mammalian cells.

[0032] In all embodiments, the cell population may be enriched for EPO-producing cells. In all embodiments, the cell population is enriched for cells that do not produce EPO. In all embodiments, the cell population may be enriched for renal tubular cells. It is possible.

[0033] In another aspect, the present invention provides erythropoietin (E) that is biologically responsive under specific culture conditions. In certain other embodiments, the bioresponsiveness is , the difference between cell populations cultured under hypoxic conditions and cell populations cultured under non-hypoxic conditions. In yet another embodiment, the biological response is induction of EPO expression when the cell is nourished. The effect of cell populations cultured under hypoxic conditions on the survival of cells compared to cell populations cultured under non-hypoxic conditions In some embodiments, the hypoxic culture conditions are: Cells cultured under conditions where oxygen levels are not reduced relative to the cell population, including but not limited to: In certain other embodiments, the available oxygen level of the culture system is reduced relative to the population. In this condition, the reduction in available oxygen levels is less than about 5%, and under conditions where oxygen levels do not decrease, In another embodiment, the condition is atmospheric oxygen levels (approximately 21%). When compared with cultures tested at levels of 21% or higher, the In another embodiment, the level of induction and / or Increased expression was observed in the cultures grown under atmospheric oxygen levels (approximately 21%), i.e., non-hypoxic culture conditions. Induction of EPO expression and / or increased EPO expression by less than about 5% compared to cells treated with This can be observed when culturing cells in oxygen-rich, i.e., hypoxic, culture conditions. In this study, EPO expression, which responds to hypoxic conditions, was regulated by the hypoxia-inducible factor HIF. In another embodiment, EPO expression in response to hypoxic conditions is regulated by H In yet another embodiment, the biological response to hypoxic conditions is regulated by IF1α. EPO expression is regulated by the hypoxia-inducible factor HIF2α.

[0034] In one embodiment, the bioresponsiveness is compared to a cell population not cultured via perfusion: Induction of EPO expression when the cell population is cultured via perfusion. Therefore, bioresponsiveness was demonstrated by increased EPO expression compared to non-cultured cell populations via perfusion. In some embodiments, perfusion conditions include, but are not limited to, power, fluid flow, Transient, intermittent or continuous circulation or agitation of the fluid to allow it to reach the cells via the In another embodiment, the perfusion culture conditions are adapted to allow the cell population to form three-dimensional structures. to be cultured in or on a material that provides a framework and / or space for To hold.

[0035] In another aspect, the present invention provides kidney cells containing the cell populations described herein. In one embodiment, the cell mixture is provided with EPO. a first cell population enriched for EPO-producing cells and a second cell population enriched for EPO-producing cells; In certain other embodiments, the second cell population comprises a second cell population that is not amplified. and a method for producing a medicament containing one or more types of kidney-derived cells, which may include one or more of the following: Can be used to identify: tubular-derived cells, glomerular-derived cells, interstitial-derived cells, collecting duct-derived cells In another embodiment, the cells are derived from connective tissue, blood, or blood vessels. In this case, the second cell population is enriched for renal tubular cells.

[0036] In all embodiments, the renal tubular cells described herein are one of the following: The expression of tubular cell markers can include, but are not limited to, one or more of: These may be signs of: hyaluronic acid synthase 2 (HAS2), CYP2D25 (vitamin A), D3 25-hydroxylase), megalin, cubilin, N-cadherin, E-cadherin Herin, aquaporin-1, aquaporin-2, RAB17, member-RAS oncogene family family (Rab17), GATA binding protein 3 (Gata3), and FXYD domains. Ion transport regulator 4 (Fxyd4), solute transporter family 9 (sodium aldehyde dehydrogenase 3 family member (Slc9a4), member 4 (Slc9a4), Member-B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member-A3 ( Aldh1a3), and calpain-8 (Capn8).

[0037] In one aspect, the present invention provides an isolated, enriched mammalian kidney tubular cell population. In one embodiment, the isolated cell population is enriched for renal tubular cells. and contains at least some EPO-producing mammalian cells. Thus, the cell population contains a greater proportion of tubular cells than the non-enriched population containing tubular cells. In certain other embodiments, an isolated, enriched collection of mammalian kidney tubular cells is provided. The population contains a concentration of renal tubular cells and at least some EPO-producing mammalian cells. In another embodiment, the enriched population contains a greater proportion of tubular cells than the unenriched population. The mammalian kidney tubular cell populations fractionated, compared to unfractionated, heterogeneous mixtures, or E Relatively depleted of EPO-producing cells compared to enriched populations of PO-producing cells It is being done.

[0038] In all embodiments, the renal cell population or the renal cell populations described herein The types of sources from which the mixture is derived include autologous or allogeneic, syngeneic (autologous or syngeneic transplant), and any combination thereof. For example, in some embodiments Thus, the mixture of cells comprises (i) a first cell population derived from an autologous source and a second cell population derived from an autologous source. or (ii) a first cell population and a second cell population derived from an autologous source. and a second cell population derived from an allogeneic source.

[0039] In another embodiment, the present invention provides a method for producing a cell population enriched for EPO-producing cells. In one embodiment, the method comprises: a) preparing a non-enriched, heterogeneous rodent population; A cell suspension containing a population of kidney cells was mechanically dissociated or enzymatically digested. prepared from mammalian kidney tissue; b) to separate one or more cellular fractions based on buoyant density. c) contacting the cell suspension with a density gradient to define one or more cell fractions; Step b) Centrifuging the cell suspension, and d) the first supernatant containing the enriched cell population and extracting a cell fraction of the enriched cell population from the non-enriched cell population. When compared, a larger proportion of cells produced EPO and a smaller proportion of cells produced EPO. In certain other embodiments, the density gradient is about 1.025 g / mL and layers with specific densities between about 1.035 g / mL and less than about 1.045 g / mL. In another embodiment, the first cell fraction of step d) has a concentration of from about 1.025 g / mL to about After centrifugation at a specific density between 1.035 g / mL or less than about 1.045 g / mL, In certain other embodiments, the centrifugation of step (c) is performed to separate the concentrated and at least one additional cell fraction that is not enriched for the selected EPO-producing cell population. In another embodiment, the method further comprises extracting at least one additional cellular fraction. In certain other embodiments, the density gradient is about 1.062 g / In another embodiment, the layer comprises a specific density between about 1.088 g / mL and about 1.088 g / mL. The cell fraction is centrifuged at a specific density between approximately 1.062 g / mL and approximately 1.088 g / mL. After separation, it exists in a gradient.

[0040] In another embodiment, the method comprises: a) determining whether or not at least some EPO is expressed or expressed; Cell suspensions from cultured non-enriched heterogeneous mammalian cell populations containing cells that can b) preparing a density gradient with the cell suspension to separate one or more cell fractions based on buoyant density; c) contacting the cell suspension of step b) to define one or more cell fractions; and d) centrifuging the cells to extract a first cell fraction containing the enriched cell population. The enriched cell population containing the ATP showed a greater proportion of ATP compared to the non-enriched cell population. The majority of cells produce EPO and a smaller proportion do not. In an embodiment, the cell suspension of step a) is cultured non-enriched heterogeneous lyophilized cereals. In such an embodiment, the density gradient is obtained from a population of rodent cells. In one embodiment, the step comprises a layer of a specific density between about 1.091 g / mL and about 1.091 g / mL. The first cell fraction of step d) has a specific ATP concentration between about 1.073 g / mL and about 1.091 g / mL. In certain other embodiments, the centrifugation of step (c) is carried out at a density of 1000 saturates. The cardiac isolation is performed using at least one cell population that is not enriched for the enriched EPO-producing cell population. In another embodiment, the method further comprises producing at least one additional cell fraction. In certain other embodiments, the method further comprises step e) extracting the added cell fraction. The layer includes a specific density between about 1.041 g / mL and about 1.062 g / mL. In morphology, the additional cell fraction is between about 1.041 g / mL and about 1.062 g / mL. After centrifugation at a specific density, they are present in the gradient.

[0041] In some embodiments, the enriched cell population is enriched for EPO-producing cells. In another embodiment, the enriched cells are enriched to remove cells that do not produce EPO. The population is enriched for interstitial fibroblasts and depleted of tubular and collecting duct cells In another embodiment, the enriched cell population is comprised of EPO-producing cells, glomerular cells, and enriched for vascular cells. In another embodiment, the centrifugation of step (c) further comprising adding at least one additional cell fraction that is not enriched for the enriched cell population. In some embodiments, the additional cell fraction is generated in a manner similar to that of the non-enriched cell population. When the cells were grown, a larger proportion of cells did not produce EPO and a smaller proportion produced EPO. In one embodiment, the at least one additional cell fraction contains cells that are viable. The cell fraction of the erythrocytes contains a smaller proportion of cells that produce EPO when compared to the other cell fractions. In this state, the additional cell fraction represents a greater proportion of the kidney's Contains tubular cells.

[0042] In some embodiments, a cell population enriched for EPO-producing cells is generated. The density gradient used in the method is an iodixanol density gradient.

[0043] In another embodiment, the present invention provides a method for detecting erythropoietin E using flow cytometry. In one embodiment, a method for generating an enriched population of PO-producing cells is provided, comprising: The method comprises: a) mechanically dissociating a cell suspension containing a non-enriched heterogeneous kidney cell population; b) one or more of the cell populations are prepared from purified or enzymatically digested mammalian kidney tissue; Towards a flow cytometry instrument capable of simultaneously measuring forward and side scatter of individual cells applying a cell suspension; c) selecting a cell subpopulation from the cell population; d) selecting cells from the cell population. e) isolating a cell subpopulation from the cell population, are characterized by low forward scatter and low side scatter compared to the overall population.

[0044] In another embodiment, the method comprises: a) determining whether or not at least some EPO is expressed or expressed; b) preparing a cell suspension from a population of cultured mammalian cells containing cells capable of Flow cytometry allows simultaneous measurement of forward and side scatter in one or more individual cells within a cluster. providing a cell suspension to a cytometry instrument; c) selecting a cell subpopulation from the cell population; d) selecting a cell subpopulation from the cell population; and e) isolating a cell subpopulation from the cell population. The cell subpopulations are characterized by low forward and side scatter compared to the total population. .

[0045] In yet another embodiment, forward scatter corresponds to cell size. Thus, side scatter corresponds to the granularity of the cells. Another embodiment of the present invention is to provide a method for producing EPO. ii) enriched for EPO-producing cells to remove non-EPO-producing cells; or The tissue is enriched for specific interstitial cortical fibroblasts, and the epithelial cells are removed. In certain other embodiments, the selection step c) provides a cell fraction containing the selected cell population. In another embodiment, the method further comprises generating at least one additional fraction that is not enriched for the target protein. The additional fractions produced a smaller proportion of EPO compared to the EPO-enriched fraction. It contains cells that

[0046] In a further aspect of the invention, the method further comprises the step of in vitro culturing. In one embodiment, the enriched cell population is cultured in vitro after isolation. In some embodiments, the culturing step is suitable to support the growth and / or maintenance of the cell population. Two-dimensional monolayers on glass or plastic surfaces suitable for the culture of mammalian cells in a controlled medium. In another embodiment, the culturing step includes the use of a layer culture. involves culturing cells on a three-dimensional (3D) scaffold suitable for growth. In the present invention, the scaffold is made of one or more biocompatible synthetic polymers or naturally occurring proteins. In another embodiment, the scaffold contains a peptide or peptides that trap mammalian cells. In another embodiment, the scaffold is configured as a porous scaffold suitable for attachment. and a gel suitable for embedding, attaching, or coating mammalian cells. do.

[0047] In another aspect, the invention provides a method comprising culturing under perfusion conditions. In one embodiment, perfusion conditions include, but are not limited to, applying force to cells via fluid flow. The method includes transient, intermittent, or continuous circulation or agitation of the fluid to transmit the fluid. In terms of morphology, perfusion culture conditions are used to induce cell populations into a framework that allows for the formation of three-dimensional structures. - To be held in or on a material that provides space and / or space for cultivation.

[0048] In another aspect, the present invention provides a method comprising culturing under hypoxic conditions. In some embodiments, the hypoxic culture conditions include, but are not limited to, the following for a cell population: The available oxygen in the culture system compared to a cell population cultured under conditions where oxygen levels are not reduced In certain other embodiments, a decrease in available oxygen levels is The lowest oxygen level is less than 5%, and the condition where the oxygen level does not decrease is the atmospheric oxygen level (about 21%). In another embodiment, the reduced oxygen conditions are less than 21% (atmospheric) oxygen. It is represented by a level.

[0049] In another additional embodiment, the present invention provides a method for detecting EPO expression in a cell population. In all embodiments, the EPO expression is EPO mRNA expression. In all embodiments, EPO expression is detectable and / or detectable. In another embodiment, EPO expression is increased when compared to a cell population not cultured via perfusion. In another embodiment, EP O expression was significantly higher in cell populations cultured under hypoxic conditions compared to non-hypoxic conditions. In another embodiment, detectable EPO expression is induced in a cell population. The cell populations cultured via perfusion showed higher numbers of cells than those not cultured via perfusion. In additional embodiments, detectable EPO expression is observed in cells cultured under non-hypoxic conditions. In certain other embodiments, the number of cells cultured under hypoxic conditions is higher than that of the total number of cells cultured under hypoxic conditions. In this case, induction of EPO expression and / or increased EPO expression is determined by the level of induction and / or Alternatively, increased expression was observed in cultures grown under atmospheric oxygen levels (approximately 21%), i.e., non-hypoxic culture conditions. Cultivate cells in hypoxic culture conditions, i.e., approximately 5% less oxygen than normal cells. In another embodiment, increased EPO expression can be observed in subjects receiving 21% oxygen. This can be observed when culturing fewer cells than in atmospheric conditions.

[0050] In yet another aspect, the present invention provides a method for the treatment of a cancer, comprising administering to a patient a therapeutically effective amount of a cancer cell comprising one or more of the cell populations described herein. In one embodiment, the present invention provides an implantable structure for a subject in need thereof. A transplantable structure for providing a cell population that produces erythropoietin (EPO) is provided. The structure comprises: a) one or more biocompatible synthetic polymers or naturally occurring proteins; a) a scaffold containing a protein or peptide; and b) a protein or peptide deposited within or on the surface of the scaffold. The first cell population is enriched for EPO-producing mammalian cells.

[0051] In another embodiment, the present invention provides a method for producing erythropoietin (EPO) in a subject in need thereof. The present invention provides an implantable structure for providing a cell population for the treatment of a disease, the structure comprising: a) one or more living cells; Biocompatible synthetic polymers or porous materials containing naturally occurring proteins or peptides a) a scaffold, and b) i) a first cell population enriched for EPO-producing mammalian cells. and ii) a cell population containing a second cell population enriched for cells that do not produce EPO. The scaffold comprises a mixture of cells, the mixture of cells being deposited on the surface and / or within the pores of the scaffold. In some embodiments, EPO expression is higher in the first cell population compared to the second cell population. In certain other embodiments, the second cell population is limited to one or more of the following: The present invention may not be limited to the use of a medicament for the treatment of kidney disease. The present invention may contain one or more kidney-derived cell types that may include, but are not limited to: Cells derived from the renal tubule, cells derived from the glomerulus, cells derived from the interstitium, cells derived from the connective tissue, cells derived from the collecting duct In another embodiment, the second cell is a native cell, a blood-derived cell, or a vascular-derived cell. The population is enriched for renal tubular cells. Characterized by expression of tubular cell markers, including, but not limited to, one or more of the following: Provides a population enriched for renal tubular cells that contains: megalin, cubilin, and N -cadherin, E-cadherin, aquaporin-1, and aquaporin-2.

[0052] In one embodiment, the mixture of kidney cells provided by the present invention comprises the cells described herein. As described above, the present invention can include cell populations derived from any type of kidney tissue source. In some embodiments, the first and second cell populations are derived from kidney tissue or cultured kidney tissue. In another embodiment, the first cell population is derived from erythropoietin (EP)-expressing cells. O) produce a greater proportion of EPO than a non-enriched population containing mammalian cells that produce EPO. In another embodiment, the first cell population contains cells that produce EPO. In yet another embodiment, the first cell contains glomerular cells and vascular cells in addition to the first cell. The cell population contains a greater proportion of EPO-producing cells than the second cell population. Further embodiments include a method for producing erythropoietin (EPO)-producing mammalian cells without enrichment. The first cell population contains a smaller proportion of renal tubular cells than the second population. In this embodiment, the first cell population contains a smaller proportion of renal tubular cells than the second cell population. In some embodiments, the cells are combined with a biomaterial. In another embodiment, the scaffold or biomaterial is configured as a three-dimensional (3-D) porous scaffold. In morphology, the 3-D porous scaffold is suitable for the entrapment or attachment of mammalian cells. In yet another embodiment, the scaffold or biomaterial is a material that embeds or attaches mammalian cells. In one embodiment, the adhesive is configured as a liquid or semi-liquid gel suitable for applying or coating. In the present invention, suitable cell populations for use in the constructs of the present invention are derived from autologous or non-autologous kidney cells. In certain other embodiments, the sample is a kidney biopsy.

[0053] In another embodiment, the present invention provides a cell line enriched for EPO-producing cells. In one embodiment, the method comprises administering to a subject in need thereof a cell population. for the treatment of erythropoietin (EPO) deficiency in subjects with EPO-producing administering to a subject a composition containing a first cell population enriched for mammalian cells that In another embodiment, the first cell population comprises EPO-producing cells, glomeruli In one embodiment, EPO deficiency is anemia. In another embodiment, the EPO deficiency or anemia occurs secondary to renal failure in the subject. In certain other embodiments, the EPO deficiency or anemia is caused by chronic renal failure, primary EPO PO deficiency, chemotherapy or antiviral treatment, non-myeloid cancer, HIV infection, liver disease, heart failure rheumatoid arthritis, or multiple organ system failure. To live.

[0054] In another embodiment, the method is for treating a kidney disease in a subject in need thereof, A composition containing a first cell population enriched for EPO-producing mammalian cells is administered. In another embodiment, the first cell population is capable of producing EPO. It is enriched for endothelial cells, glomerular cells, and vascular cells.

[0055] In some embodiments, the composition administered to a subject in need thereof produces EPO. In another embodiment, the method further comprises the step of: The composition may comprise one or more biocompatible synthetic polymers and / or naturally occurring proteins. Alternatively, the method further comprises the step of: providing a porous scaffold containing a peptide; and the first cell population is located on the surface of the scaffold. and / or deposited within the pores. In additional embodiments, the composition comprises one or more biocompatible materials. Contains biocompatible synthetic polymers and / or naturally occurring proteins or peptides The method further comprises the step of: providing a porous scaffold, the first cell population and the second cell population being present on a surface of the scaffold and In another embodiment, the first cell population and / or the second cell population are deposited within the pores. The two cell populations are derived from mammalian kidney tissue or cultured mammalian kidney cells. In embodiments, the first cell population and / or the second cell population may be autologous or non-autologous. In one embodiment, the sample is a kidney biopsy.

[0056] In one embodiment, the second cell population is enriched for renal tubular cells. In embodiments, the renal tubular cells may include one or more of the following: Characterized by expression of tubular cell markers, including but not limited to: megalin, cubilin , N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2.

[0057] In all embodiments, the second cell population is selected from the group consisting of tubular-derived cells, glomerular-derived cells, cells of interstitial origin, cells of collecting duct origin, cells of connective tissue origin, cells of blood origin, or blood vessels The present invention relates to a method for treating kidney disease, the method comprising administering to a patient a kidney-derived cell type selected from the group consisting of: In some embodiments, the second cell population is a heterogeneous unfractionated population or a population of cells that is more homogeneous than the first cell population. This results in a relative elimination of glomerular cells, vascular cells, and oxygen-responsive EPO-producing cells. It is being done.

[0058] In another aspect, the present invention includes a method of providing red blood cell homeostasis to a subject in need thereof. In one embodiment, the method comprises: a) providing a first antibody enriched for mammalian cells that produce EPO; a) administering to a subject a composition containing one or more cell populations; and b) increasing the level of an erythropoietic functional indicator. A test sample from a subject is measured to determine whether the level of the indicator is different from the level of the control. The difference in the indicator levels indicates red blood cell homeostasis in the subject.

[0059] In another embodiment, the method comprises: a) enriching for mammalian cells that produce EPO; and a second cell population that is not enriched for EPO-producing cells. and b) administering to the subject a composition comprising the erythropoietic functional indicator, wherein the level of the erythropoietic functional indicator is higher than the control level. measuring in a test sample from the subject that the index level is different from the index level; The difference in levels indicates red blood cell homeostasis in the subject.

[0060] In another aspect, the present invention includes a method of improving renal function in a subject in need thereof. In one embodiment, the method comprises: a) providing a first sample enriched for mammalian cells that produce EPO; a) administering to a subject a composition containing the cell population of any one of the preceding claims; and b) determining whether the level of a renal function index is higher than that of a control group. measuring in a test sample from the subject that the level of the indicator is different from that of the indicator of A difference in the indicator levels indicates improved renal function in the subject. The composition comprises one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides. The method further comprises the step of: providing a porous scaffold containing a peptide; and wherein the first cell population is present on the surface of the scaffold and / or or deposited in the pores.

[0061] In another embodiment, the method comprises: a) enriching for mammalian cells that produce EPO; a first cell population enriched for EPO-producing cells and a second cell population not enriched for EPO-producing cells; administering the composition to the subject; and b) determining whether the level of the indicator of renal function is elevated relative to the level of the indicator in a control. the difference in the indicator levels being: In another embodiment, the composition comprises one or more biocompatible Porous membranes containing synthetic polymers and / or naturally occurring proteins or peptides The method further comprises the step of: providing a scaffold; and wherein the first cell population and the second cell population are located on a surface of the scaffold and / or It is deposited in the pores.

[0062] In other embodiments, the first cell population and / or the second cell population are derived from mammalian kidney cells. In another embodiment, the first cell is derived from kidney tissue or cultured mammalian kidney cells. The cell population and / or the second cell population may be derived from an autologous or non-autologous kidney sample. In other embodiments, the sample is a kidney biopsy.

[0063] In one embodiment, the first cell population is a hypoxia-responsive EPO-producing cell. In another embodiment, the first cell population is enriched for hypoxia-responsive EPO-producing cells. It is enriched for endothelial cells, glomerular cells, and vascular cells.

[0064] In one embodiment, the second cell population is enriched for renal tubular cells. In embodiments, the renal tubular cells may include one or more of the following: Characterized by expression of tubular cell markers, including but not limited to: hyaluronan synthase 2 (HAS2), CYP2D25 (vitamin D3 25-hydroxylase), megalin , cubilin, N-cadherin, E-cadherin, aquaporin-1, aquaporin-2 and aquaporin-4. In another embodiment, the second cell population is renal tubular cells. In another embodiment, the second cell collection is enriched for collecting duct epithelial cells. The clusters are relatively concentrated in relation to tubular cells, contain collecting duct epithelial cells, and are hypoxia-responsive. There is a relative decrease in EPO-producing cells, glomerular cells, and vascular cells. [Brief explanation of the drawings]

[0065] [Figure 1] Gomori trichrome staining highlighting fibrosis in diseased kidney tissue compared to normal kidney tissue is shown. [Figure 2] 1 shows oxygen-regulated expression of the erythropoietin gene in cells from pigs with chronic kidney disease. [Figure 3] Figure 1 shows tubule-like structures in the expanded cultures. [Figure 4] Figure 1 shows receptor-mediated uptake of albumin by cultured functional tubular cells. [Figure 5] 1 shows relatively higher EPO expression after isolation than in primary human kidney tissue. [Figure 6] 1 shows the retention of EPO gene expression in cultured cells from human kidney. [Figure 7] Figure 1 shows the results of (+)EPO expression in dynamic 3D cultures at atmospheric (21%) oxygen levels. [Figure 8] Figure 1 shows dynamic 3D culture (+)EPO expression at low (2%) oxygen levels. [Figure 9] Dynamic cultured (+) tubule gene expression during extended culture. [Figure 10] Figure 1 shows EPO expression in hypoxic versus normoxic cultures. [Figure 11] Figure 1 shows stimulation of EPO expression by dynamic 3D culture in vitro. [Figure 12]ELISA results of target proteins quantified in both cell lysates (top panel) and conditioned medium (bottom panel) collected from 2D and 3D cultures are shown. [Figure 13] H&E stained, seeded OPLA and Col1 scaffolds (stained after 7 days of perfused or static culture) that were fixed in 10% buffered formalin and paraffin-embedded using standard methods are shown. H&E staining was performed to examine cell presence and morphology. Cellularity was greater in perfused versus static scaffolds and was more pronounced in Col1 scaffolds compared to OPLA scaffolds. More striking was the presence of cellular organization in perfused Col1 scaffolds compared to static Col1 scaffolds. [Figure 14] Figure 1 shows SEMs of OPLA and Col1 scaffolds after 7 days of culture (static and perfused). Note the greater cellularity in perfused than static, as well as the superior cellular organization and interconnectivity in Col1 perfused than static conditions. [Figure 15] RT-PCR results of mRNA isolated from scaffolds or 2D cultures by adding lysis buffer (Qiagen) are shown. For 3D scaffolds, electrical homogenization (Polytron) was used to ensure complete lysis and release of RNA. Purified mRNA was subjected to RT-PCR analysis using intron-spanning primers specific for the target gene of interest. (Lanes 1-7: various 3D configurations (perfused) / 5-day culture; Lanes 8-10: various 2D configurations / 5-day culture; Lanes 11-17: various initial cell populations (before seeding); Lanes 18-19: 2D cultures / 4-day culture; Lanes 21: macroscopically dissected fresh tissue.) [Figure 16] 1 depicts the metabolic activity of three different seeded scaffolds. [Figure 17] 1 shows glucose and glutamine consumption by perfused and static 3D cultures of primary kidney cells. [Figure 18] The mean survival time (days) of rodents after treatment is shown. [Figure 19] The body weights of the rodents during the study are shown. [Figure 20] Weight gain from pre-treatment to sacrifice is plotted. [Figure 21] Weekly average serum BUN concentrations are shown. [Figure 22] Weekly mean serum creatinine concentrations are shown. [Figure 23] Relative BUN at midpoint / individual rat data is shown. [Figure 24] Relative serum creatinine at midpoint / individual rat data are shown. [Figure 25] Average weekly HCT compared to HCT at midpoint (individual rat data) is shown. [Figure 26A] Relative HCT at midpoint (individual rat data) is shown. [Figure 26B] Serum creatinine after nephrectomy is shown. [Figure 26C] Hematocrit after nephrectomy is shown. [Figure 27] Weekly average RBC# is shown. [Figure 28] Relative RBC# at midpoint / individual rat data are shown. [Figure 29] Indicates the terminal serum electrolyte concentration. [Figure 30] Relative serum phosphite (final) / individual rat data is shown. [Figure 31] Terminal serum protein concentrations are expressed. [Figure 32] Relative serum albumin & total protein (final) / individual rat data are shown. [Figure 33] Represents terminal serum liver function. [Figure 34] Terminal serum cholesterol, triglycerides, and glucose are shown. [Figure 35] Relative serum cholesterol, triglycerides, glucose (final) per individual rat data are shown. [Figure 36] Represents terminal Hb, MCH, &MCHC. [Figure 37] Terminal relative [Hb] / individual rat data are shown. [Figure 38] Indicates terminal white blood cell count. [Figure 39] Terminal leukocyte composition is shown. [Figure 40] Represents terminal reticulocytes. [Figure 41] Terminal platelet data are shown. [Figure 42] The swimming test results are shown. [Figure 43] Representative H&E-stained histological sections of bone marrow from each test group are shown. The top panel shows equal bone marrow cellularity and myeloid to erythroid ratios in NX+ cell-injected animals compared to controls. In contrast, the bone marrow of NX+ constructs and untreated animals showed moderate and markedly reduced bone marrow cellularity, respectively (200x magnification). The bottom panel shows a magnification of the boxed image in the top panel (magnification unknown). [Figure 44] Representative H&E-stained histological sections of spleens from each test group are shown. The top panel shows that no major histological differences or changes were observed between the control and NX+ cell-injected groups. In contrast, the immediate subcapsular red pulp space in NX+ constructs and untreated animals showed moderate and marked reductions in adult red blood cells (RBCs) and RBC precursors (magnification 200x), respectively. The bottom panel shows a magnification of the boxed image in the top panel (magnification unknown). [Figure 45] Representative H&E-stained histological sections of livers from each test group are shown. No appreciable changes were observed in the liver parenchyma and / or NX+ structures or portal triads of untreated NX animals compared with the control group. In contrast, focal areas of sinusoidal hematopoiesis (circles) were noted in the NX+ cell-injected group (magnification 200x). The lower panel shows a magnified view of the portal triad shown in the upper panel (magnification unknown). [Figure 46]Representative H&E-stained histological sections of kidneys from each test group are shown (1x magnification). The top panel shows that, compared with controls, kidney sections from the other three groups showed progressive glomerular and tubular degeneration with loss of structure in all groups, characterized by scant hemosiderin pigmentation and focal tubular regeneration (200x magnification). Arrows indicate the site of structural treatment. The bottom panel shows a magnification of the boxed image in the panel above (magnification unknown). [Figure 47] Individual rat data / HCT% versus serum creatinine are shown: Group 5 = filled circle, Group 6 = circle number "3", Group 4 = circle number "1", Group 1 = circle number "4", Group 2 = circle number "2". [Figure 48] Enrichment of EPO-producing cell fractions from freshly dissociated kidney tissue using a multilayer step gradient technique (left panel) or a single layer mixing gradient technique. Both methods result in partial removal of non-EPO-producing cellular components (mainly tubular cells) from the EPO band appearing between 1.025 and 1.035 g / mL. [Figure 49a] Quantitative real-time PCR (QRTPCR) results confirming E-cadherin expression are shown. [Figure 49b] Quantitative real-time PCR (QRTPCR) results confirming Epo enrichment using a density step gradient are shown. [Figure 50] Relative gene expression in step gradient fractions in culture at the time of isolation is graphically depicted. Panels (a) and (b) show two separate batches of primary kidney cells subjected to density gradient fractionation immediately after tissue dissociation. Erythropoietin (Epo) mRNA expression is reproducibly enriched in band 1 of the step gradient in both batches. Epo expression in all samples was normalized to the freshly digested heterogeneous kidney cell population. Panel (c) clearly shows that band 1, in addition to enrichment for Epo expression, is relatively depleted of tubular cells, as indicated by low expression of tubular markers N-cadherin, E-cadherin, aquaporin 1 and 2. In contrast, tubular markers are enriched in bands 2 and 3, highlighting an additional feature of the step gradient—concomitant enrichment of the tubular cell fraction. [Figure 51] Western blot analysis of aquaporin-1, a tubular cell marker. In further support of the tubular cell enrichment of fractions 2 & 3, panel (d), as illustrated in Figure 2, this Western blot shows a clear and specific enrichment of aquaporin-1 protein in bands 2 & 3. [Figure 52] Hematocrit levels at 16 weeks post-treatment are shown. [Figure 53] Hemoglobin levels at 12 weeks post-treatment are shown. [Figure 54] Hematocrit levels over time are shown in 5 / 6 nephrectomized rats treated with two different doses of rEPO compared to control and untreated groups. [Figure 55] Hematocrit levels over time are shown in 5 / 6 nephrectomized rats treated with high and low doses of EPO-enriched cells compared with tubule-enriched cells and rEPO. [Figure 56] a and b show serum creatinine concentration at 16 weeks post-treatment and creatinine as a percentage of controls pre-treatment to 16 weeks post-treatment. [Figure 57] Shown are swimming endurance results at 12 weeks post-treatment. [Figure 58] Weight gain at 16 weeks post-treatment is shown as a percentage of initial weight. [Figure 59] Serum creatinine is expressed as a percentage of healthy controls among untreated uremic rats and uremic rats treated with high doses of EPO-enriched cells (Prototype #2) or tubule-enriched cells (Prototype #4). [Figure 60] 1 shows the albumin / globulin ratio at 12 weeks after treatment in untreated uremic rats and in uremic rats treated with high doses of EPO-enriched cells (Prototype #2) or tubule-enriched cells (Prototype #4). [Figure 61] Phosphorus:calcium ratios are shown in untreated uremic rats and in uremic rats treated with high doses of EPO-enriched cells (Prototype #2) or tubule-enriched cells (Prototype #4). [Figure 62] Serum triglyceride levels in untreated uremic rats and in uremic rats treated with high doses of EPO-enriched cells (Prototype #2) or tubule-enriched cells (Prototype #4) are shown. [Figure 63] Serum cholesterol levels in untreated uremic rats and in uremic rats treated with high doses of EPO-enriched cells (Prototype #2) or tubule-enriched cells (Prototype #4) are shown. [Figure 64] Serum hemoglobin levels are shown in untreated uremic rats and in uremic rats treated with high doses of EPO-enriched cells (Prototype #2) or tubule-enriched cells (Prototype #4). [Figure 65] Serum hematocrit levels in untreated uremic rats and in uremic rats treated with high doses of EPO-enriched cells (Prototype #2) or tubule-enriched cells (Prototype #4) are shown. [Figure 66] Figure 1 shows the uptake of rhodamine-conjugated albumin by functional rodent kidney tubular cells. [Figure 67] 1 shows maintenance of serum creatinine (an index of kidney function) at near-normal levels in uremic rats treated with neo-kidney cells (heterogeneous EPO-producing cells) compared to untreated uremic rats. [Figure 68] 1 shows the maintenance of phenotypic characteristics of tubular and glomerular cells isolated and expanded from normal human kidneys. [Figure 69] Figure 1 shows increased expression of the tubule marker cadherin in tubule-enriched populations compared to tubule-depleted populations cultured in 3D dynamic cultures. [Figure 70] Through flow cytometry, the isolation of EPO-producing cells is demonstrated. [Figure 71] Shown are step gradients of "normoxic" (21% oxygen) and "hypoxic" (2% oxygen) rodent cultures collected separately and subjected side-by-side to identical step gradients. [Figure 72]Step gradients of "normoxic" (21% oxygen) and "hypoxic" (2% oxygen) canine cultures collected separately and applied side-by-side to identical step gradients are shown. [Figure 73] 1 shows the effect of hypoxic culture on gene expression in B4. [Figure 74] Figure 1 shows the results of compositional analysis of the B2 and B4 subfractions. Quantitative real-time PCR (qRTPCR) was used to assess the expression of renal cell type-specific genes in the B2 and B4 subfractions. (a, b) Subfraction B2 was enriched for proximal tubule cells based on the relative expression of vitamin D hydroxylase (CYP2R1) and cubilin compared with B4 and all other subfractions. (c) The distal tubule marker E-cadherin was also enriched in subfraction B2 compared with B4. (e, f) The glomerular markers nephrin and podocin were enriched in subfraction B4, as was the vascular marker kdr (d). (g, h) The oxygen-regulated interstitial markers Hif2α and Epo were also enriched in B4. (i) Results are shown quantitatively for B2 and B4. (j) Robust expression of proximal (N-cadherin / green) and distal (E-cadherin / red) tubule markers by B2 was confirmed through immunocytochemistry on cells cultured after subfractionation. (j) In contrast, E-cadherin- or N-cadherin-positive cells were rare in B4 cultures. [Figure 75]Functional characteristics of B2 and B4 subfractions are shown. (a) B2 and B4 cells were subcultured and assessed for cubilin expression and receptor-mediated albumin uptake. Panels show: i. B2 cells stained with an isotype-matched IgG control antibody against cubilin; ii. B2 cells pretreated with the competitive inhibitor, RAP, and pulsed with rhodamine-conjugated human serum albumin (HSA-rhodamine) for 15 min (10 μg / ml); iii. B2 cells pulsed with HSA-rhodamine for 15 min (10 μg / ml) (red) and labeled with anti-cubilin antibody (green); iv. Overlay image of (iii) showing colocalization of cubilin cells (green) with uptake of HSA-rhodamine (red); v. Same as (iii / iv), but with very few cubilin cells (green) and little to no albumin uptake (red). In all conditions, nuclei were counterstained with Hoechst (blue). (b) Epo expression was examined in each fraction (B1–B4) after 24 h of exposure to ambient (21%) or low (2%) oxygen tension (both at 37°C). The relative proportion of Epo-expressing cells capable of upregulating Epo in response to reduced oxygen tension was greatest in subfraction B4. Note the consistency of the relative Epo expression in B2 and B4 with the results illustrated in Figure 1(i). [Figure 76]The effects of in vivo transplantation of B2 and B4 on survival time, body weight, and heart weight in uremic / anemic rats are shown. (a) Percent survival is represented for each control and treatment group over the study duration (30 weeks after nephrectomy, 6 months after treatment). Among treatment groups, only B2-treated rats had 100% survival, equivalent to that of healthy controls. (b) The percent change in body weight over time was calculated individually for each rat as ((body weight at sacrifice) - (body weight at study start)) / (body weight at study start). The 14.3% increase in body weight in B2-treated animals compared with the 5% loss in body weight in Nx control animals was considered highly significant (P value < .0001). Additionally, B4 (P value = 0.0239) and vehicle (P value = 0.0125) showed significantly greater body weight gain than Nx animals. (c) Relative heart weight was calculated as a percentage of body weight at the time of necropsy. Untreated Nx rats had significantly enlarged hearts (150% of healthy controls) at the time of necropsy. B2 showed only a 25% increase in relative heart weight after 6 months of treatment (Fig. 3c) compared with Nx animals (P value < 0.0001). B4 (P value = 0.0002) and vehicle (P value < 0.0001) were also statistically significant compared with Nx controls. [Figure 77] Temporal and statistical evaluation of treatment effects on filtration and erythropoiesis are shown. One-way analysis of variance (ANOVA) was performed on serum chemistry results from all 10- to 20-week data sets using JMP version 7.0 from SAS Institute Inc. (Cary, NC). (a, b, c) Significant differences in erythropoiesis were observed between treatment groups (HCT, p = 0.0005; RBC, p = 0.0029), with B2- and B4-treated rats showing the greatest improvement over untreated Nx, UNFX-treated, and rEPO-treated rats. (d, e, f) Significant differences in filtration function were observed between treatment groups (sCREAT, p < 0.0001; BUN, p < 0.0001), with the effect of B2 treatment being most significant, followed by B4. [Figure 78]Results of clinical assessments at study midpoint (Wk 12-14) are shown. Blood was collected for clinical chemistry at study midpoint (Wk 12-14), and parameters highlighting differences between healthy controls and Nx, or Nx vs. B2-, B4-, or UNFX treatment are shown in panel (a). Two significant effects unique to B2 treatment were (b) enhanced protein retention, as indicated by increased serum albumin (sALB) and albumin / globulin (A / G) ratio in B2 vs. Nx (p<0.001), and (c) reduced serum triglycerides and cholesterol in B2 vs. Nx (p<0.001). [Figure 79] Kidney mass correlates with kidney function. Kidney weights (right) were collected at necropsy. (a) Hemi-renal mass in B2-treated kidneys was comparable to that of healthy controls. Examination of sCREAT in serum collected at necropsy revealed a clear inverse correlation between kidney mass and sCREAT (panel a, axis 2). (b) Linear regression analysis of kidney mass and sCREAT for all rats in the study confirmed a significant inverse correlation between kidney weight and kidney function (r = 0.38, P < 0.001). [Figure 80] PCR-based DNA analysis using a probe for SRY is shown. [Figure 81]Histopathological analysis of kidneys and bone marrow is shown. (a) Representative optical micrographs of sham control and remnant kidneys (Masson's trichrome and PAS staining). Sham (control) kidneys have normal parenchymal architecture, characterized by a well-defined cortico-medullary junction and the absence of tubular or glomerular damage. Nx-untreated animals showed progressive glomerular and tubular degeneration consisting of moderate to marked tubulointerstitial fibrosis and glomerulosclerosis (stained blue by Masson's trichrome), tubular dilation with luminal casts (eosinophilic staining by PAS), and reduced bone marrow cellularity (myeloid to erythroid ratio). In contrast to Nx animals, B2-treated rats showed evidence of treatment efficacy, characterized by reduced tubulointerstitial fibrosis and glomerulosclerosis, and near-normal bone marrow cellularity with myeloid to erythroid ratios comparable to those of healthy controls. (b) H&E stained bone marrow revealed ample evidence of bone resorption in untreated Nx rats, with prominent osteoclasts and the formation of lacunae associated with bone erosion. Like healthy controls, the endosteal surface of B2-treated rats was smooth without any evidence of osteoclasts, lacunae, or bone erosion. (c) Terminal serum phosphite (mg / dl) and calcium (mg / dl, corrected for total protein [Ca] -0.4 [TP] +3.3) provided systemic support for histological observation of bone resorption in the model and amelioration of resorption with B2 treatment. [Figure 82a] Survival over 12 weeks post-treatment is shown. [Figure 82b] Survival over 25 weeks post-treatment is shown. [Figure 83a] Total body weight from initial weight to sacrifice is shown. [Figure 83b] 1 shows the percent weight change from initial weight to sacrifice in rodents treated with cell populations of the delivery system. [Figure 84] Serum creatinine levels over 12 weeks for all treatment groups are shown. [Figure 85] Serum creatinine levels over 12 weeks are shown for B2 and B4 treatment groups along with NX, healthy controls, and sham NX. [Figure 86]One-way analysis of variance of serum creatinine across all time points is presented. [Figure 87] Blood urea nitrogen (BUN) levels are shown. [Figure 88] HCT percentages over 12 weeks for all treatment groups are shown. [Figure 89] HCT percentages over 12 weeks are shown for B2 and B4 treatment groups along with NX, healthy controls, and sham Nx. [Figure 90] One-way ANOVA of HCT percentages across all time points is presented. [Figure 91] Heart weight is expressed as a percentage of total body weight. [Figure 92] Total serum protein levels at study endpoints are shown. [Figure 93] Histological evaluation at 3 months is shown. [Figure 94] Survival data at 12 weeks post-treatment are shown. [Figure 95] The percentage change in body weight from pre-treatment to 12 weeks post-treatment is shown. [Figure 96] Serum creatinine levels at 12 weeks are shown. [Figure 97] Serum BUN levels at 12 weeks are shown. [Figure 98] Expressed urinary protein levels compared to healthy controls are presented. [Figure 99] Serum A / G ratios are shown 6 weeks after treatment. [Figure 100] HCT percentage at 12 weeks is shown. [Figure 101] RBC counts at 12 weeks are shown. [Figure 102] Mean systemic blood pressure at 6 and 2 weeks after treatment is presented. [Figure 103] Histological evaluation at 3 to 5 months is shown. [Figure 104] The effect of the prototype on serum creatinine levels is shown. [Figure 105] The prototype effect of no effect on serum creatinine levels is shown. [Figure 106]Figure 1 shows the effect of B3 and B2 / B4 on serum creatinine levels. [Figure 107] 1 shows the effects of B2, B2 / B4 and B2 / B3 on urinary protein levels. [Figure 108] Shows the effects of B2 / B4, B2 / B3 and B3 / B4 on blood pressure. [Figure 109] The difference between baseline creatinine levels and the end of the study is shown. [Figure 110] The difference between baseline BUN levels and the end of the study is shown. [Figure 111] Represents the difference between baseline and end of study ALB levels. [Figure 112] The difference between baseline and end of study TPRO levels is shown. [Figure 113] Represents the difference between baseline PHOS levels and the end of the study. [Figure 114] The difference between baseline and end of study calcium levels corrected for total protein is represented. [Figure 115] 1 shows canine kidney cell growth. [Figure 116] 1 shows porcine kidney cell growth. [Figure 117] Human kidney cell growth. [Figure 118] 1 depicts cell proliferation results from human biopsies. [Figure 119] 1 shows the results of immunocytochemistry on cells. [Figure 120] Flow cytometry results are shown showing that EPO-expressing cells contained in rat cultures were distinct from both proximal and distal tubule cells (Fig. 120a, b). Functionality of cubilin-positive proximal tubule cells in culture was assessed via uptake of fluorescently conjugated albumin, and the specificity of uptake was confirmed by the addition of the competitive inhibitor, RAP (Fig. 120c-f). [Figure 121] Monthly monitoring of filtration function is shown (sCREAT is shown in (a), HCT is shown in (b), and comparative clinical chemistry results are shown in (c)). [Figure 122]1 depicts histological analyses performed to identify tissue level effects in both kidney and bone marrow. [Figure 123] 1 shows the histopathological features of CKD samples compared to those of non-CKD kidney samples from both pigs and humans. [Figure 124] Expansion capacity between CKD and non-CKD-derived cultures is shown (a); tubular cell function was confirmed by observing receptor-mediated uptake of albumin in a subset of cubilin-positive cells in established cultures (b-d). [Figure 125] EPO mRNA expression in CKD versus non-CKD tissue samples (a). Isoelectric focusing and Western blot analysis of these samples confirmed that gene expression patterns were generally recapitulated at the protein level (b); EPO-expressing cell cultures established from both CKD and non-CKD kidney samples responded to hypoxic stimulation with a variable upregulation of EPO gene transcription within 24 hours of stimulation (c). [Figure 126] Figure 1 shows cell bands in an adult rodent step gradient. [Figure 127] 1 shows gene expression patterns of erythropoietin, nephrin, podocin, cubilin, and Cyp in adult rodent cell preparations. [Figure 128] A cell preparation isolated from a diseased adult rodent kidney is shown (5X). [Figure 129] 1 shows the expression of B4-specific genes in cells isolated from adult rats with end-stage renal failure. [Figure 130] 1 depicts a direct comparison of creatinine levels in juvenile and adult B2 cell preparations. [Figure 131] 1 depicts a direct comparison of serum BUN values ​​in juvenile and adult B2 cell preparations. [Figure 132] Expression of Has-2 in B2 and B4 cell preparations is shown. [Figure 133] In vivo expression of HAS mRNA (qRTPCR, bottom graph) and protein (top panel, Western blot) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention provides an isolated kidney comprising renal tubules and erythropoietin (EPO)-producing kidney cells. and methods for isolating and culturing the same, as well as enriched tubular and A method of treating a subject in need with bioactive kidney cells, including a cell population that produces EPO, is disclosed. Let's call it an elephant.

[0067] definition Unless otherwise defined, technical and scientific terms used herein are defined by the principles of the art to which this invention belongs. (R Lanza, R Lan) ger, & J Vacanti (eds.) Principles of Tissue En Engineering, 3rd Ed., 2007, provides a basis for many of the terms used in this application. The present invention provides general guidance to those skilled in the art that may be used to practice the invention. Many methods and materials similar or equivalent to those described herein will be readily apparent to those skilled in the art. Indeed, the present invention is in no way limited to the methods and materials described.

[0068] As used herein, a "cell population" refers to a cell population obtained directly from a suitable tissue source, usually from a mammal. The term refers to a large number of cells obtained by contact isolation. The isolated cell population is then cultured in vitro. Those skilled in the art will appreciate the ease with which cell populations can be isolated and cultured for use with the present invention. It will be appreciated that various methods and various numbers of cells in cell populations suitable for use in the present invention may be used. The cell population can be an unfractionated, heterogeneous cell population derived from the kidney. For example, Heterogeneous cell populations can be isolated from kidney biopsies or from whole kidney tissue. , heterogeneous cell populations arising from in vitro culture of mammalian cells, kidney biopsies or whole kidneys Unfractionated heterogeneous cell populations can be established from liver tissue. It can also be referred to as.

[0069] As used herein, the term "mixture" refers to a mixture of two or more cells resulting from an unfractionated, heterogeneous population of cells. According to certain embodiments, the present invention relates to a combination of one or more isolated, enriched cell populations. The bright cell population is a kidney cell population.

[0070] An "enriched" cell population or preparation is one in which the proportion of a particular cell type is greater than that of the starting population. A starting kidney cell population (e.g., an unfractionated, heterogeneous cell population) containing a proportion of that cell type For example, a starting kidney cell population may be divided into a first, second, third, fourth, or As used herein, "cell" refers to a cell population that is enriched for a fifth, etc., cell population of interest. The terms "cell population," "cell preparation," and "cell prototype" are used interchangeably.

[0071] In one embodiment, an "enriched" cell population, as used herein, is a cell population that is enriched for EPO from a starting population. The proportion of cells capable of producing EPO is greater than the proportion of cells capable of producing EPO. A starting kidney cell population containing IgG (e.g., a cell suspension from a kidney biopsy or cultured mammalian cells) For example, "B4" refers to a cell population derived from a starting population of: Starting kidney containing a greater proportion of EPO-producing cells, glomerular cells, and vascular cells The cell populations of the present invention are cell populations derived from a stem cell population. The EPO-enriched cells can be enriched and depleted of one or more other cell types. For example, The resulting cell population is enriched for interstitial fibroblasts and is not enriched for non-interstitial fibroblasts, i.e. i.e., the starting cell population from which the enriched cell population was derived, interstitial fibroblasts and tubular cells. Compared to conventional methods, it is possible to remove renal tubular cells and collecting duct epithelial cells. In all embodiments that refer to a "B4" population, the enriched cell population is endogenous Oxygen regulation is essential as demonstrated by oxygen-regulatable EPO expression from the native EPO gene. It is a heterogeneous population of cells containing cells that can produce EPO in a controlled manner. .

[0072] In another embodiment, the enriched cell population is enriched for one or more tubular cell markers of the starting population. The proportion of cells expressing one or more tubular cell markers is greater than the proportion of cells expressing It can also refer to a cell population derived from the starting kidney cell population described above, containing, for example, "B2" represents starting kidney cells containing a greater proportion of tubular cells compared to the starting population. It refers to a cell population arising from a tubular cell population. In addition, it is possible to identify one or more tubular cell markers (or "B The cell population enriched for cells expressing α-glucan 2′) contains some epithelial cells from the collecting duct system. A cell line enriched for cells expressing one or more renal tubule cell markers may be included. The cytoplasmic population (or "B2") is composed of EPO-producing cells, glomerular cells, and vascular cells. Although the cells are relatively depleted, the enriched population contains a smaller proportion of these cells compared to the starting population. It can contain vesicles (EPO-producing glomeruli and blood vessels). The homogeneous cell population is the cell type of the removed cell population that was present in the heterogeneous cell population before removal. One or more cell types are removed to contain a smaller proportion of the cell type compared to the proportion of the The cell type that can be removed is any type of kidney cell. For example, in certain embodiments In the present study, the cell type that can be removed is approximately 1.045 g / m, referred to as "B1". Contains cells of the collecting duct and tubular system with a density less than L and large granularity. In an embodiment, the cell type that can be removed is a cell type designated as "B5" that is approximately 1.0 Contains debris and small cells of low granularity and viability with a density greater than 95 g / mL. In some embodiments, the cell population enriched for tubular cells is relatively rich in all of the following: Removed by: "B1", "B5", oxygen-regulatable EPO-expressing cells, glomerular cells , and vascular cells.

[0073] As used herein, the term "hypoxic" culture conditions refers to conditions in which cells are cultured in an atmosphere containing oxygen levels (approximately 100% oxygen). 21%), which reduces the available oxygen levels in the culture system compared to standard culture conditions. Non-hypoxic conditions refer to the culture conditions of cells. Non-hypoxic conditions are referred to herein as normal or normoxic culture conditions. It is called.

[0074] As used herein, the term "oxygen tunable" refers to a cell that is oxygen-tunable based on the amount of oxygen available to the cell. "Hypoxia-inducible" refers to the ability of a cell to regulate gene expression (increase or decrease). Upregulation of gene expression in response to a decrease in oxygen tension (regardless of pre-induction or onset of oxygen tension) Refers to...

[0075] As used herein, a "biomaterial" refers to a natural or synthetic biological material suitable for introduction into living tissue. Natural biomaterials are materials made by living systems. Synthetic biomaterials A biomaterial is a material that is not produced by a biological system. The biomaterials disclosed herein are naturally occurring materials. and synthetic biocompatible materials. Biomaterials include, for example, polymer matrices and scaffolds. Those skilled in the art will appreciate that biomaterials can be used in a variety of ways. It can be configured in various forms, for example, as a liquid hydrogel suspension, a porous foam, etc. It will be appreciated that the present invention may comprise one or more natural or synthetic biocompatible materials.

[0076] As used herein, the term "anemia" refers to a functional anemia by cells that produce EPO in a subject. Inadequate production of EPO protein and / or inadequate delivery of EPO protein to the systemic circulation Red blood cells due to inability of the bone marrow erythroblasts to respond to EPO protein properly. Refers to a deficiency in blood cell count and / or hemoglobin levels. A subject with anemia has impaired red blood cell homeostasis. Anemia is generally associated with decreased kidney function (e.g., chronic renal failure) or loss, anemia associated with related EPO deficiency, anemia associated with congestive heart failure, chemotherapy, or anemia associated with myelosuppressive therapy such as antiviral therapy (e.g., AZT), and non-myeloma cancers anemia associated with viral infections such as HIV, and autoimmune diseases (e.g., Occurs with anemia, liver disease, and multiple organ system failure in chronic diseases such as rheumatoid arthritis .

[0077] The term "EPO deficiency" refers to the use of erythropoietin receptor stimulating drugs (e.g., EPO refers to any condition or disease that is treatable with EPO (e.g., recombinant EPO or EPO analogs).

[0078] As used herein, the term "kidney disease" refers to the loss of blood filtration and excess fluid, electrolytes, and and acute or chronic conditions that cause an inability of the kidneys to perform their function of removing waste products from the blood. Refers to a condition associated with any stage or degree of kidney failure. Kidney disease can be caused by anemia (erythropoietin) or Also includes endocrine dysfunction such as ethine deficiency and mineral imbalances (vitamin D deficiency) Kidney disease can begin in the kidneys or can be caused by heart failure, high blood pressure, diabetes, autoimmune diseases, or It can be secondary to a variety of conditions, including but not limited to liver disease.

[0079] The term "treatment" refers to a condition that affects kidney disease, anemia, EPO deficiency, tubular transport deficiency, or Refers to both therapeutic treatment and prophylactic or preventative measures against glomerular filtration deficiency, The goal is to reverse, prevent, or slow (reduce) the target disease. What is needed is kidney disease, anemia, EPO deficiency, tubular transport deficiency, or glomerular Those with pre-existing deficiencies in systemic filtration, as well as those with kidney disease, anemia, EPO deficiency, and renal tubular transport those prone to deficiency of glomerular filtration, or kidney disease, anemia , EPO deficiency, tubular transport deficiency, or glomerular filtration deficiency is prevented. As used herein, the term "treatment" includes stabilizing and / or improving renal function. include.

[0080] The term "construct" refers to a structure composed of one or more synthetic or naturally occurring biocompatible materials. refers to one or more cell populations deposited on or within the surface of a scaffold or matrix. one or more cell populations in a culture medium containing one or more synthetic or naturally occurring biocompatible polymers, proteins, or or by coating, depositing, or embedding in a biomaterial composed of a peptide. One or more cell populations can be attached to, or entrapped within the In vitro and in vivo, it can be combined with biomaterials or scaffolds or matrices. Typically, one or more biocompatible materials are used to form the scaffold / biomaterial. The method comprises forming at least one multicellular, three-dimensional, organized cell population deposited thereon. Selected to direct, facilitate, or enable. Used to generate structures. The one or more biomaterials used may be used to construct and / or integrate into endogenous host tissue. directing, facilitating, or enabling cellular components of structures, including tissues; or Directing the survival, engraftment, tolerance, or functional capacity of the construct or its cellular components To make, facilitate, or enable.

[0081] The term "subject" refers to a subject suffering from one or more of kidney disease, anemia, or EPO deficiency who is suitable for treatment. Patients who are experiencing or have experienced any of the above signs, symptoms, or other indicators "kidney disease" means any single human subject, regardless of cause, including any single human subject having kidney disease, Newly diagnosed or previously diagnosed with a disease, anemia, or EPO deficiency This includes, but is not limited to, subjects experiencing or at risk of developing or experiencing a flare-up or relapse. Subjects must not have been previously treated for kidney disease, anemia, or EPO deficiency. They may or may not have been so treated.

[0082] The term "patient" refers to any single animal, more preferably a mammal, for whom treatment is desired. animals (e.g., dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non- More preferably, the patient herein refers to a human. It is.

[0083] The term "sample," or "patient sample," or "biological sample" generally refers to a sample of a subject or or any material obtained from a patient, body fluid, body tissue, cell line, tissue culture, or other source. "biological sample" means a biological sample. This term includes, for example, a tissue biopsy, such as a kidney biopsy. The term includes, for example, cultured cells such as cultured mammalian kidney cells. Methods for obtaining tissue biopsies and cultured cells from tissues are well known in the art. When the term "sample" is used alone, it is understood that the "sample" is a "biological sample" or "patient sample" is still meant to be a "patient sample," i.e., the terms are used interchangeably. It is used for the purpose.

[0084] The term "test sample" refers to a sample from a subject that has been treated by the methods of the present invention. Test samples include, but are not limited to, blood, semen, serum, urine, bone marrow, mucosa, tissue, etc. The cellular components of the present invention may be derived from a variety of sources in mammalian subjects.

[0085] The terms "control" or "control sample" refer to a sample in which a negative or positive result correlates with the result of a test sample. This refers to a negative or positive control that is expected to aid in the association of The controls included samples known to exhibit indices characteristic of normal red blood cell homeostasis, samples known to exhibit indices characteristic of anemia, and samples known to exhibit indices characteristic of anemia. samples known to exhibit indicators of anemia, obtained from subjects known not to be anemic Examples of samples obtained from subjects known to be anemic include, but are not limited to, samples obtained from subjects known to be anemic. Additional controls suitable for use in the methods of the present invention include, but are not limited to, red Pharmacological agents known to regulate blood cell production (e.g., recombinant EPO or E These include, but are not limited to, samples from subjects who have been treated with PO analogs. Additionally, the control may be a sample obtained from the subject prior to treatment with the method of the present invention. Further suitable subjects may have any type or stage of kidney disease. Test samples obtained from subjects known to have kidney disease of any type or stage are also included. The control may be a sample from a subject known to be free of the disease. Those skilled in the art will recognize other targets suitable for use in the present invention.

[0086] cell population The present invention provides specific bioactive components or cell types for use in the treatment of kidney disease. concentrated against and / or containing certain inactive or undesirable components or To provide a cell type-depleted, isolated, heterogeneous renal cell population and mixtures thereof, i.e. That is, it provides stabilization and / or improvement and / or regeneration of kidney function.

[0087] Bioactive cell populations In one aspect, the present invention provides a method for preparing a composition enriched for a bioactive ingredient, which is inactive or undesirable. Specific subfractions of the heterogeneous renal cell population, from which unwanted components have been removed, may provide superior therapeutic benefits compared to the starting population. This is based on the unexpected discovery that the drug can produce both inactive and regenerative results. or undesired components, e.g., B1 and B5, have been removed from the bioactive components of the present invention, e.g., For example, B2, B4, and B3, alone or in combination, may have unexpected effects on renal function. In a preferred embodiment, the invention provides stabilization and / or improvement and / or regeneration. The bioactive cell population is B2. In certain embodiments, the B2 cell population is B4. In another embodiment, the B2 cell population is mixed with B3.

[0088] The B2 cell population expresses megalin, cubilin, hyaluronan synthase 2 (HAS2), and vitamin D. Cytochrome P450 (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aq p2), RAB17, member of the RAS oncogene family (Rab17), GATA binding Protein 3 (Gata3), FXYD domain-containing ion transport regulator 4 (F xyd4), solute transporter family-9 (sodium / hydrogen exchanger), member-4 (Slc 9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde Aldh1a3, member A3 of the aldehyde dehydrogenase 1 family, and calpain a renal tubule cell marker selected from the group consisting of Capn8 (Capn8), and a collecting duct marker Characterized by aquaporin-4 (Aqp4) expression, greater than B3 and / or B4 Furthermore, because it is granular, the concentration is approximately 1.045 g / mL to approximately 1.063 g / mL (rodents), Approximately 1.045 g / mL to 1.052 g / mL (human), and approximately 1.045 g / mL to approximately It has a buoyant density of 1.058 g / mL (dog).

[0089] The B3 cell population expresses vascular markers, glomerular markers, and proximal tubule markers. and some EPO-producing cells, compared with B2 and B4. Due to its medium size and granularity, it is approximately 1.063 g / mL to approximately 1.073 g / mL ( rodents), approximately 1.052 g / mL to 1.063 g / mL (humans), and approximately 1.058 g / mL B3 has a buoyant density of approximately 1.063 g / mL (dog). B3 is one or more of the following: Characterized by expression of a marker selected from the group consisting of: aquaporin 7 (Aqp7) , FXYD domain-containing ion transport regulator 2 (Fxyd2), solute transporter phagocytosis Milli-17 (sodium phosphate), member-3 (Slc17a3), solute transporter family -3, member-1 (Slc3a1), claudin 2 (Cldn2), napsin A, and asparagine Napsa, solute transporter family 2 (facilitated glucose transport) membrane), 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-bisphosphatase 1 (Fbp1), and alanine aminotransferase 2 (Agx t2). B3 binds to the vascular expression markers platelet endothelial cell adhesion molecule (Pecam) and The glomerular-expressed marker podocin (Podn) is also characterized.

[0090] The B4 cell population contains one or more of the following: PECAM, VEGF, KDR, and HIF1a The vascular markers podocin (Podn) and nephrin (Neph) A glomerular marker set containing one or more of unfractionated (UNFX), B2, and Compared to B3, B4 is characterized by an oxygen-tunable EPO-enriched population. They are also characterized by the expression of one or more of the following receptors: chemokine (CXC motif) receptor 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 protein Protein receptor (Kdr), secreted protein, acidic, cysteine-rich (osteone) Cutin (Sparc), Serglycin (Srgn), TIMP metallopeptidase inhibitor Timp3, Wilms' tumor 1 (Wt1), wingless type MMTV integration site family Wnt4, regulator of G-protein signaling 4 (Rgs4) , platelet endothelial cell adhesion molecule (Pecam), and erythropoietin (Epo). It is also characterized by being smaller and less granular than either B2 or B3. Approximately 1.073 g / mL to approximately 1.091 g / mL (rodents), approximately 1.063 g / mL ~ Buoyant density of approximately 1.091 g / mL (human and dog).

[0091] Hyaluronic acid production by B2 and B4 Hyaluronan (also called hyaluronic acid or hyaluronate) is a glycosaminoglycan Glycans (GAGs) containing non-sulfated disaccharide units, particularly N-acetylglucosamine and glutamic acid. It consists of a regular repeating sequence of uronic acid. Its molecular weight is between 400 daltons (disaccharide) and It can range from 100,000 to over 1 million daltons. All tissues, such as skin, cartilage, and eyes of adult animals It is found in varying amounts in tissues and most, if not all, body fluids. is particularly abundant in early embryos. Hyaluronan, in fact, is formed by GAGs in general. The space created by these membranes is a critical space for cell migration, cell attachment, wound repair, organogenesis, immune cell adhesion, and intracellular signaling. This suggests that hyaluronan plays a role in the activation of cellular signaling and tumor metastasis. Their role is to bind specific proteins and proteoglycans to hyaluronan. Cell motility and adhesion of immune cells are mediated by the cell surface receptor RHAMM (R eceptor for Hyaluronal-Mediated Motility ; Hardwick et al., 1992) and CD44 (Jalkenan e t al., 1987; Miyake et al., 1990). Hyaluronan is synthesized directly in the inner membrane of the cell surface, and the growing polymer translocates through the membrane once synthesized. The synthesis is carried out by a gene family with at least three members. It is mediated by a single protein enzyme, hyaluronan synthase (HAS), which is composed of can be.

[0092] Hyaluronan interacts with CD44, and such interaction may, among other effects, induce damage Mobilize non-resident cells (mesenchymal stem cells (MSCs) etc.) to damaged kidney tissue to promote kidney regeneration (Kidney International (2007) 72, 430- 441) has recently become apparent.

[0093] Unexpectedly, B2 and B4 cell preparations were more specifically enriched in the B2 cell population. In vivo, hyaluronan synthase-2 (HAS-2) is a marker of hyaluronan synthesis. Able to express high molecular weight species of hyaluronic acid (HA) both in vitro and in vivo Treatment with B2 in the 5 / 6Nx model resulted in strong HA in vivo. Concomitant with the expression of S-2 and the expected production of high molecular weight hyaluronan in the treated tissues , which have been shown to reduce fibrosis. Notably, the untreated 5 / 6Nx model showed AS-2 was detected only slightly, resulting in fibrosis, and high molecular weight HA was hardly produced. Without wishing to be bound by theory, it is primarily due to B2 (and to some extent to B4) This anti-inflammatory high molecular weight species of hyaluronan, produced by the inflammatory process, is thought to reduce renal fibrosis and promote kidney regeneration. It is hypothesized that this compound acts synergistically with the cell preparation in supporting the The invention includes the delivery of the cell prototypes of the invention in biomaterials comprising hyaluronic acid. Providing biomaterial components that stimulate regeneration through direct production or stimulation of production by transplanted cells , are contemplated by the present invention.

[0094] In one aspect, the present invention provides a method for treating kidney disease, anemia, and / or The present invention provides an isolated heterogeneous population of EPO-producing renal cells for use in treating EPO deficiency. In one embodiment, the cell population is derived from a kidney biopsy. In certain other embodiments, the cell population is derived from a kidney biopsy or are derived from in vitro cultures of mammalian kidney cells established from whole kidney tissue. In morphology, these populations are unfractionated cell populations and are not enriched herein. Also referred to as a cell population.

[0095] In another embodiment, the present invention provides a method for producing EPO in a starting or initial cell population. The proportion of EPO-producing cells in the enriched subpopulation is higher compared to the proportion in the control group. To further enrich for erythropoietin (EPO)-producing renal cells, an isolated population was prepared. In one embodiment, the enriched EPO-producing cell fraction is not enriched. A higher proportion of interstitial fibroblasts and tubular cells compared to the initial population In certain embodiments, the renal cell membrane contains stromal fibroblasts and a lower proportion of tubular cells. The enriched EPO-producing cell fraction is a fraction of glomerular cells contained in the initial non-enriched population. , a higher proportion of glomerular and vascular cells compared with vascular cells, and collecting duct cells, In such embodiments, these collecting duct cells contain a lower proportion of collecting duct cells. The group is referred to herein as the "B4" cell population.

[0096] In another embodiment, the present invention provides an EPO-producing kidney cell that is mixed with one or more additional kidney cell populations. In one embodiment, the EPO-producing cell population is provided. In another embodiment, the first cell population is enriched for cells that The EPO-producing cell population is not enriched for EPO-producing cells. In another embodiment, the first cell population is a second renal cell population, e.g., B2. In some embodiments, the second cell population is enriched for renal tubular cells. In another embodiment, the renal tubule cell phenotype may be indicated by the presence of the renal tubule cell phenotype. The phenotype can be indicated by the presence of tubular cell markers. The tubular cell phenotype can be indicated by the presence of one or more tubular cell markers. Cell markers include megalin, cubilin, hyaluronic acid synthase 2 (HAS2), and vitamin Cysteine ​​D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E -cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp 2) RAB17, a member of the RAS oncogene family (Rab17), a GATA-binding protein Gata3, FXYD domain-containing ion transport regulator 4 (FXYD domain-containing ion transport regulator 4) yd4), solute transporter family-9 (sodium / hydrogen exchanger), member-4 (Slc9 a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde Aldhyl dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8). In another embodiment, the first cell population cells of interstitial origin, tubular cells, collecting duct-derived cells, glomerular-derived cells, and / or The cysts contain several types of kidney cells, including, but not limited to, blood or vascular derived cells. It is mixed with at least one of these.

[0097] In one embodiment, the EPO-producing renal cell population of the present invention is characterized in that a decrease in oxygen tension in the culture system induces EPO production. EPO expression and the biological response to oxygen were characterized, resulting in induction of EPO expression. In one embodiment, the EPO-producing cell population is characterized by the presence of EPO-producing cells. In one embodiment, the cell population is enriched for standard atmospheric levels of availability (approximately 21%). The level of oxygen available to cells in the culture system is higher than that of a cell population cultured in the available oxygen. When cells are cultured under conditions where they are exposed to a decrease in the level of EPO, expression of EPO is induced. In this study, EPO-producing cells cultured under lower oxygen conditions were found to be more efficient than those cultured under normoxic conditions. These cells express higher levels of EPO compared to cultured EPO-producing cells. , culturing cells at lower available oxygen levels (also called hypoxic culture conditions) The decreased oxygen level is due to the reduction in oxygen levels compared to standard atmospheric levels of available oxygen (standard or normoxic culture). In one embodiment, the cell culture temperature is lower than that of a cell culture in a nutrient-rich environment (also called nutrient conditions). Hypoxic cell culture conditions include those containing less than about 1% oxygen, less than about 2% oxygen, less than about 3% oxygen, or less than about In another embodiment, the cells are cultured at less than 4% oxygen, or at less than about 5% oxygen. In this study, standard or normoxic culture conditions were defined as approximately 10% oxygen, approximately 12% oxygen, and approximately 13% oxygen. of oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% Cultivating cells at about 10% oxygen, about 19% oxygen, about 20% oxygen, or about 21% oxygen Includes.

[0098] In certain other embodiments, cells are cultured with less than about 5% available oxygen and the production of EPO is By comparing the present levels of EPO to cells cultured in atmospheric (approximately 21%) oxygen, Induction or increased expression can be obtained and observed. Induction occurs when cell cultures are grown in atmospheric oxygen (approximately 21%) for some period of time. In the first culture step, the available oxygen level is reduced so that the cells grow to less than about 5% of the available oxygen. and a second culture step in which the cells are cultured in an oxygen-rich atmosphere. In another embodiment, the present invention provides a method for producing a cellular marker that is responsive to hypoxic conditions. The expression of certain EPOs is regulated by HIF1α. Other oxygen-manipulated culture conditions known in the art may be used with the cells described herein. You will understand that.

[0099] In one embodiment, the enriched population of EPO-producing mammalian cells is a biological response to perfusion conditions. In one embodiment, the perfusion conditions are characterized by transient responsiveness (e.g., EPO expression). In one embodiment, the cells are cultured in a manner that includes constant, intermittent, or continuous fluid flow (perfusion). The medium is circulated intermittently or continuously in a manner that transfers power to the cells via flow. In one embodiment, the expression of EPO is induced transiently when the cells are heated or stirred. Cells exposed to intermittent or continuous fluid flow exhibit frame-like behavior for the formation of three-dimensional structures. - Such three-dimensional structures in or on the material that provide work and / or space In one embodiment, the cells are cultured in a manner that allows them to express themselves. Cultured on a rocking platform, orbital platform, or spinner flask In another embodiment, the cells are exposed to intermittent or continuous fluid flow by The cells are cultured on a 3D scaffold and placed in a device, which stabilizes the scaffold and allows fluid The skilled artisan will appreciate that the flow of fluid through or across the scaffold is well known in the art. It will be understood that certain other perfusion culture conditions may be used for the cells described herein. Deaf.

[0100] Inactive cell population As described herein, the present invention provides a method for preparing a pharmaceutical composition that is enriched for bioactive ingredients and is inactive or The specific subfraction of a heterogeneous population of kidney cells, from which undesired components have been removed, is more abundant than the starting population. This is based in part on the unexpected discovery that endothelial cell carcinoma (EOC) therapy provides superior therapeutic and regenerative outcomes compared to conventional endothelial cell carcinoma (EOC). In a preferred embodiment, the cell populations of the present invention are depleted of B1 and / or B5 cell populations. It has been done.

[0101] The B1 cell population contains the large granular cells of the collecting duct and tubular system, and the cells of this population are approximately The B5 cell population has a buoyant density of less than 1.045 g / m. The B5 cell population is characterized by debris, granularity, and viability. It is composed of small cells with low solubility and has a buoyant density of greater than approximately 1.091 g / mL.

[0102] Methods for isolating and culturing cell populations In one aspect, the present invention provides a method for treating kidney disease, anemia, EPO deficiency, renal tubular transport deficiency, and For therapeutic uses, including the treatment of glomerular filtration deficiency, kidney cell components, e.g., concentrated kidney cells, A method for separating and isolating a cell population is provided. In one embodiment, the cell population comprises: , from freshly digested, i.e., mechanically or enzymatically digested kidney tissue, or It is isolated from a heterogeneous in vitro culture of mammalian kidney cells.

[0103] A heterogeneous mixture of kidney cells is cultured in hypoxic culture conditions before separation on a density gradient. This results in an enrichment of the cellular distribution and composition in both the B4 and B2 fractions. was unexpectedly discovered. Kidney tissue isolated from both diseased and unaffected kidneys For cells, an enrichment of oxygen-dependent cells in B4 from B2 was observed. Although not a guarantee, this may be due to one or more of the following phenomena: 1) Low 2) selective survival, death, or proliferation of specific cellular components during oxygen culture; Depending on the cell granularity and / or size, the buoyant density and subsequent to achieve changes in localization during density gradient separation; and 3) in response to periods of hypoxic incubation. Changes in the expression of genes / proteins in cells, thereby affecting any given fraction of the gradient. Thus, in one embodiment, the differentiation characteristics of cells within the renal tubule cells are Enriched cell populations, such as B2, are hypoxia tolerant.

[0104] Exemplary techniques for separating and isolating cell populations of the present invention include determining the specific cell types contained within the population. This involves separation on a density gradient based on the different specific gravities of the different cell types being sampled. The specific gravity of a cell can be affected by the degree of granularity within the cell, the intracellular volume of water, and other factors. In one aspect, the present invention relates to a method for the treatment of rhesus mastitis in animals, including, but not limited to, humans, dogs, and rodents. For the isolation of cell preparations of the present invention, e.g., B2 and B4, across multiple species, In a preferred embodiment, the density gradient provides optimal gradient conditions for heterogeneous renal cell populations. A novel enriched population of renal tubular cells, i.e., a B2 cell population, was obtained from the B2 cell population. In one embodiment, the density gradient is used to separate a population of renal cells derived from a heterogeneous population of renal cells. This results in a novel enriched population of EPO-producing cells, i.e., a B4 cell population. In another embodiment, the density gradient is used to separate renal tubular cells, glomerular cells, and / or and is used to obtain an enriched subpopulation of endothelial cells. Both PO-producing cells and tubular cells are separated from red blood cells and cellular debris In one embodiment, the EPO-producing cells, glomerular cells, and vascular cells are different from other cells. A subpopulation of renal tubular and collecting duct cells separated from cytoplasmic and red blood cells and cellular debris. The aggregates are simultaneously separated from other cell types as well as red blood cells and cellular debris.

[0105] The present invention provides a 60% non-ionic iodized iodine-containing ... OPTIPREP® (Axis-Shie) containing the compound iodixanol in water ld) New cell populations were generated in part by using density gradient media. However, one of skill in the art will appreciate that any density gradient or other characteristics necessary to isolate the cell populations of the present invention can be readily obtained. Other means, such as using cell surface markers, are known in the art. It will be appreciated that immunological separation may also be used in accordance with the present invention. The same cellular characteristics (size and granularity) that contribute to the separation of cell subpopulations by flow cytometry It will also be understood by those skilled in the art that the method may be used to separate cell subpopulations by tri- (Forward scatter = reflection of size by flow cytometry, and side scatter = particle size) Importantly, the density gradient medium should have low toxicity to the specific cells in question. The density gradient medium should have low toxicity to the specific cells in question. While the present invention should have a role in the selection process of the relevant cells, The use of a gradient medium is contemplated. Without being bound by theory, the loading step and the recovery step Substantial cell loss was observed during the gradient, identifying exposure to iodixanol under gradient conditions. This suggests that iodixanol-containing gradients cause the removal of cells. The cell population of the present invention recovered by this method is considered to be iodixanol resistant. Cells appearing in a specific zone after the iodixanol gradient were induced by iodixanol and / or density gradient. Therefore, the present invention provides a method for producing a cell population of the present invention that is resistant to any adverse effects of exposure to the cell population. The use of additional contrast agents that are mild to moderate nephrotoxicants in isolation and / or selection is also contemplated. Furthermore, the density gradient medium should not bind to proteins in human plasma. Failure to do so can have detrimental effects on the vital functions of the affected cells.

[0106] In another aspect, the present invention provides a method for three-dimensionally culturing a renal cell population. In one embodiment, the present invention provides a method for culturing a cell population by continuous perfusion. In this study, cell populations cultured by three-dimensional culture and continuous perfusion were compared to those cultured statically. The cells exhibit greater cellularity and interconnectivity when compared to the cell populations of interest. In this embodiment, cell populations cultured by three-dimensional culture and continuous perfusion are Higher expression of EPO, as well as cadherins, in the kidney when compared to static cultures of the group. Figure 1 shows enhanced expression of renal tubule-related genes.

[0107] In yet another embodiment, the cell population cultured by continuous perfusion is a cell population cultured by static perfusion. Higher levels of glucose and glutamine consumption compared to control cell populations Shows.

[0108] Those skilled in the art will appreciate that other isolation and culture methods known in the art may be incorporated herein. It will be understood that the cells described may also be used.

[0109] Biomaterials (polymer matrices or scaffolds) Bertram et al., U.S. Patent Application Publication No. 20070276507 (incorporated herein by reference thereto). a polymer matrix or scaffold, as described in can be designed to satisfy any number of overall system, geometric, or spatial constraints. In one embodiment, the matrix or matrix of the present invention may be formed into any number of desired configurations. The scaffold may be three-dimensional and adapted to fit the dimensions and shape of the organ or tissue structure. For example, kidney disease, anemia, EPO deficiency, renal tubular transport deficiency, have demonstrated the use of polymer scaffolds to treat glomerular filtration deficiency through the development of three-dimensional (3D) matrices. Various different shapes of 3D scaffolds may be used. The polymer matrix is ​​available in different sizes and shapes to accommodate different sized patients. The polymer matrix may also be shaped to accommodate the specific needs of the patient. In another embodiment, the polymer matrix or The scaffold may be a biocompatible, porous polymer scaffold. The scaffold may have an open cell structure. Polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose acetate esters, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polya Crylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxa Zoll, polycarbonate, polycyanoaryl ether, polyester, polyester Carbonate, Polyether, Polyether ether ketone, Polyetherimide, Poly Ether ketone, polyether sulfone, polyethylene, polyfluoroolefin, poly Imide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene Polyethylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, poly Tetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl alcohol Nyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, Lagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid , agarose, or polymeric or physical blends thereof. The scaffold may be formed from a variety of synthetic or naturally occurring materials. They may range from liquid, to soft porous scaffolds, to rigid shape-retaining porous scaffolds.

[0110] Hydrogels may be formed from a variety of polymeric materials and are used in a variety of biomedical applications. Hydrogels are physically described as three-dimensional networks of hydrophilic polymers. Depending on the type of hydrogel, it contains various percentages of water, but the total Despite their high water content, hydrogels are insoluble in water due to the presence of hydrophilic residues. Hydrogels are capable of binding large amounts of liquid due to their gelatinous The basic physical properties of hydrogels are that they can expand to large extents without changing their structure. The properties of the polymer and the product can be specifically modified by additional specialized equipment.

[0111] Preferably, the hydrogel is biologically inert and physiologically compatible with mammalian tissue. polymers, biologically occurring materials, synthetically derived materials, or combinations thereof. The hydrogel material preferably does not induce an inflammatory response. Examples of other materials that can be used to make the membrane include (a) modified alginates, (b) modified alginates, (c) modified alginates, (d) modified alginates, (e) modified alginates, (f) modified alginates, (g) modified alginates, (h) modified alginates, (i) modified alginates, (j ... b) Polysaccharides that gel upon exposure to monovalent cations (e.g., gellan gum and and carrageenan), (c) are very viscous liquids or are thixotropic , polysaccharides (e.g., hyaluronan) that form gels over time by gradually developing structures. (d) polymer hydrogel precursors (e.g., polyethylene oxide-poly U.S. Patent No. 6, No. 224,893 B1 describes various polymers suitable for making hydrogels according to the present invention. The paper provides a detailed description of the polymers and their chemical properties.

[0112] The properties of the scaffold or biomaterial must be such that cells can attach to and interact with the scaffold or biomaterial. and / or provide a porous space in which cells can be trapped. In one embodiment, the porous scaffold or biomaterial of the present invention can The cell population or mixture may be placed on a porous scaffold (e.g., by cell attachment) and / or a scaffold Attachment or deposition onto biomaterials configured as pores (e.g., by entrapment of cells) In another embodiment, the scaffold or biomaterial allows for the growth of cells within the scaffold. allowing or promoting cell and / or cell:biomaterial interactions, as described herein The structures described are formed.

[0113] In one embodiment, the biomaterial used in accordance with the present invention is a hydrogel. Contains hydroxybenzoates (HA) and contains HA molecules ranging in size from 5.1 kDa to over 2 x 106 kDa In another embodiment, the biomaterial used in accordance with the present invention is a porous foam biomaterial. Contains hyaluronic acid, as well as HA molecules ranging in size from 5.1 kDa to over 2 x 106 kDa In yet another embodiment, the biomaterial used in accordance with the present invention is an open-porous Polylactic acid (PLA) having a structure and pore sizes of about 50 microns to about 300 microns In yet another embodiment, the foam comprises a specific cell population (preferentially B2). B4) specifically regulates hyaluronan synthase-2 (HAS-2) after intrarenal transplantation. The present invention provides for the direct and / or simultaneous synthesis of high molecular weight hyaluronic acid by

[0114] Those skilled in the art will recognize that other types of synthetic or naturally occurring materials known in the art may be used in the present invention. It will be understood that the present invention may be used to form scaffolds as described herein. .

[0115] In one aspect, the present invention provides a scaffold or biomaterial as described herein, which is made from the scaffold or biomaterial referred to above. Provide a structure as described.

[0116] structure In one aspect, the present invention provides a method for treating kidney disease, anemia, or EPO deficiency in a subject in need thereof. Transplantable cells having one or more of the cell populations described herein for treating deficiencies. In one embodiment, the structure is made of a biocompatible material or a biomaterial. , a scaffold or matrix comprising one or more synthetic or naturally occurring biocompatible materials; and the present invention, deposited on or embedded in the surface of the scaffold by attachment and / or entrapment. In certain embodiments, the cell populations or mixtures of cells described herein may be used. In the present invention, the structure is made of a biomaterial and a biomaterial component (a plurality of biomaterial components) coated thereon. deposited on the biomaterial component(s), deposited in the biomaterial component(s), attached to, entrapped in, or embedded in biomaterial component(s) one or more cells as described herein in combination with the biomaterial component(s). In another embodiment, the deposited structure comprises a population or mixture of cells. The cell population or cell component is enriched for oxygen-tunable EPO-producing cells. In another embodiment, the first renal cell population is an oxygen-tunable renal cell population. In addition to cells that produce EPO, they contain glomerular cells and vascular cells. In this embodiment, the deposited cell population or cell component(s) of the structure is / are first concentrated. In some embodiments, the renal cell population comprises both the first renal cell population and the second renal cell population. The cell population is not enriched for oxygen-tunable EPO-producing cells. In another embodiment, the second cell population is enriched for renal tubular cells. The second cell population is enriched for renal tubular cells and contains collecting duct epithelial cells. In another embodiment, the renal tubular cells express megalin, cubilin, hyaluronic acid Synthase 2 (HAS2), Vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp 1), aquaporin-2 (Aqp2), RAB17, member of the RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), and FXYD domain-containing On transport regulator-4 (Fxyd4), solute transporter family-9 (sodium / hydrogen exchanger), member-4 (Slc9a4), aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh 1a3), and calpain-8 (Capn8), It is characterized by the expression of the above tubular cell markers.

[0117] In one embodiment, the method comprises depositing the biomaterial or scaffold onto the biomaterial or scaffold to form the structure of the present invention. The isolated or combined cell populations may be autologous, allogeneic, or syngeneic (autologous or syngeneic). They originate from a variety of sources, including transplant sources.

[0118] Those skilled in the art will appreciate that cell populations can be deposited or otherwise combined with biomaterials to form structures. It will be appreciated that there are several suitable methods for forming the body.

[0119] In one aspect, the constructs of the present invention are suitable for use in the methods of use described herein. In one embodiment, the structure is suitable for treating kidney disease of any cause, anemia, or any It is suitable for administration to a subject in need of treatment for a cause of EPO deficiency. Thus, the construct is suitable for administration to a subject in need of improvement or restoration of red blood cell homeostasis. In another embodiment, the structure is suitable for administration to a subject in need of improved renal function. It is suitable.

[0120] How to use In one aspect, the present invention provides the renal cell populations and mixtures of renal cells described herein. Method for treating kidney disease, anemia, or EP deficiency in a subject in need thereof In one embodiment, the present invention provides a method for producing EPO. In another embodiment, the method comprises administering to a subject a composition comprising the first renal cell population. The first cell population is enriched for EPO-producing cells, glomerular cells, and vascular cells. In another embodiment, the composition may further comprise one or more additional renal cell populations. In one embodiment, the additional cell population is enriched for EPO-producing cells. In another embodiment, the additional cell population is a second cell population that does not produce EPO. A second cell population that is not enriched for vascular cells, glomerular cells, or vascular cells. In embodiments, the compositions are used in the treatment of diseases or disorders described herein. deposited in a biomaterial to form an implantable structure as described herein. , deposited on a biomaterial, embedded in a biomaterial, coated with a biomaterial, or biomaterial Also included are renal cell populations or mixtures of renal cells supplemented in the material component(s). In this context, the cell populations can be used alone to stimulate regeneration in acute or chronic disease states. or other cells or biomaterials, such as hydrogels, porous scaffolds, or natural or in combination with synthetic peptides or proteins.

[0121] In another embodiment, the method of the present invention is used to treat kidney disease, anemia, or EPO in a subject. Effective treatment of deficiency is monitored by various indicators of erythropoiesis and / or renal function. In one embodiment, the indicator of red blood cell homeostasis is hematocrit (HCT). , hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC), retinal reticulocyte count, reticulocyte percentage, mean corpuscular volume (MCV), and red blood cell distribution width (RD) In certain other embodiments, the indicator of renal function includes, but is not limited to, Serum albumin, albumin to globulin ratio (A / G ratio), serum phosphorus, serum sodium, Kidney size (measurable by ultrasound), serum calcium, phosphorus / calcium ratio, serum potassium , proteinuria, urinary creatine, serum creatine, blood urea nitrogen (BUN), cholesterol including, but not limited to, blood glucose levels, triglyceride levels, and glomerular filtration rate (GFR) Furthermore, some indicators of relative health and well-being are related to weight gain or loss, Survival rate, blood pressure (mean systemic, diastolic, or systolic), and physical endurance capacity Including, but not limited to:

[0122] In another embodiment, effective treatment is evidenced by stabilization of one or more indicators of renal function. The stabilization of renal function is comparable to that in subjects not treated with the methods of the present invention. Observing changes in indicators in subjects treated using the methods of the present invention compared to indicators Alternatively, stabilization of renal function may be demonstrated by the same index in the same subject before treatment. Observation of changes in indicators in subjects treated using the methods of the present invention compared to controls. The change in the first index can be an increase or decrease in value. In embodiments, the treatment provided by the present invention involves increasing blood urea nitrogen (BUN) in a subject. The BUN levels observed in the subject may include stabilization of BUN levels, and the BUN levels observed in the subject may be stabilised by the methods of the present invention. In certain other embodiments, the pulmonary circulation is slower than in subjects with a similar condition who are not treated with the method. In the method, the treatment may include stabilizing serum creatine levels in a control, The serum creatine levels observed in subjects not treated with the methods of the present invention were significantly higher than those observed in subjects not treated with the methods of the present invention. In another embodiment, the treatment reduces the level of hematoma in the subject. This may include stabilization of hemoglobin (HCT) levels, which may reduce the HCT levels observed in the subject. The level is higher compared to subjects with a similar condition who are not treated using the methods of the present invention. In another embodiment, the treatment includes stabilizing red blood cell (RBC) levels in the subject. The RBC levels observed in a subject may be measured using the methods of the present invention. Those skilled in the art will appreciate that the level of urinary tract infections described herein or by other methods may be increased compared to subjects with similar conditions who do not have the condition. One or more additional indicators known in the art may be used to assess the presence of kidney disease in a subject. It will be appreciated that the present invention may be measured to determine effective treatment.

[0123] In another aspect, the present invention relates to a method of providing red blood cell homeostasis to a subject in need thereof. In one embodiment, the method includes (a) administering a renal cell population as described herein, e.g. , B2 or B4, or a mixture of kidney cells, e.g., B2 / B4 and / or B2 (b) administering to a subject / B3; and (b) determining whether the level of an indicator of erythropoiesis is higher than the level of an indicator of control. and determining in the biological sample from the subject that the nucleotide sequence is different from that of the nucleotide sequence; The difference in the indicator level indicates that (i) the subject is responsive to administering step (a). or (ii) exhibiting red blood cell homeostasis in a subject. The method includes (a) providing a composition comprising a renal cell population or mixture of renal cells as described herein; (b) administering to a subject a level of an indicator of erythropoiesis compared to the level of the indicator in a control. and determining in the biological sample from the subject that the index level is different compared to the index level. Does the difference in level indicate that (i) the subject is responsive to administering step (a)? or (ii) indicating red blood cell homeostasis in the subject. In another embodiment, the method comprises: (a) providing a biomaterial or biocompatible polymer scaffold; and (b) providing an implantable The renal cell populations or renal cells of the invention may be cultured in the manner described herein to form the desired structures. (c) depositing the mixture of cells onto or into a biomaterial or scaffold; (d) determining whether the level of the indicator of erythropoiesis is different from the level of the indicator in a control. and determining a difference in the biological sample from the subject by comparison; A difference in the levels indicates that (i) the subject is responsive to administering step (a). or (ii) exhibiting red blood cell homeostasis in the subject.

[0124] In another aspect, the present invention provides a method for treating a patient requiring both stabilization of renal function and restoration of red blood cell homeostasis. The method of providing a subject with impaired renal function and anemia and / or In one embodiment, the method comprises administering to at least one of the following cell types: a renal cell population or mixture of renal cells as described herein, comprising at least one Administering: tubule-derived cells, glomerular-derived cells, interstitial-derived cells, aggregation Cells derived from ducts, cells derived from stromal tissue, or cells derived from the vasculature. In this state, the population or mixture contains both EPO-producing cells and renal tubular epithelial cells. The tubular cells are identified by at least one of the following markers: : Megalin, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25- Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin ( Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB1 7, member of the RAS oncogene family (Rab17), GATA-binding protein 3 (G ata3), FXYD domain-containing ion transport regulator 4 (Fxyd4), solute Transporter family-9 (sodium / hydrogen exchanger), member-4 (Slc9a4), aldehyde Aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8) In this embodiment, treatment of the subject can be either a treatment-naive subject or a subject with a pre-treatment indication. Compared with either steroid or steroid therapy, there was an improvement in at least one measure of renal function, as well as at least one improvement in erythropoiesis. This is shown by an improvement in one indicator.

[0125] In one aspect, the present invention provides a method for enriching cells that produce EPO as described herein. containing a renal cell population enriched for EPO-producing cells, By administering a mixture of kidney cells, (i) kidney disease, anemia, or EPO deficiency can be prevented. (ii) a method for stabilizing renal function; and (iii) a method for restoring red blood cell homeostasis. or (iv) any combination thereof, wherein the beneficial effects of administration are The effect of administering a cell population that is not enriched for O-producing cells is greater than that of administering a cell population that is not enriched for O-producing cells. In this manner, the enriched cell population contributes to serum blood urea nitrogen (BUN). In embodiments, the enriched cell population results in improved protein retention in serum. In an embodiment, the enriched cell population has improved levels of serum cholesterol and and / or triglyceride levels. The population provides improved levels of vitamin D. In one embodiment, the enriched cells The cell population provides an improved phosphorus:calcium ratio compared to non-enriched cell populations. In another embodiment, the enriched cell population has improved cell viability compared to a non-enriched cell population. In a further embodiment, the enriched cell population results in improved levels of serum creatine compared to non-enriched cell populations. In yet another embodiment, the enriched cell population has improved cell viability compared to a non-enriched cell population. In a further embodiment, the concentrated cells The cell populations produce improved levels of red blood cell (RBC) counts compared to non-enriched cell populations. In one embodiment, the improved level of hematocrit is 95% of normal healthy individuals. In a further embodiment, the enriched cell population is In other embodiments, the enriched cells provide improved reticulocyte counts compared to unenriched cell populations. The enriched cell population has an improved reticulocyte percentage compared to the non-enriched cell population. In yet other embodiments, the enriched cell population has a lower cell density than the non-enriched cell population. In yet another embodiment, the present invention provides improved levels of red blood cell volume distribution width (RDW) compared to the conventional method. In the enriched cell population, improved levels of In yet another embodiment, the enriched cell population is derived from bone marrow. The bone marrow was examined for red blood cells, with near-normal cellularity and near-normal myeloid:erythroid ratios. Produces a hematopoietic response.

[0126] In another aspect, the present invention provides a method for administering an enriched cell population to: (i) treat kidney disease; (ii) a method for treating renal disease, anemia, or EPO deficiency; and (iii) a method for stabilizing renal function. (iii) a method for restoring red blood cell homeostasis, or (iv) any combination thereof. and administering a renal cell population or mixture of renal cell populations described herein. The beneficial effects of administration are due to the effects of administering recombinant EPO (rEPO). In one embodiment, the beneficial effect is characterized by improved red blood cell homeostasis compared to the beneficial effect. wherein the population or mixture, when administered to a subject in need thereof, produces a recombinant EPO protein. Improved red blood cell homeostasis (hematocrit, hemoglobin, and (as determined by RBC count). In one embodiment, the population or mixture , which, when administered, results in a hematocrit that is about 10% lower or higher than the control hematocrit. Provides improved levels of hematocrit, RBC, or hemoglobin compared to EPO In a further embodiment, a single dose or delivery of the population or mixture is administered. When administered, a single dose or delivery of recombinant EPO protein results in improved red blood cell homeostasis. Improved red blood cell homeostasis (hematocrit) in treated subjects for a period significantly exceeding that observed in In another embodiment, the effect is an increase in the number of RBCs in the blood (as measured by an increase in RBC count, hemoglobin, or RBC count). In the present invention, the population or mixture, when administered at the doses described herein, has a corresponding health effect. Hematocrit, hemoglobin, or RBC count above approximately 110% of normal levels in normal controls In a further embodiment, the population or mixture does not produce the desired product. When administered in amounts, the recombinant EPO protein delivered in the doses described herein In comparison, superior red blood cell homeostasis (measured by hematocrit, hemoglobin, or RBC count) In another embodiment, the recombinant EPO is provided at a concentration of about 100 IU / kg, about 200 IU / kg, about 300 IU / kg, about 400 IU / kg, or about 500 The recombinant E. coli is delivered at a dose of 1.5 IU / kg. It will be appreciated that other dosages of PO may be suitable.

[0127] Another embodiment of the present invention provides a method for treating kidney disease, anemia, or EPO deficiency in a subject in need thereof. treating a kidney deficiency, providing red blood cell homeostasis to a subject in need, or and (c) administering to a subject a compound of formula (I) or (II) ... Contemplated are uses of the cell populations or implantable structures described herein.

[0128] Another embodiment of the present invention is the selection of specific cell subpopulations based on specific validated therapeutic properties. Specific enriched cell population(s) for the treatment of kidney disease of a specific cause based on ) (described herein).

[0129] Administration method and route The cell preparations of the present invention may be administered alone or in combination with other bioactive ingredients. can be done.

[0130] A therapeutically effective amount of the renal cell population or mixture of renal cell populations described herein is administered to a subject. Therefore, the maximum number of cells that can be safely tolerated is sufficient to treat kidney disease, e.g., to prevent one or more kidney functions. The number of cells required to stabilize, slow, or improve the rate of decline may range from 0.2 to 1.0. In some embodiments, the methods of the present invention involve culturing cells at a concentration of about 10,000 cells / kg, about 20,000 cells / kg, Approx. 30,000 cells / kg, approx. 40,000 cells / kg, approx. 50,000 cells / kg, approx. 100,000 cells / kg, approximately 200,000 cells / kg, approximately 300,000 cells / kg , about 400,000 cells / kg, about 500,000 cells / kg, about 600,000 cells / kg, about 700,000 cells / kg, about 800,000 cells / kg, about 900,000 cells cells / kg, approx. 1.1x106 cells / kg, approx. 1.2x106 cells / kg, approx. 1.3x10 6 cells / kg, approx. 1.4x106 cells / kg, approx. 1.5x106 cells / kg, approx. 1.6x 106 cells / kg, about 1.7x106 cells / kg, about 1.8x106 cells / kg, about 1. 9x106 cells / kg, approx. 2.1x106 cells / kg, approx. 2.1x106 cells / kg, approx. 1.2x106 cells / kg, about 2.3x106 cells / kg, about 2.4x106 cells / kg , about 2.5x106 cells / kg, about 2.6x106 cells / kg, about 2.7x106 cells / kg, about 2.8x106 cells / kg, about 2.9x106 cells / kg, about 3x106 cells / kg kg, about 3.1x106 cells / kg, about 3.2x106 cells / kg, about 3.3x106 cells / kg cells / kg, approx. 3.4x106 cells / kg, approx. 3.5x106 cells / kg, approx. 3.6x10 6 cells / kg, approx. 3.7x106 cells / kg, approx. 3.8x106 cells / kg, approx. 3.9x 106 cells / kg, about 4x106 cells / kg, about 4.1x106 cells / kg, about 4.2x 106 cells / kg, about 4.3x106 cells / kg, about 4.4x106 cells / kg, about 4. 5x106 cells / kg, approx. 4.6x106 cells / kg, approx. 4.7x106 cells / kg, approx. 4.8x106 cells / kg, approximately 4.9x106 cells / kg, or approximately 5x106 cells / kg g of a renal cell population or mixture of renal cell populations described herein. In another embodiment, the dosage of cells to a subject is a single dose, or a single dose and additional doses. In other embodiments, the doses may be any of the doses described herein. In another embodiment, the amount of cells administered to a subject may be may be calculated based on estimated kidney mass or functional kidney mass.

[0131] A therapeutically effective amount of the renal cell populations or mixtures thereof described herein may be administered in a pharmaceutically acceptable form. The drug may be suspended in an acceptable carrier or excipient. Such a carrier may be 1% serum albumin or 1% ethanol. Basic medium supplemented with ethanol, saline, buffered saline, glucose, water, collagen, Glycine, hyaluronic acid, fibrin glue, polyethylene glycol, polyvinyl alcohol Examples of suitable cellulose acetates include, but are not limited to, cellulose acetate, carboxymethylcellulose, and combinations thereof. The dosage form should be adapted to the mode of administration. Thus, the present invention provides a method for treating renal disease in a subject. and the use of a renal cell population or a mixture thereof (e.g., B2 cell population alone or mixed with B3 and / or B4 cell populations. In some embodiments, the drug is a growth factor, a chemokine, or a cytokine, etc. In a further embodiment, the drug further comprises a recombinant polypeptide of human kidney-derived The cells used to manufacture the drug may be derived from a cell population derived from a cell line or a cell line derived from a cell line, or a cell line ... The method can be used to isolate, derive, or enrich for any of the variants provided. can.

[0132] The kidney cell preparation(s), or mixtures thereof, or compositions are adapted for administration to humans. The pharmaceutical compositions are usually formulated according to routine procedures for intravenous administration, ... Compositions for intravenous administration or administration in renal capsules are prepared by dissolving the compound in a sterile isotonic aqueous buffer. Optionally, the composition may be administered topically to relieve any pain at the injection site. An anaesthetic may also be included. Generally, the ingredients are presented separately or together in a unit dosage form, e.g., Supplied as a cryopreserved concentrate in a sealed container such as an ampoule indicating the quantity of active agent. If the product is administered by infusion, it should be administered with pharmaceutical-grade sterile water or saline. When the composition is administered by injection, it can be dispensed using an intravenous drip bottle having a provides an ampoule of sterile water for injection or saline so that the ingredients may be mixed prior to administration. may be provided.

[0133] Pharmaceutically acceptable carriers will vary depending on the particular composition being administered and the nature of the agent by which the composition is administered. The degree of accuracy is determined in part by the particular method used to obtain it. Suitable pharmaceutical composition dosage forms exist (see, for example, Alfonso R Gennaro (ed), Remington:The Science and Practice of Pharmacy, former ly Remington's Pharmaceutical Sciences 2 0th ed., Lippincott, Williams & Wilkins, (See, e.g., 2003). Pharmaceutical compositions generally comply with the U.S. Food and Drug Administration's Drug and Pharmaceutical Ingredients Regulations. Sterile and virtually non-toxic in full compliance with all GMP (Good Manufacturing Practice) regulations. It is formulated as an isotonic solution.

[0134] One embodiment of the invention is, for example, a B2 cell preparation alone, or a B3 and / or B4 cell preparation. a renal cell preparation of the present invention in combination with a renal cell preparation, and a pharmaceutically acceptable carrier. Pharmaceutical formulations are further provided. In some embodiments, the formulations contain 10 to 10 Includes cells derived from mammalian kidneys.

[0135] In one aspect, the present invention provides a method for administering to a subject in need thereof the cells described herein, including the mixture. In one embodiment, the present invention provides a method for providing one or more of the cell populations. The source of the cells (multiple cells possible) can be autologous, allogeneic, syngeneic (autologous or syngeneic transplant), and their If not an autologous source, the method may include administering immunosuppressive drugs. Suitable immunosuppressants include azathioprine, cyclophosphamide, mizoram, and fluticasone. Bottle, cyclosporine, tacrolimus hydrate, chlorambucil, lobenzarit di-sodium Urum, auranofin, alprostadil, gusperimus hydrochloride, biosynsorb, Romonab, Alefacept, Pentostatin, Daclizumab, Sirolimus, Mycophenone- mofetil, leflunomide, basiliximab, dornase alfa, bindalide arid), cladribine, pimecrolimus, ilodecaquine, cedelizumab, efalizumab Mab, everolimus, anisperimus, gavilimomab, faralimomab, clofarabine , rapamycin, siplizumab, saireito, LDP-03, CD4, SR-43551, S K&F-106615, IDEC-114, IDEC-131, FTY-720, TSK -204, LF-080299, A-86281, A-802715, GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT- 1345, CNI-1493, CBP-2011, J-695, LJP-920, L-7 32531, ABX-RB2, AP-1903, IDPS, BMS-205820, BM S-224818, CTLA4-1g, ER-49890, ER-38925, ISAt x-247, RDP-58, PNU-156804, LJP-1082, TMC-95A , TV-4710, PTR-262-MG, and AGI-1096 (See U.S. Patent No. 7,563,822.) Those skilled in the art will recognize other suitable immunogens. You will be well aware of pharmaceuticals.

[0136] The therapeutic methods of the present invention involve delivering an isolated renal cell population or mixture thereof to an individual. In one embodiment, it is preferred to administer the cells directly to the intended site of benefit. In one embodiment, the cell preparation or mixture of the present invention is individually administered in a delivery vehicle. delivered to the body.

[0137] Various means for administering cells to a subject will be apparent to those of skill in the art in light of this specification. Such methods include injecting cells into a target site in a subject. The cells can be inserted into a delivery device or vehicle that facilitates introduction by transplantation. In certain embodiments, the delivery vehicle can include natural materials. In certain embodiments, the delivery vehicle may comprise a synthetic material. In this case, the delivery vehicle has a structure that mimics the structure of the organ or fits appropriately within the structure. In other embodiments, the delivery vehicle has liquid-like properties. The delivery device may be a catheter or other device for injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tube further comprises a tube containing the cells of the present invention. In some embodiments, the needle may be inserted into a desired location, such as a syringe. In this study, a cell population derived from the mammalian kidney was administered intravascularly through a catheter. (The term "catheter" refers to a variety of tube-like systems for delivering substances to blood vessels.) (The term "cell" is intended to include any of the above.) Alternatively, the cells may be woven, knitted, braided, meshed, or the like. and fibers such as nonwoven fabrics, perforated films, sponges and foams, and porous beads. Biomaterials or scaffolds, including but not limited to beads, microparticles, nanoparticles, etc. Cells may be inserted into or onto the surface of a cell. Cells may be prepared in a variety of different forms for delivery. For example, the cells may be suspended in a solution or gel. The cells may be mixed with a pharmaceutically acceptable carrier or diluent that maintains the cells in a viable state. Pharmaceutically acceptable carriers and diluents include saline, buffered aqueous solutions, solvents, and / or The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid, and often isotonic. It is stable under the conditions of manufacture and storage and does not contain, for example, parabens, chlorobutanol, phenols, -By using chlorine, ascorbic acid, thimerosal, etc., contamination of microorganisms such as bacteria and mold can be prevented. Those skilled in the art will appreciate that the delivery of the cell populations and mixtures thereof of the present invention is protected against contamination. It is understood that the delivery vehicle used in the present invention may comprise a combination of the above properties. It would be.

[0138] For example, isolated B2 cell populations alone or mixed with B4 and / or B3. The mode of administration of the renal cell population(s) may be systemic, intrarenal (e.g., parenchymal), intravenous, or including, but not limited to, intra-arterial injection and injection directly into the tissue at the intended site of activity. An additional mode of administration for use in accordance with the present invention is by direct laparotomy, direct laparoscopy. This includes single or multiple injection(s) via intraperitoneal, intraperitoneal, or transdermal route. Additional modes of administration that may be used include, for example, retrograde infusion and ureteral infusion. Surgical means of administration include partial nephrectomy and construct transplantation, nephrectomy, partial pyelectomy (partial nephrectomy), and tial pyelectomy), vascularization using omentum ± peritoneum, multifocal biopsy needle tracking, A one-step procedure including conical or pyramidal to cylindrical and renal polar displacement, if and, for example, two-step procedures involving organoids in vivo for reimplantation. In one embodiment, the mixture of cells is cultured in the same manner. In another embodiment, each of the cell compositions comprising the controlled mixture is delivered through are simultaneously or temporally controlled by one or more of the methods described herein. delivered separately to a specific location or by a specific method in any of the formats described above. It will be delivered.

[0139] The appropriate cell transplant dosage in humans is determined based on known cell activities, e.g., EPO production. This can be determined from existing information or from dose studies performed in preclinical studies. The amount of cells can be estimated from in vitro culture and in vivo animal experiments. This can be used to calculate the appropriate dose of implant material. to determine whether or not the implant material can be reduced accordingly. The patient can be monitored.

[0140] One or more types of collagen or hyaluronic acid known in the art selected extracellular matrix components and / or growth factors, platelet-rich plasma, and One or more other components, including drugs, may be added to the cell populations and mixtures thereof of the present invention. good.

[0141] All patents, patent applications, and references cited herein are incorporated by reference. It is incorporated herein in its entirety.

[0142] [Section 1] A mixture of human kidney cells comprising a first cell population, B2, comprising an isolated, enriched population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, and a second cell population comprising erythropoietin (EPO)-producing cells, glomerular cells, and vascular cells, wherein the mixture does not include a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density less than 1.045 g / mL, or a B5 cell population comprising debris and small cells of low granularity and viability having a density greater than 1.091 g / mL. [Section 2] Item 1, wherein B2 further comprises collecting duct epithelial cells. [Section 3] Item 1. The mixture according to Item 1, wherein the second cell population is a B4 cell population having a density of 1.063 g / mL to 1.091 g / mL. [Section 4] Item 1. The mixture according to Item 1, wherein the second cell population is a B3 cell population having a density of 1.052 g / mL to 1.063 g / mL. [Section 5] Item 10. The mixture of cells according to Item 1, wherein the mixture of cells is capable of receptor-mediated albumin uptake. [Section 6] Item 10. The mixture of cells according to Item 1, wherein the mixture of cells is capable of oxygen-tunable erythropoietin (EPO) expression. [Section 7] The mixture exhibits high molecular weight hyaluronic acid (HA) both in vitro and in vivo. HAS-2-expressing cells capable of producing and / or stimulating the production of endothelial cells Item 1. The mixture according to item 1, comprising: [Section 8] Item 14. The mixture of item 1, wherein the mixture is capable of providing a regenerative stimulus upon delivery in vivo. [Section 9] Item 1. The mixture according to item 1, wherein the mixture, upon delivery in vivo, can reduce, stabilize, or ameliorate a decline in glomerular filtration, tubular reabsorption, urine production, and / or endocrine function. [Section 10] Item 1. The mixture of item 1, wherein the first and second cell populations are derived from kidney tissue or cultured kidney cells. [Section 11] B2 expresses megalin, cubilin, hyaluronan synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, a member of the RAS oncogene family (Rab17), GATA-binding protein 3 (Gata3), and an ion-binding domain containing the FXYD domain. Item 1. The mixture according to Item 1, characterized by the expression of a renal tubule cell marker selected from the group consisting of acetylcholine 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). [Section 12] An isolated, enriched population of human kidney cells comprising a B2 cell population, wherein B2 comprises an isolated, enriched population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, and the enriched population does not comprise a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density less than 1.045 g / mL, or a B4 cell population comprising erythropoietin (EPO)-producing cells, glomerular cells, and vascular cells having a density of 1.063 g / mL to 1.091 g / mL. [Section 13] Item 1. The B2 cell population described in item 1, wherein the B2 cell population is capable of producing and / or stimulating the production of high molecular weight species of hyaluronic acid (HA) both in vitro and in vivo via expression of HAS-2 (hyaluronan synthase-2). [Section 14] The B2 cell population of paragraph 12, further comprising collecting duct epithelial cells. [Section 15] Item 13. The B2 cell population according to Item 12, which is capable of receptor-mediated albumin uptake. [Section 16] Item 13. The B2 cell population of item 12, which is capable of providing a regenerative stimulus upon delivery in vivo. [Section 17] Item 13. The B2 cell population of item 12, which upon delivery in vivo, is capable of reducing, stabilizing, or ameliorating the decline in glomerular filtration, tubular reabsorption, urine production, and / or endocrine function. [Section 18] 13. The B2 cell population of paragraph 12, derived from kidney tissue or cultured kidney cells. [Section 19] Megalin, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 13. The B2 cell population of claim 12, characterized by expression of a tubular cell marker selected from the group consisting of 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB17, a member of the 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 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8). [Section 20] Item 13. The B2 cell population according to Item 12, which does not include the B3 cell population comprising erythropoietin (EPO)-producing cells having a density of about 1.052 g / mL to about 1.063 g / mL, glomerular cells, and vascular cells. [Section 21] 13. The B2 cell population of paragraph 12, which does not include a B5 cell population comprising debris and small cells of low granularity and viability having a density greater than about 1.091 g / mL. [Section 22] 1. A method for preparing a human B2 cell population, comprising: a) exposing a cell suspension containing a non-enriched heterogeneous kidney cell population to hypoxic culture conditions; b) contacting the cell suspension with a density gradient to separate one or more cell fractions, wherein a first cell fraction is present in the gradient after centrifugation at a specific density between 1.045 g / mL and 1.052 g / mL; c) extracting a first cell fraction comprising the B2 cell population. [Section 23] 23. The method of claim 22, wherein the B2 cell population comprises a greater proportion of tubular cells and a smaller proportion of EPO-producing cells, glomerular cells, and vascular cells when compared to a non-enriched cell population. [Section 24] 1. A method for preparing a human B4 cell population, comprising: a) exposing a cell suspension containing a non-enriched heterogeneous kidney cell population to hypoxic culture conditions; b) contacting the cell suspension with a density gradient to separate one or more cell fractions, wherein a first cell fraction is present in the gradient after centrifugation at a specific density between 1.063 g / mL and 1.091 g / mL; c) extracting a first cell fraction comprising the B4 cell population. [Section 25] 25. The method of claim 24, wherein the B4 cell population comprises a greater proportion of EPO-producing cells, vascular cells, and glomerular cells, and a smaller proportion of non-EPO-producing cells, non-vascular cells, and non-glomerular cells, when compared to a non-enriched cell population. [Section 26] 1. A method for producing a B4 cell preparation, comprising: a) exposing a cell suspension containing a non-enriched heterogeneous kidney cell population to hypoxic culture conditions; b) applying the cell suspension to a flow cytometry instrument; c) selecting a subpopulation of cells from said cell population using vascular, glomerular, and endocrine cell markers; d) selecting a subpopulation of cells from said cell population; e) isolating said subpopulation of B4 cells from said cell population; The method, wherein the subpopulation of B4 cells is characterized by expression of the vascular, the glomerular, and the endocrine cell markers relative to the majority of the population. [Section 27] 1. A structure for implantation into a subject in need of improved renal function, comprising: a) a biomaterial comprising one or more biocompatible synthetic polymers or naturally occurring proteins or peptides; b) a mixture of mammalian kidney cells, comprising a first cell population, B2, comprising an isolated, enriched population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, and a second cell population comprising erythropoietin (EPO)-producing cells, glomerular cells, and vascular cells, said mixture excluding a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density less than 1.045 g / mL, or a B5 cell population comprising low granularity and viability debris and small cells having a density greater than 1.091 g / mL, said structure coated on, deposited on or within, entrapped within, suspended within, embedded within, and / or otherwise combined with said biomaterial. [Section 28] Item 28. The structure according to Item 27, wherein the second cell population is a B4 cell population having a density of 1.063 g / mL to 1.091 g / mL. [Section 29] Item 28. The structure according to Item 27, wherein the second cell population is a B3 cell population having a density of 1.052 g / mL to 1.063 g / mL. [Section 30] 28. The structure of paragraph 27, wherein the mixture is derived from mammalian kidney tissue or cultured kidney cells. [Section 31] Item 28. The structure of item 27, wherein the biomaterial is configured as a three-dimensional (3-D) porous biomaterial suitable for entrapment and / or attachment of the mixture. [Section 32] Item 28. The structure of item 27, wherein the biomaterial is configured as a liquid or semi-liquid gel suitable for embedding, adhering, suspending, or coating mammalian cells. [Section 33] Item 28. The structure of item 27, wherein the biomaterial comprises predominantly high molecular weight species of hyaluronic acid (HA) in the form of a hydrogel. [Section 34] 28. The structure of claim 27, wherein the biomaterial comprises predominantly high molecular weight species of hyaluronic acid in the form of a porous foam. [Section 35] Item 28. The structure according to Item 27, wherein the biomaterial comprises a polylactic acid foam having pores of about 50 microns to about 300 microns. [Section 36] 28. The construct of paragraph 27, wherein the cell population is derived from an autologous kidney sample. [Section 37] 37. The construct of paragraph 36, wherein the sample is a kidney biopsy. [Section 38] 28. The construct of paragraph 27, wherein the cell population is derived from a non-autologous kidney sample. [Section 39] 28. The construct of paragraph 27, wherein the improved renal function is red blood cell homeostasis. [Section 40] 1. A composition for use in treating kidney disease in a subject in need thereof, comprising: a) administering to the subject a composition comprising a mixture of mammalian kidney cells, the mixture comprising a first cell population, B2, comprising an isolated, enriched population of tubular cells having a density of 1.045 g / mL to 1.052 g / mL, and a second cell population comprising erythropoietin (EPO)-producing cells, glomerular cells, and vascular cells, wherein the mixture does not comprise a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density less than 1.045 g / mL, or a B5 cell population comprising debris and small cells of low granularity and viability having a density greater than 1.091 g / mL; b) determining in a test sample from the subject that the level of an indicator of renal function is different compared to the indicator level in a control, wherein the difference in indicator level indicates a reduction, stabilization, or improvement in decline of one or more renal functions in the subject. [Section 41] Item 41. The composition according to Item 40, wherein the second cell population is a B4 cell population having a density of 1.063 g / mL to 1.091 g / mL. [Section 42] Item 41. The composition according to Item 40, wherein the second cell population is a B3 cell population having a density of 1.052 g / mL to 1.063 g / mL. [Section 43] Item 41. The composition of claim 40, wherein the kidney disease is accompanied by erythropoietin (EPO) deficiency. [Section 44] Item 44. The composition according to Item 43, wherein the EPO deficiency is anemia. [Section 45] 44. The composition of paragraph 43, wherein the EPO deficiency or anemia occurs secondary to renal failure in the subject. [Section 46] 44. The composition of claim 43, wherein the EPO deficiency or anemia occurs secondary to a disease selected from the group consisting of chronic renal failure, primary EPO deficiency, chemotherapy or antiviral therapy, non-myeloadenomatous cancer, HIV infection, liver disease, heart failure, rheumatoid arthritis, or multiple organ system failure. [Section 47] 41. The composition of claim 40, further comprising a biomaterial comprising one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, wherein the mixture is coated on, deposited on or within, entrapped within, suspended within, embedded within, and / or otherwise combined with the biomaterial. [Section 48] 41. The composition of claim 40, wherein the mixture is derived from mammalian kidney tissue or cultured mammalian kidney cells. [Section 49] 41. The composition of claim 40, wherein the mixture is derived from an autologous kidney sample. [Section 50] 50. The composition of claim 49, wherein the sample is a kidney biopsy. [Section 51] 41. The composition of paragraph 40, wherein the mixture is derived from a non-autologous kidney sample. [Section 52] A selected population of renal cells is isolatable by density gradient centrifugation using a gradient containing a portion with a density of 1.045 g / mL to 1.052 g / mL after exposure to an oxygen level of about 1% to about 5% for 12 to 24 hours, wherein the cell population (i) is retained in the gradient after centrifugation at a density of 1.045 g / mL to 1.052 g / mL; (ii) comprises a renal tubular cell population characterized by expression of at least one renal tubular cell marker; (iii) comprises a subpopulation of renal tubular cells capable of receptor-mediated albumin transport; (iv) does not comprise a B1 cell population comprising large granular cells of the collecting duct and tubular system having a density less than 1.045 g / mL, or a B5 cell population comprising low granularity and viability debris and small cells having a density greater than 1.091 g / mL; and (v) is capable of modulating one or more renal functions when delivered to a subject at risk for or with kidney disease. [Section 53] 52. A selected population of renal cells isolatable by density gradient centrifugation using a gradient comprising a portion with a density of 1.063 g / mL to 1.091 g / mL after exposure to an oxygen level of about 1% to about 5% for 12 to 24 hours, wherein the cell population (i) is retained in the gradient after centrifugation at a density of 1.063 g / mL to 1.091 g / mL; (ii) comprises oxygen-tunable erythropoietin (EPO)-expressing cells, glomerular cells, and vascular cells; (iii) is capable of modulating one or more renal functions when delivered to a subject at risk for or having kidney disease; and (iv) is capable of enhancing the modulation of one or more renal functions by the population of renal cells of 52 upon co-administration. The following examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Example]

[0143] Example 1 - Isolation and Characterization of Bioresponsive Kidney Cells from Adult Pigs with Renal Failure For evaluation and characterization of cellular composition by direct comparison with age-matched normal porcine kidney tissue, Cases of sudden progressive chronic kidney disease (CKD) with anemia in adult pigs (Sus scrofa) Fresh diseased kidney tissue was provided from each case.

[0144] Histological examination of the kidney tissue at the time of collection revealed severe, diffuse, chronic interstitial fibrosis with multiple fibrosis. Renal disease characterized by chronic fibrosis and crescentic glomerulonephritis was identified. The bed chemistry revealed azotemia (elevated blood urea nitrogen and serum creatinine) and mild Anemia (mildly decreased hematocrit and decreased hemoglobin levels) was confirmed. Cells were isolated from both metastatic and normal kidney tissue, expanded, and characterized.

[0145] Figure 1 shows a diseased kidney as revealed by Gomori trichrome staining compared with normal kidney tissue. The tissue shows fibrosis (blue staining indicated by arrows). Cubilin: Megalin is expressed and is a receptor. Functional tubular cells capable of receptor-mediated albumin transport were isolated from normal and diseased kidney tissue. The cultures were propagated from both the erythropoietin (EPO)-expressing cells. These cells were then maintained through multiple passages and freeze-thaw cycles. EPO-expressing cells derived from both the normal and diseased tissues were found to express HIF1α-induced EPO. In vitro hypoxia using induction of EGFR and other hypoxia-regulated gene targets, including vEGF It was confirmed that the animals responded to the prime conditions.

[0146] Cells were isolated from porcine kidney tissue by enzymatic digestion using collagenase and dispase. It was also isolated in another experiment by simple mechanical digestion and explant culture. At passage 2, explant-derived cell cultures containing EPO-expressing cells were cultured in air (21% ) and various hypoxic (<5%) culture conditions, and exposure to hypoxia increased EPO We determined whether this leads to upregulation of gene expression. Previously (see Example 3), normal pigs showed oxygen-dependent expression and regulation of the EPO gene. Surprisingly, the uremia / anemic state of CKD pigs (hematocrit < 34 Despite the high serum creatinine (creatinine > 9.0), EPO-expressing cells were easily isolated from tissues and These mice were then bred and propagated to maintain EPO gene expression under hypoxic control, as shown in Figure 2.

[0147] As shown in Figure 3, cells in propagated cultures self-organize into tubule-like structures. demonstrated ability.

[0148] As shown in Figure 4, receptor-mediated uptake of FITC-conjugated albumin by cultured cells was observed. Observation confirmed the presence of functional tubular cells in the culture (passage 3). The green dots (indicated by thin white arrows) are receptors specific to renal tubule cells. Uptake of fluorescein-conjugated albumin mediated by megalin and cubilin This indicates that protein reabsorption by functional tubular cells was achieved. The blue staining (indicated by the thick white arrow) is Hoechst-stained nuclei.

[0149] Taken together, these data suggest that even in severely damaged kidney tissue due to CKD, Functional tubular and endocrine cells can also be isolated and propagated from porcine kidney tissue. Furthermore, these findings support the development of autologous cell-based therapies for the treatment of CKD. This supports the advancement of therapeutic drugs.

[0150] Example 2 - Isolation of bioresponsive EPO cells from human kidney EPO-producing cells from normal adult human kidney (as described in Example 1 above) As shown in Figure 5, the isolation procedure resulted in a relative abundance of the original tissue. As shown in Figure 6, EPO gene expression was maintained after isolation. In this manner, it is possible to maintain human EPO-producing cells in culture. Untreated tissue culture treated plastic or, for example, fibronectin or collagen -Cultured / grown on plastic coated with a specific extracellular matrix such as Gen et al. All were found to support EPO expression over time.

[0151] Example 3 - Bioresponsive EPO-expressing cells and tubular cells from rodent kidneys culture In this study, primary kidney cells isolated from Lewis rats were used to investigate the effects of hypoxia and seizures. The expression of EPO and tubular markers in vitro in response to shear force was analyzed.

[0152] Isolate primary kidney cells from Lewis rats using standard methods adapted from mice. (Aboushwareb et al.,2008.World J.Urol.Au g;26(4):295-300), in hypoxic / hyperoxic or static / dynamic 3D culture and bred them.

[0153] The oxygen dependency of EPO-producing cells was investigated under "normoxic" culture conditions in air (21% O The cells were cultured in a 37°C incubator equilibrated with 2.5% CO2, and then EPO To activate hypoxia-dependent gene transcription in , the oxygen tension is lowered to hypoxic culture (2 The cells were then incubated in a 37°C incubator equilibrated with 5% O2 and 5% CO2. Eventually, switching to normoxic conditions inhibits gene transcription that occurs under hypoxic conditions. To allow cells to attach, two-dimensional (2D) plates and Primary kidney cells, both on 3D (see Cultisphere example below) structures The cells were then cultured (usually for 48 hours). The attached cells were then transferred to a hypoxic incubator and incubated for 4 After the final time point of 48 hours of hypoxic culture, the cells were Cells from triplicate plates were cultured initially in normoxic conditions, followed by The cells were harvested at the designated time points under hypoxic culture and then returned to normoxic culture. The samples were flash frozen in liquid nitrogen and stored at -80°C before analysis. A was isolated and gene expression analysis was performed by synthesizing cDNA from total mRNA. Relative gene expression was determined using real-time quantitative PCR (qRTPCR). In addition to the control replicate, two technical replicates were analyzed using qRTPCR, and the time of each culture condition was A total of six replicates were obtained per point.

[0154] Cultisphere-S Gelatin Microcarrier Beads Primary kidney cells were isolated from young rats using standard methods. Approximately 200,000 cells were isolated. Cells were cultured in sterile Cultisphere-S (Sigma-Aldrich) on low-attachment 6-well plates. Aldrich, Cat. No. M9043) Macroporous Gelatin Microcarrier Beads Place in a culture containing 200 μl of a 50% (v / v) slurry of beads (130-380 μm). Under static conditions (the plate containing the cells is not subjected to any movement during the experiment) or Cells were placed in either a 2% or 21% oxygen chamber under dynamic conditions (constant movement). During the test (7 days), samples were collected from each condition at regular intervals. Three samples were collected per well. Gene expression in all samples was analyzed using the primary cell line from the initial isolation. Normalization was performed to the starting material, which was an unfractionated cell suspension. QRTPCR was performed to measure tubular expression, and endocrine cell markers, E-cadherin and EPO, respectively.

[0155] Results: EPO expression in cultured endocrine cells was significantly increased by dynamic culture, unlike static culture. and / or hypoxia. The results of (+)EPO expression in dynamic culture at low oxygen levels (2%) are shown in Figure 7. Figure 8 shows the results of dynamic culture at low oxygen levels (2%). , (+) EPO expression in 3D dynamic culture. Figure 9 also shows EPO expression in long-term dynamic culture. (+) indicates tubular gene expression. Both hypoxic and dynamic 3D cultures are associated with hyperoxia, respectively. The EPO expression was significantly increased (p<0.05) compared with the control and static culture. Figure 11 shows EPO expression in hypoxic cultures compared to normoxic cultures. We show that dynamic 3D culture in vitro stimulates EPO expression. Increased expression of HIF1α, a regulator of HIF1α, and other HIF1α target genes such as VEGF Therefore, EPO expression in cultured neonatal kidney cells was associated with HIF1α-mediated regulation. It is clear that the oxygen level was controlled by the oxygen and mechanical Bioresponsive primary rodent kidney cells that retain the control of transfected EPO expression are useful in in vitro studies. It has been shown that it can be isolated and propagated in vitro.

[0156] Example 4 - 3D Structures and Comparative Cultures In vivo functionality (e.g., therapeutic potential) of the neo-tissue / neo-organ constructs containing cells, scaffolds, and media To determine the best in vitro adaptation of the new cell line, multiple three-dimensional culture configurations were designed. The tissue / neo-organ construct was designed as follows: primary renal cell cultures (cells producing EPO and The cells (containing both human and renal tubular cells) were placed in a porous circular cavity 5 mm in diameter and 5 mm in height. Cells were seeded onto the columnar scaffolds at a density of 500,000 to 1,000,000 cells / scaffold, and the prototype Culture and experiment were performed using a multi-well perfusion system (MPS, BD Technologies). The medium was DMEM + 10% FBS (Culture Medium). A 1:1 mixture of either DMEM + 10% FBS and KSFM medium (Medium A) or DMEM + 10% FBS and KSFM medium (Medium B). The scaffolds evaluated in these experiments included open-cell polylactic acid (OPLA). ) and collagen 1 scaffolds (both from BD), and were prepared using standard methods. The fabricated polyglycolic acid-based scaffold (PGA) was included.

[0157] Properties include: sufficient pores to allow fluid to flow through the cell-scaffold composite Size and structure; providing a microenvironment that allows cell-scaffold and cell-cell interactions the structure and composition of the scaffold that provides the necessary proteins and express and / or produce and / or transport or express and / or The presence of cells with the potential to produce and / or transport For example, a preferred 3D scaffold comprising open-cell polylactic acid (OPLA) is used to A bioreactor device seeded with kidney cells and providing continuous perfusion of culture medium throughout the scaffold The in vitro conditioned scaffold + cell complex was subjected to in vitro culture. The cells were characterized as follows: presence of viable, metabolically active cells; cell-cell and Cell-material interactions: megalin, gamma-glutamyltransferase (GGT), E Renal tubule markers, including but not limited to, α-cadherin and N-cadherin -expression of renal endocrine markers, including but not limited to erythropoietin; -expression.

[0158] Results: As described, conditioned medium and all proteins were collected from 2D and 3D cultures. Cell lysates were collected. Cell lysates (top panel of Figure 12) and conditioned medium (bottom panel of Figure 12) were collected. ELISA analysis was performed to quantify the target protein in both the IgG and IgG subunits (bottom panel). Ta.

[0159] After 7 days of perfusion or static culture, the seeded OPLA and The Col1 and Col1 scaffolds were fixed in 10% buffered formalin and embedded in paraffin. - Eosin (HE) staining was performed to examine the presence and morphology of cells. The distribution of cells was higher in perfusion culture than in culture, and the distribution of cells was higher in OPLA scaffolds compared to Col1 scaffolds. The field was distributed throughout the entire scaffold (see Figure 13).

[0160] Figure 14 shows the scans of OPLA and Col1 scaffolds after 7 days of culture (static and perfused). Scanning electron microscope (SEM) images are shown. Perfusion culture resulted in higher cellularity and and cellular organization.

[0161] mRNA was isolated from scaffolds or 2D cultures by addition of lysis buffer (Qiagen). and removed from the 3D scaffold by electrical homogenization (Polytron) in lysis buffer. RNA was isolated using intron-spanning primers specific for the target gene of interest. Purified mRNA analyzed by RT-PCR showed that 3 / 7 of the 3D constructs examined were The results showed that the target gene (i.e., EPO) was expressed over a 5-day period.

[0162] In contrast, the 2D configuration (lanes 8-10 in Figure 15) showed no detectable target genes within 5 days. In Figure 15, lane 1 shows the expression of the target gene (lane 21). The expression levels approach those seen in freshly dissected macroscopic tissues, which are known to This shows the 3D configuration that will be achieved in 5 days at the current level.

[0163] Figure 16 shows the CellTiter Bl used to assess metabolic activity in scaffolds. Results of UE® (resazurin metabolism) are shown. From scaffold configuration C, perfusion and static The best metabolic response was obtained under both conditions, followed by scaffold B and scaffold A, respectively. In all scaffold configurations, perfusion culture was superior to static culture in terms of resazurin metabolism.

[0164] Investigating glucose and glutamine consumption by perfused and static 3D cultures of primary renal cells To perform the assay, the conditioned medium was collected and analyzed using Nova BioProfile® 400. Glucose consumption was significantly enhanced under perfusion conditions compared with static conditions. Glutamine was consumed to some extent in all 3D conditions, with perfusion conditions consuming more glutamine than static conditions. The production of glutamate, a by-product of glutamine metabolism, was found to be slightly higher than that of glutamate. was higher under perfusion conditions than under static conditions for all scaffold configurations tested, and The same was true for the production of lactate, a by-product of the course (see Figure 17).

[0165] Example 5 - Isolation of a Heterogeneous Population of an Unfractionated Mixture of Renal Cells (Test Article No. 1) Contains primarily tubular cells, but also small numbers of collecting duct, glomerular, endocrine, vascular, and other cell types Enriched populations of unfractionated mixtures of kidney cells (UNFX) containing subpopulations were prepared from whole kidney tissue as follows: Isolated from:

[0166] Cell donors: Twenty 2-week-old male Lewis rats were sacrificed and kidneys were collected. The excised kidneys were placed in chilled (4°C) Hypothermasol (Biolife S Solutions, Inc., Bothell, WA) 50 mL cone containing 50 mL The kidneys were placed in tubes (10 kidneys per tube) and kept on ice. The next day, the kidney-containing tubes were Rinse with 0% ethanol and place in a biological safety cabinet (BSC) for processing. Ta.

[0167] Renal cell isolation process: 50 μg / mL gentamicin (Sigma, St. Louis, MO) s, MO) in 1X PBS (Gibco, Grand Island, NY). The kidneys were rinsed and the connective tissue and calyces were manually removed using forceps and a scalpel. The kidneys were minced using forceps and a scalpel into a cell / tissue slurry.

[0168] Cell / tissue preparations were incubated with dispase (4 U / mL) (No. 07193, Stem Cell Technologies, Vancouver, BC)+5mM CaCl 2+ collagenase type IV (300 U / mL) (Worthington, Newark, Enzymatic incubation was performed in Krebs buffer containing 100% NJ for 30 min at 37°C on a rocking platform. The resulting cell suspension was then strained through a cell strainer with 70 μm pores. (BD Biosciences, Franklin Lakes NJ) and place in a sterile conical 50 mL polypropylene tube containing 50:50 kidney cell growth medium. The suspension was then incubated at 300x for 5 minutes in a 1:1 high glucose DMEM:KSFM. The cells were centrifuged at 400 x g and resuspended in 10 mL of 50:50 kidney cell growth medium.

[0169] 10 mL of cell suspension was aliquoted into two 15 conical tubes (5 mL each). 0% w / v Optiprep (Sigma, St. Louis, MO) was added to each tube. Invert 6 times. After mixing, carefully layer 1 ml of 1X PBS on top of each suspension. The tubes were centrifuged at 800 x g for 15 minutes (room temperature) without interruption. Sterile 10 mL Remove the cell band (containing viable neo-renal cell prototype No. 1) with a pipette and add 5 ml of Dilute 5x with 0:50 kidney cell growth medium (made with low glucose DMEM) and centrifuge at 300 x g. After centrifugation, the supernatant was carefully removed and the cell pellet was placed in 10 mL of 5 The cells were resuspended in 0:50 (low glucose) kidney cell growth medium and counted. A sample of 0.5 million cells was collected. The total yield of viable cells was 230 x 10 (94% viability). It was.

[0170] A total of 46.8 × 106 cells (1.17 × 106 cells per plate) were cultured in 50 40 p100 tissue culture treated polystyrene plates in 50 (low glucose) medium (BD Biosciences, Franklin Lakes NJ) and After 48 hours, the cells were placed in a standard CO2 incubator (21% O2) at a 50:50 ratio (low group The culture medium was 100% exchanged with course medium and placed in a 2% O environment at 37°C. After 24 hours, cells were harvested for transplantation in 2% O2. Each plate was washed once with sterile PBS. The solution was washed, followed by warmed 0.25% Trypsin w / EDTA (Sigma) 5.0 5 mL of growth medium was added to each plate and the mixture was returned to 37°C for 5-7 minutes. The cell suspension was removed and stored in a sterile conical 50 mL polypropylene tube. The cells were pelleted by centrifugation at × g for 5 min, washed twice in sterile PBS, and chilled. The cells were resuspended in cold sterile PBS (4°C) and counted using a hemacytometer. An aliquot of 10 x 106 cells / 100 μL was prepared. The yield of viable cells after culturing was The cell proliferation rate (from seeding to harvesting) was 3.91 times. Ta.

[0171] Example 6 - Isolation of a Heterogeneous Population of Unfractionated Mixture of Renal Cells and Scaffold Seeding (Test Article No. 2) Cell donors: Ten 2-week-old male Lewis rats were sacrificed and their kidneys were collected. The newly excised kidneys were placed in chilled (4°C) Hypothermasol (Biolife Solutions, Inc., Bothell, WA) 50 mL bottle containing 50 mL of The kidneys were placed in conical tubes (10 kidneys per tube) and kept on ice. The next day, the kidney-containing tubes were Rinse with 70% ethanol and place in a biological safety cabinet (BSC) for processing. Placed.

[0172] Renal cell isolation process: 50 μg / mL gentamicin (Sigma, St. Louis, MO) s, MO) in 1X PBS (Gibco, Grand Island, NY). The kidneys were rinsed and the connective tissue was manually removed using forceps and a scalpel. The kidneys were minced using a scalpel and made into a cell / tissue slurry.

[0173] Cell / tissue preparations were incubated with dispase (4 U / mL) (No. 07193, Stem Cell Technologies, Vancouver, BC)+5mM CaCl 2+ collagenase type IV (300 U / mL) (Worthington, Newark, , NJ) in Krebs buffer containing the enzyme at 37 °C for 30 min on a rocking platform. The resulting cell suspension was then strained through a cell strainer with 70 μm pores. (BD Biosciences, Franklin Lakes NJ) and place in a sterile conical 50 mL polypropylene tube containing 50:50 kidney cell growth medium. The cell suspension was then incubated at 300°C for 5 minutes in a 1:1 high glucose DMEM:KSFM. × g and resuspended in 10 mL of 50:50 kidney cell growth medium.

[0174] 10 mL of cell suspension was aliquoted into two 15 conical tubes (5 mL each). % w / v Optiprep (Sigma, St. Louis, MO) was added to each tube and 6 After mixing, carefully layer 1 ml of 1X PBS on top of each suspension. The tubes were centrifuged at 800 x g for 15 minutes (room temperature) without interruption. The cell band (containing viable neo-renal cell prototype No. 1) was removed by centrifugation and 50: Dilute 5x with kidney cell growth medium (prepared with low glucose DMEM) and centrifuge at 300 x g for 5 minutes. After centrifugation, the supernatant was carefully removed and the cell pellet was placed in 10 mL of 50: The cells were suspended in 50 (low glucose) kidney cell growth medium and counted. The total yield of viable cells was 71 x 10 6 (survival rate 97%).

[0175] Sterile packaged 24 open-cell polylactic acid (Open-Cell Polylactic Acid) Actic Acid:OPLA® scaffold (BD Biosciences, (Franklin Lakes, NJ) was pre-moistened with 50:50 (low glucose) medium. 1 x 10 RK42 neo-kidney cells were added to each scaffold in 50 μL of 50:50 (low glucose) medium. Cell prototype No. 1 was seeded. The scaffolds were acclimated at 37°C / 21% O for 2 hours, and then , filled MPS (Multiwell Perfusion System) unit (BD Technology The cells were transferred to a 50:50 (low glucose) medium (RTP NC) and incubated at 2% O2 for 1 hour. The scaffolds were maintained at room temperature for 5 days. The medium was changed every 2 days (50% of the volume was changed). The cells are carefully removed from the MPS, rinsed in sterile PBS, and transported to prepare them for transplantation. The mixture was then kept at ambient temperature for approximately 2 hours to allow for the desired reaction.

[0176] Example 7 - Transplantation of an Unfractionated Mixture of Renal Cells into a Rat Model of Renal Failure and Anemia A neorenal cell prototype containing a heterogeneous mixture of cells including EPO-producing interstitial fibroblasts. To evaluate the therapeutic potential and safety of No. 1 (UNFX), Renal tubular epithelial cells, glomerular cells, and endothelial cells were isolated from rat kidneys as described above and The ability of Renal Cell Prototype No. 1 to delay or reverse renal failure and / or anemia after surgical resection The study design is shown in Table 1 below. Non-limiting results Success factors include: 1) Significant favorable effect on HCT and / or RBC counts 2) Significant decrease in serum BUN and / or creatinine 3) Histological evidence of erythrocyte stimulation 4) Histological evidence of kidney regeneration 5) Improvements at the biological level (weight gain, survival time)

[0177] According to the study design, 24 adult female Lewis rats (8–10 weeks old) were placed in Ch arles River Laboratories (Wilmington, MA) The recipients were procured from the following sources and assigned to the study as shown in Table 1 below. Daily health assessments, Subsets of each group were screened for the development of disease by weekly serology and cytology. All recipient animals were monitored for anemia / uremia before being treated with the neo-kidney prototype. Before the start of the study, serum creatinine levels in nephrectomized rats were measured for two consecutive weeks. Nephrectomized rats were divided into one of four groups: Group 1 rats were assigned to receive the following 1000-mg doses (see Table 1 below): Ten million Neo-Kidney Cell Prototype No. 1 (test substance) delivered by direct injection into the corticomedullary region Group 2 rats received the Neokia Construct Prototype No. 1 (1 × 10 Six neo-renal prototypic No. 1 cells were seeded and cultured for 4 days on an OPLA scaffold. Group 3 rats were treated by attaching an empty OPLA scaffold to the distal pole of the remnant kidney. Group 4 rats received no treatment. Unmanipulated age-matched controls. The rats were group 5 and age-matched controls that underwent sham nephrectomy but no further manipulation were group 6. I chose 6.

[0178] Renal function (creatinine and BUN) and erythropoiesis (HCT, RBC, and plasma) All animals were bled weekly via the tail vein for evaluation of nuclear RBCs (nRBCs) (50 During the study, health observations were conducted weekly and body weights were measured. On day 84, surviving rats were subjected to a swimming endurance test. At autopsy, femurs, kidneys, and liver were examined. The liver, spleen, heart, and lungs were harvested, weighed, and formalin-fixed and prepared for histological examination. Part of the kidney was embedded in OCT medium and processed for paraffin embedding (FFPE). The FFPE tissue was frozen and processed using cryosections and processed for HE staining.

[0179] Table 1: Study design TIFF0007625287000001.tif116165

[0180] time Day 0, first nephrectomy (50%) Day 7: Second partial nephrectomy (removal of the remaining 2 / 3 of the kidney) Day 43, first blood draw / serology / hematology day Days 83-91, treatment days Day 170, Sacrifice / End of Study

[0181] Test substance: Test Substance No. 1 - A subpopulation of EPO-producing cells isolated as described above in Example 3 New Renal Cell Prototype No. 1 (UNFX), which contains a heterogeneous mixture of cultured kidney cells. Test Article No. 2 - Contains a subpopulation of cells that produce EPO, as described above in Example 4 Neokidneys containing OPLA® scaffolds seeded with a heterogeneous mixture of cultured kidney cells Structure prototype No.1. Test Article No. 3 - OPLA® Scaffold

[0182] Recipient 5 / 6 nephrectomized Lewis rat females: 13 Lewis rat females (8-10 weeks old) were cultured at Charles Rivers Laboratories as described above. All rats underwent a two-stage surgical 5 / 6 nephrectomy at the University of Wisconsin Hospital in Wilmington, PA.

[0183] Briefly, during the first phase of the procedure, a ventral midline incision is made to the abdomen and a sterile drain is placed. A suction cap was applied. The intestines were retracted laterally to expose the right kidney. The kidney was dissected free from surrounding tissue. One-third of the suture was wrapped around each pole of the kidney. The suture was loosely tied around the kidney. On each end, one-third of the kidney was resected just beyond the ligature. The abdominal incision was closed with a suction cup and wound clips. During the second phase of the procedure and 1 week after the first step, Afterwards, the animals were anesthetized and prepared as described above. The dorsal lumbar region was shaved. The entire thorax was A craniocaudal skin incision was made on the left side of the animal's spine, with the cranial end immediately below. The kidney was dissected free from surrounding tissue and gently pulled away from the incision. The adrenal glands, loosely attached to the anterior pole of the kidney by fat, were gently removed by peeling away the attachments. The renal vessels and ureter were cauterized. The vessels immediately distal to the cauterized area were then cauterized. The kidney was removed by cutting the ureter and the incision. The incision was closed with sutures and wound clips. Closed.

[0184] As controls, six age-matched controls underwent sham nephrectomy (surgical equipment for both procedures). Anesthesia, laparotomy, and abdominal closure were performed. Five additional age-matched unoperated controls were obtained. After recovery, all laparotomies were performed. The rats were quarantined for 5 days. Each rat was assigned to a group and given a numerical identifier, study number, The rats were placed in cages with the other rats and the supervisor. Purina Certified Regular Rodent Cho The rats were fed No. 5002 of the w. Water was available ad libitum.

[0185] Survival assessment of clinical parameters: Body weight was observed and recorded weekly using a calibrated analytical balance. Recorded.

[0186] Hematology / Clinical Chemistry: Beginning on day 43, blood and serum will be sent to Antech weekly. The collected data includes hematocrit (HCT), red blood cell count (RBC), nucleated red blood cell count (nRBC), Creatinine (CRE) and blood urea nitrogen (BUN) were measured in the tail or saphenous vein. Blood was collected aseptically from the rats and placed into blood or serum collection tubes (both from BD, Franklin, IL). (Lin Lakes, NJ). To control for diurnal variation, the temperature was maintained between 8:00 AM and 12:00 PM. Blood was drawn between 00:00 and 00:00.

[0187] Surgical Procedure: Anesthesia / sedation / analgesia: 0.05cc (0.3ml) syringe attached to a 26 gauge needle (IP) By first giving 100 mg / mL buprenorphine (Buprenex) Before preparation, rats were sedated. They were then placed in an isoflurane chamber at 4–5% isoflurane. Anesthesia was then maintained throughout the procedure with inhaled isoflurane (3%) via a nose cone. After surgery, the rats were anesthetized with isoflurane inhalation anesthesia. Buprenex was administered, followed by a third dose the next day.

[0188] Surgical preparation: After achieving an adequate level of anesthesia (assessed by toe pinch), place the animal in the dorsal position. Using a No. 5 clipper, betadine (3 times) and ethanol (4 times) were injected concentrically. The right dorsolateral area was shaved and a sterile, transparent The area was prepared for sterile surgery using suitable adhesive drapes.

[0189] Direct injection of neorenal cell prototype No. 1 (Test Substance No. 1, or UNFX): Right dorsolateral region The area was incised longitudinally to expose the peritoneal cavity. The remaining right kidney was isolated and wrapped in sterile gauze and a sharp edge. The kidney was partially extracted from the peritoneal cavity using blunt surgical forceps. Gently resuspend 1 (prep RK40, test article 1) and dispense into a sterile 1 cc syringe (B D, Franklin Lakes, NJ) was filled and then fitted with a 23G needle. Inject 100 μL of cell suspension (10 million cells) through the needle, targeting the corticomedullary junction. ) was slowly delivered into the renal parenchyma. A new sterile 23G needle was used for each animal. Once the bleeding has been reduced, a collagen disc (GelFoam, 2mm x 2mm) is placed in the area. ) was placed at the injection site. The kidney was returned to the abdominal cavity and warmed sterile saline was added to provide hydration. One mL of saline was added. The muscle wall was closed using 4.0 Vicryl sutures and wound clips were attached. The skin was closed using a tape (both items are from Ethicon Inc., Somerville, VA, USA). Postoperatively, oxygen was administered (via inhalation / nose cone) and the patient was awake and conscious. The animals were monitored until a condition occurred.

[0190] Scaffold-based surgical delivery of test articles (No. 2 and No. 3): Right dorsolateral region was surgically incised longitudinally. The remaining right kidney was isolated and inserted into the peritoneal cavity using sterile gauze and blunt surgical forceps. The distal pole of the right kidney was located and partially extracted from the peritoneal cavity using a pointed Using non-surgical techniques or forceps, remove the adherent abdominal fat until dark red kidney tissue is observed. The area was torn away with a Sklar No. 10 scalpel (Sklar Scientific c, West Chester, PA) to expose the kidney, and approximately 4 × 4 mm of renal parenchyma was extracted. The area was debrided with the scalpel until it was exposed and showed minor bleeding. Single neo-kidney construct The prototype No. 1 (Test Article No. 2) or the scaffold alone (Test Article No. 3) was bisected (exposed (to provide a large, flat point of attachment to the renal parenchyma), fibrin glue (Ethicon Inc., Somervile, NJ) on the debrided area (the debrided surface was The abdominal fat was folded over the scaffold and then attached using fibrin glue. By sealing the fat to the kidney surface, a supplemental blood supply was provided to the attached scaffold. The kidney is then observed for approximately 1 minute for signs of bleeding, and if any bleeding is present, it is removed with fibrin glue. After hemostasis, the remnant kidney with the scaffold attached was returned to a neutral position in the abdominal cavity. 4.0 mL of warmed sterile saline was added to the abdominal cavity to aid hydration. The muscle wall was closed using sutures and the skin was closed using wound clips (both items Et (Hicon Inc., Somerville, NJ). Postoperative oxygen was administered (inhaled / non-inhaled). -Zukon-) and monitored the animals until they were awake and conscious.

[0191] Post-operative recovery: After surgery, animals were placed on absorbent pads (unfixed bedding) placed on a heating pad. The rats were allowed to recover in an operating room equipped with cages housing rats (no rats). They were then returned to their individual cages and observed for the remainder of the day and first thing the next day.

[0192] Pre-necropsy procedures: Swimming endurance test: A large circular metal tub (140 cm diameter, 45 cm deep) was filled with water. The tank was filled with 100ml of water and maintained at 32°C. The rats (one at a time) were gently placed facing the side of the tank. The rat was placed in water and released. A stopwatch was used to measure the time it took for the rat to stop swimming from the time of release. The time until the fish could no longer stay above the water surface was measured and recorded. The rats were dried and allowed to recover on a heating pad before being returned to their cages. The excrement was removed from the tank and the temperature was checked and recorded.

[0193] Final weight: Animals were transported to the necropsy room. Final weight was measured using an analytical balance and recorded in grams. It was recorded.

[0194] Euthanasia: Rats were placed in a CO2 chamber. Deep pain reflex and eye examination confirmed the presence of steroids. The patient was confirmed dead.

[0195] Autopsy Procedure: Cardiac puncture / blood collection: Euthanized rats were placed on the necropsy table and blood samples were collected directly into serum and blood collection tubes. The serum samples were spun and serum was collected for a complete serum chemistry panel. Hematology Panel Blood samples were collected for

[0196] Tissue collection: The remnant kidney (or the entire right kidney for controls and sham-treated animals) was carefully removed. One half was placed in 10% buffered formalin and embedded in paraffin. The specimens were then processed for HE staining. The other half was placed in a mold and frozen. The frozen kidney halves were kept on dry ice and frozen. Sections were stored at -80°C until ready for Y chromosome analysis. Remove and weigh the heart and lungs, make a small incision in each, and encase them in paraffin. The femur was then removed and placed in 10% buffered formalin for embedding and HE staining. The proximal condyle was scraped with a scalpel blade to expose the bone marrow and the entire femur, and then They were placed in 10% buffered formalin before paraffin embedding and HE staining procedures.

[0197] result Survival rate: Most of the nephrectomized rats did not survive to the end of the study period. and 6 survived until sacrifice on day 84. The mean survival time of untreated nephrectomized rats in group 4 ( The mean age (days) was 48.25 ± 29.8 days. Nephrectomized rats receiving only the scaffold (Group 3) Rats receiving Neo-Kidney Construct Prototype No. 1 (Group 2) survived 25 days after treatment. They lived an average of 57.5 ± 14.3 days, or 9.25 days longer than untreated nephrectomized rats. Group 1 rats treated with new kidney cell prototype No. 1 survived the study period and survived until day 84 ( The rats were sacrificed together with groups 5 and 6 (arrow) (Figure 18).

[0198] Body weight: Body weight was measured weekly from the start of the study (day 14) until sacrifice (day 170). The weight data (g) are shown in Figure 19. Rats in Group 5 (control) weighed an average of 5g throughout the study period. Group 6 (sham-operated) rats increased by an average of 30.3% (±5.3%). All nephrectomized rats (groups 1-4) lost a significantly lower percentage of their body weight over the study period. Group 1 (new kidney cell prototype No. 1) showed an average increase of 13.5%, and Group 2 (new kidney structure prototype No. Type No. 1) increased by an average of 13.3%, Group 3 (empty scaffold) increased by 7.8%, and Group 4 (untreated) ) increased by 13.7%. When examining the weight gain between the time of treatment and the time of slaughter or death, The differences between the groups were even more pronounced (Figure 20). During the treatment period (days 83 to 170), Rats in groups 5 and 6 gained 11.9% and 8.8% body weight, while those left untreated Nephrectomized rats (group 4) lost an average of 4.8% of their body weight. Rats receiving the scaffold alone (groups 1, 2, and 3) also gained weight during the treatment period. The decrease was not as great as in Group 4 untreated nephrectomized rats.

[0199] Weekly assessment of renal function (BUN and creatinine): Beginning at week 4 and continuing through week 17 Both BUN and creatinine were measured weekly from serum until sacrifice on day 0. The average data for the rats are shown in Tables 2 and 3 below. Figures 21 and 22 show the BUN data, respectively. Temporal measurements of creatinine and creatinine between groups are shown as weekly means. The blood urea nitrogen (BUN) was 19.1±1.9 on average throughout the study. At 7 and 8 weeks (arrows), serum BUN increased in all nephrectomized rats. The mean [BUN] among rats was 53.3±19.9. Treated nephrectomized rats showed persistently elevated [BUN] levels, reaching values ​​above 100 before death. After treatment with the new kidney cell prototype No. 1, rats in Group 1 showed a serum [BUN] of 14 weeks. The new kidney structure showed a stabilization of [BUN] from that point until sacrifice at 19 weeks. Group 2 rats administered with Prototype No. 1 showed lower serum [BUN] than Group 4. However, rats in Group 3, which received only the scaffold, did not show the same degree of stabilization as those in Group 1. Rapid decline after the procedure, as evidenced by elevated [BUN] and death at 10 weeks That was all.

[0200] The serum creatinine results for each group showed the same trend in treatment effect as described for [BUN]. Throughout the study (weeks 7 to 19), the control animals in Group 5 and the control animals in Group 6 showed a tendency to Serum creatinine in sham-operated animals remained stable at 0.4 ± 0.5. In contrast, The untreated nephrectomized rats were tested at week 7 with a mean creatinine of 1.38 ± 0.75. The experimental setting reached a maximum value of 2.7, and the mean value at the time of death was 2.6 ± 0.15. The mean creatinine of the inoculated animals was 2.3 (std. 0.92) at the time of death. , representing a mild improvement in Group 4. Interestingly, the neo-renal cell prototype No. 1 (Group 1) in nephrectomized rats, the cells were transplanted (at week 7) and the nephrectomized rats had a 0.8±0.0 to 1 Maintain stable creatinine levels up to 1.0 ± 0.0 at week 5, and then at the end of the study. Over the course of the study (16-19 weeks), the mean score increased only slightly to 1.3±0.18.

[0201] Data from each individual rat are expressed as a percentage of (control + sham-operated - groups 5 and 6). Examination of BUN and creatinine values ​​at midpoints (12 and 13 weeks) of the study provides a means to examine the variability between rats within each group (Figures 23 and 24). At both weeks 11 and 13, 3 / 5 of the Group 2 Neo-Kidney Construct Prototype No. 1 rats were in Group 4. The BUN and creatinine levels in the nephrectomized rats were significantly lower than those in the untreated nephrectomized rats. 2 / 2) Rats treated with Group 1 neo-renal cells had significantly lower BUN and erythrocyte counts than Groups 2 or 4. and creatinine were shown.

[0202] TIFF0007625287000002.tif243140

[0203] TIFF0007625287000003.tif242163

[0204] Weekly assessment of erythropoiesis (HCT, RBC, nRBC): All data are shown in Table 4 and 5. From week 7 (treatment) to week 19 (end of study), the control group 5 and the control group 6 The mean HCT for sham surgery was 46.9 ± 0.8 and 46.2 ± 1.2, respectively. In contrast, nephrectomized animals in Group 4, which received no treatment, had a mean HCT of 40 ± 2.6 at week 7. Thereafter, the disease declined rapidly until the animals were sacrificed due to morbidity, with a mean HCT of 35. The mean HCT at death for Group 2 neo-kidney construct prototype No. 1 animals was 3. The mean HR was 8.4±4.3, which was an improvement compared with Group 4, but did not reach the control HCT in Group 5. Interestingly, animals in Group 1 who received the new kidney cell prototype No. 1 had a mean pre-transplant ( Week 7) 42.8 ± 0.2 to Week 14 (48.9 ± 0.4) equal to Groups 5 and 6 The HCT value in Group 1 gradually decreased from Weeks 15 to 19. At the time of sacrifice, the average weekly data is shown in Figure 25. Variability between rats in treatment groups was sometimes high, so individual rat data could be evaluated. It is useful to look at the HCT data in an additional format. Figure 26a shows the percentage of controls. Data from all treatment groups at weeks 12 and 13, expressed as (%), are provided (vs. (Reference values ​​= mean values ​​from all control and sham-operated animals on these days).

[0205] RBC and nucleated RBC (nRBC) counts were also measured weekly. Nucleated RBC counts were measured throughout the study. The mean data are shown in Table 5 below. Week 7 (at the time of treatment) From week 19 (end of study) to week 19 (end of study), the mean RBC counts for the control group 5 and the sham group 6 were The mean R of untreated nephrectomized rats at the beginning of the seventh week was 8.32 ± 0.2. The BC count was 7.58 ± 0.58 and decreased rapidly until death, with a mean RBC count of 0.58 ± 0.58 at death. The mean RB at death for Group 2 neo-kidney construct prototype No. 1 animals was 6.5 ± 0.5. The C count was 6.94 ± 0.7. Group 1 animals receiving New Kidney Cell Prototype No. 1 had 8 The results were stable and consistent, ranging from 6.71 ± 2.53 at week 10 to 8.03 ± 0.32 at week 18. There was no significant decrease in RBC counts at week 19 (end of study) (6 Overall, mean weekly RBC counts were followed at HCT (Figure 27 Similarly, interanimal variability in the various treatment groups can be visualized by adding graphs of intermediate time points. This makes it useful to compare the RBC counts of individual animals (at 12-13 weeks) with the percentage of controls ( %) (Figure 28).

[0206] TIFF0007625287000004.tif242155

[0207] TIFF0007625287000005.tif242149

[0208] Hematology and clinical chemistry at time of sacrifice Electrolyte balance: Serum levels of calcium, potassium, chloride, and phosphorus were measured at the time of sacrifice. (Exceptions were made when the animal died and blood could not be collected.) The results are shown in Tables 7a to 7c. The results are summarized in Figure 29. The normal range of each measured parameter is The double dotted line on the graph indicates the normal range (reference range is Gad (ed.), Animal Models in Toxicology,2nd edition,(2008), (Reference: Informa Healthcare USA, New York, NY) Renal failure usually manifests as elevated potassium, sodium, and phosphorus, as well as elevated calcium and Nephrectomized rats in Group 4 showed decreased sodium and chloride levels compared to controls. Nephrectomized rats showed elevated levels of phosphate (but not potassium) and decreased chloride levels. The most consistent electrolyte changes in the 2010 study were decreased chloride levels and increased phosphorus levels. Treatment with Neo-Kidney Cell Prototype No. 1 (Group 1) significantly increased phosphorus and calcium levels compared to Group 4. This resulted in a decrease in chlorine and a slight increase in chlorine (see Figure 30).

[0209] When considering serum phosphorus levels in the analyzed rats individually, 2 / 4 rats were in Group 5. and 6 control and sham-operated rats, as well as group 1 neo-renal cell-treated rats. Low phosphorus levels were observed, as were BUN, creatinine, HCT, and RBC values. , it is clear that the responses of rats in Group 2 were heterogeneous.

[0210] Serum proteins: At the time of sacrifice, unless the rat was dead and blood could not be collected. Serum total protein, serum albumin, and serum globulin levels in all rats For control and sham-operated rats in groups 5 and 6, serum albumin and Nephrectomized rats in Group 4 were compared with control and sham-operated rats. Serum albumin and total protein were significantly lower in the new kidney cell prototype No. 1 compared to the new kidney cell prototype No. 1. Treatment with (Group 1) resulted in a mild recovery of serum albumin and serum total protein. Treatment with Neokidney Construct Prototype No. 1 (Group 2) also resulted in a mild increase in serum total protein. Individual rat data for serum albumin and total protein are shown in Figure 32. The results are expressed as a percentage of the control.

[0211] Liver function: by measuring bilirubin, AST, ALT, GGT, and ALP Liver function was assessed. All data from these tests are shown in Tables 7a-7c. Mean serum A ST was observed in sham-operated rats in Group 6 and in rats treated with Nx + Neo-Kidney Prototype No. 1 in Group 1. The control group in group 5 was within the normal range, and the control group in group 6 was above the normal range reported for the control group (Figure 33). Group 4 and 2 were similar, but with a higher degree of variability. Group 2 (New Kidney Structure Prototype No. 1) With the exception of 1, all groups had mean serum ALT and IL-1 levels higher than the reported normal range for rats. Bilirubin levels were within the reported normal range and were significantly higher between treatment groups. No differences were observed.

[0212] Lipids and sugars: Cholesterol, triglycerides, and and glucose were also measured. All data are tabulated in Appendix F. Regardless, in the nephrectomy groups (1, 2, and 4), mean serum cholesterol and triglyceride levels were significantly higher. The levels of both glycerides were significantly increased in the neo-renal cell prototype No. 1 treated rats in Group 1. The mean triglyceride levels were significantly higher than those in the control and sham-operated groups (groups 5 and 6). Note that mean serum glucose was also higher than Groups 4 and 2 (Figure 34). The serum levels were higher than the normal range reported for all groups. The vesicle-treated rats showed serum glucose levels comparable to those of the control and sham-operated groups. Rats in groups 4 and 2 showed slightly lower serum glucose. Individual rat data are presented as a percentage of control + sham-operated to allow for understanding of variability. is shown in Figure 35.

[0213] TIFF0007625287000006.tif242146

[0214] TIFF0007625287000007.tif188149

[0215] TIFF0007625287000008.tif176150

[0216] Haematology at slaughter Hemoglobin: At the time of sacrifice, blood was collected to measure hemoglobin (Hb), mean corpuscular hemoglobin (MHC), and Used to measure mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin (MCHC) All data are shown in Tables 7a-7c. Nephrectomized untreated rats in Group 4 were compared with control and The [Hb] in Group 4 was significantly lower than in the sham-operated group (Figure 36). A decrease in both HCT and RBC count was observed in Group 1 and Group 2. Mean [Hb] Both were high, and most rats returned to the normal range (see individual data in Figure 37). MCH was relatively equal in all rats, and hemoglobin concentrations per RBC were similar between treatments. These results suggest that the kidneys are similar to those of the kidneys treated with Neo-Kidney Cell Prototype No. 1 or Neo-Kidney Structure Prototype No. 1. The increase in [Hb] observed in rats in groups 1 and 2 after treatment with steroids was due to the This was consistent with the observed increases in RBC count and hematocrit in the control group. The data therefore support the stimulation of erythropoiesis and oxygen transport in the treated rats. This is suggestive.

[0217] WBC and RBC counts and composition: When the rat died and blood could not be collected, White blood cell (WBC) and red blood cell (RBC) counts were measured from all rats at the time of sacrifice, except for All hematological data are presented in Tables 7a-7c. In addition, the WBC population from each rat was The total number of lymphocytes, monocytes, basophils, neutrophils, eosinophils, and large unstained cells (L As shown in Figure 38, the relative proportion (%) of WBC decreased in the group with WBC. The mean WBC was within the normal range in all groups except for the un-nephrectomized treated rats in group 4. reveals differences in WBC composition between groups. New Kidney Structure Prototype No. In nephrectomized untreated rats of Group 4 and nephrectomized rats of Group 2 administered 1, the relative Note the relatively low percentage of lymphocytes and the relatively high percentage of neutrophils.

[0218] Regarding the red blood cell population, the number and percentage (%) of reticulocytes were measured at the final blood draw. (See Figure 40). RBC and HCT have already been reported (Figures 26-2 9). The number and percentage of reticulocytes in nephrectomized untreated rats in Group 4 ( % was decreased compared to the control and sham-operated groups in groups 5 and 6. and 2) showed slight improvements compared to Group 4, but the differences were not statistically significant. RDW (measures RBC width variation) and MCV (mean corpuscular volume) were measured, but there were no significant differences between groups. No significant differences were observed in the blood sugar level (data not shown). All hematological data are shown in Table 7a-c. Shown in 7c.

[0219] Platelets: Platelet count and mean platelet volume (MPV) were measured in blood collected at the time of sacrifice. Platelet counts were slightly above the reported normal range in all groups. No significant differences were observed between treatment groups in MPV or MPV (see Figure 41). .

[0220] Swimming endurance: At the end of the test just before sacrifice on day 84, the remaining rats were The rats were subjected to a swimming endurance test. The rats treated with neo-renal cells in Group 1 and the control and sham-operated rats (Group 5) were As shown in Figure 42, healthy rats completed a swimming test in an average of 2 minutes. The swim time was 2 seconds, with reasonable variation between rats (±33 seconds). It was confirmed that the functional level of the neo-renal cell-treated rats in Group 1 was equivalent to that of the control group.

[0221] histopathology Representative photographs of each tissue examined can be seen in Figures 43 to 46. A summary of findings on this is provided below.

[0222] Liver: Group 4 (Nx) or Group 2 (Nx) compared with controls and sham-operated groups 5 and 6 No notable changes were observed in the liver parenchyma and / or portal triad (+Neo-renal structures). In contrast, intrasinusoidal hematopoiesis was not observed in rats in Group 1 (Nx+Neo-renal cells). Localized areas of

[0223] Kidney: Compared with groups 5 and 6, all other test groups showed an unclear boundary between the cortex and medulla. cystic spaces, juxtaglomerular fibrosis, replacement of the glomerular tuft with avascular vitreous material, hemoglobin kidneys with structural loss characterized by phosphorus pigmentation and multifocal tubular regeneration showed progressive degeneration of the glomeruli and tubules.

[0224] Spleen: Between control and sham-operated groups 5 and 6 and group 1 (Nx+Neo-kidney cell injection) No major histological differences or changes were observed. Examination of the pulp emptying below shows that adult red blood cells (RBCs) and RBC precursors showed moderate and significant reductions.

[0225] Bone marrow: Cellularity of bone marrow in rats in group 1 compared with controls and sham-operated rats in groups 5 and 6 The sex and bone marrow to erythroid ratios appeared to be equal. In contrast, the bones of animals in groups 2 and 4 The marrow was characterized by a moderate and a marked decrease in marrow cellularity, respectively. As shown in 26, in vivo delivery of neo-renal cells to uremic / anemic rats resulted in bone marrow transplantation. Figure 26 further demonstrates that uremic / anemic rats were stimulated by erythrocytes, especially the red blood cell population. These results demonstrate that delivery of neo-renal cells in vivo also stimulates overall cellularity. , which persisted for at least three months after delivery.

[0226] As shown above, the neo-renal cell prototype No. 1 ( UNFX) as determined by RBC count and temporal analysis of HCT, and other Confirmed by final blood test with parameters (Hb, MHC, reticulocyte) As such, it has a regenerative effect on erythropoiesis and red blood cell homeostasis in a surgical 5 / 6 nephrectomy model. Among animals, especially in rats treated with New Kidney Construct Prototype No. 1 in Group 5, Some variability in efficacy was observed, which may be due to variations in scaffold retention by the kidney. Or it may be due to the concomitant occurrence of unidentified scaffolding breakdown within the renal parenchyma. The results showed that in any given rat, improvement in HCT or creatinine was related to the RBC The observation that the results translated into improved blood glucose and BUN levels, resulting in an overall positive effect. Figure 47 shows the HCT data for each rat's serum creatinine. All groups 5 and 6, control and sham-operated, showed higher HC Clustering in the upper right quadrant indicates lower T and creatinine. Groups 1 and 2 2 / 2 and 2 / 5 of the rats in Group 2 are separated into the upper right quadrant along with Groups 5 and 6. One-third of treated rats in group 1 showed improvement in creatinine compared to group 4, but no improvement in HCT. No improvement was observed and is shown in the upper right quadrant. The remaining two Group 2 rats Little or no improvement was observed in any of the parameters. Therefore, they are clustered in the lower left quadrant with rats from Group 4 (high creatinine, low HCT). .

[0227] In conclusion, the above results show that transplantation of neo-renal cells (Group 1) significantly improved the survival rate of 5 / 6 nephrectomized rats. 2 / 2 Group 1 rats reached the 3-month post-treatment time point and showed improvement in both control and The mice were sacrificed on day 84 along with the sham operation. Neo-renal cell transplantation (Group 1) was performed using HCT and RBC This model was performed within approximately 12 weeks, as evidenced by the return of the values ​​to the normal range. Furthermore, Figure 47 shows that administration of the new kidney cell prototype No. 1 to uremic / anemic rats reversed the anemia. Delivery in vivo restores hematocrit to normal levels and reduces untreated uremic toxins. The survival rate of the anemic / anemic rats was increased to a level exceeding that of the anemic / anemic rats. As evidenced by the failure of treatment to overcorrect the blood, resulting in polycythemia vera, Renal cell transplantation regulated erythroid homeostasis for a period of 12 weeks. The new kidney cells were activated, as evidenced by the stabilization of serum BUN and CREA levels from the first 12 weeks. Transplantation resulted in stabilization of kidney function. The new kidney construct prototype No. 1 was This treatment resulted in both improved HCT and stabilization of renal function in 2 / 5 rats. At the time of death of rats due to nephrectomy, renal and red blood cell function were significantly higher than those of nephrectomized untreated rats in Group 4. These results were somewhat better than those obtained when the patients died from morbidity. The heterogeneous nature of the cultured neo-renal cells was observed, which may explain the clear therapeutic effect observed after transplantation. In conjunction with this, the specific cellular component(s) within the population that are responsible for the therapeutic effect This facilitated the identification of

[0228] The above results were also confirmed by the New Kidney Cell Prototype No. 1 (2 / 2) and the New Kidney Structure Prototype No. 1 ( 3 / 5) was delivered, a clear and significant favorable effect was observed on HCT and RBC counts. The positive effects of the new kidney cell prototype No. 1 were rapid (from treatment onward). This study also demonstrated that the new kidney cell prototype No. 1 ( 2 / 2) and Neo-Kidney Construct Prototype No. 1 (3 / 5) were compared with untreated nephrectomized rats. In comparison, it has been shown to stabilize renal function and slow the progression of the disease. , clear histological evidence showed that neorenal vesicle prototype No. 1 (2 / 2) had normal cellularity and Provides sufficient stimulation of erythroblasts in the bone marrow to produce an M:E ratio at the time of sacrifice (3 months after treatment). Furthermore, the rats of the new kidney construct prototype No. 1 (group 2) were sacrificed. Although normal bone marrow histology was occasionally not demonstrated, cellularity and the presence of erythropoietic cells were observed in the group. The histological evidence from this study also demonstrated that the nephrectomy effect of 100 mg / kg ... Group 2 neo-renal cells were found to be repopulating the kidney as judged by the regeneration of tubular vesicles near the explant site. It has been suggested that it stimulated a mild histological improvement in some aspects. These changes are mild and not widespread throughout the kidney. Although not a significant improvement in renal function overall, any improvement seen in renal function was due to individual differences in Group 2 rats. These effects are due to the effects at the cellular level of the steroid hormone, and are not fully detected histologically at the tissue level. Finally, we hypothesize that the cells treated with the new kidney cell prototype No. 1 may not be able to express the same phenotype. Given that the rats survived the test period (3 months), the above test was .1 and New Kidney Construct Prototype No.1 have been shown to extend lifespan in nephrectomized rats. All groups failed to gain weight between the treatment and sacrifice dates, but the New Kidney Cell Prototype No. 1 The group treated with α-glucan had less weight loss.

[0229] Example 8 - Isolation of Renal Cells from Heterogeneous Cell Populations and Enrichment of Specific Bioreactive Renal Cells Renal cell isolation: Briefly, 10 batches of 2-week-old Lewis rat kidneys were purchased from a commercial supplier. (Hilltop Lab Animals Inc.) and Viaspan stock medium. The samples were transported underground overnight at a temperature of approximately 4°C. All steps described here were performed in sterile conditions. The procedure was performed in a biological safety cabinet (BSC) to maintain the kidneys in Hank's Wash three times in balanced salt solution (HBSS) to rinse off the Viaspan storage medium. After the third wash, the remaining kidney capsule and any remaining interstitial tissue were removed. The major calyx was also removed using dissection techniques. The kidney was then minced using a sterile scalpel. The slurry was then transferred to a 50 mL conical centrifuge tube and weighed. A small sample was withdrawn for RNA and placed in a sterile, RNAse-free 1.5 mL microcentrifuge. The cells were placed in a cardiac tube and flash-frozen in liquid nitrogen. After freezing, they were then stored at -80°C until analysis. The tissue weight of 10 young kidneys was approximately 1 gram. Prepare digestion medium to deliver 20 mL of digestion medium per gram of tissue, based on weight. The digestion buffer for this procedure was 4 units of dispase 1 (S) in HBSS. 300 units containing 5 mM CaCl2 (Sigma) The solution contained 100 mg / mL of type IV collagenase (Worthington).

[0230] An appropriate volume of pre-warmed digestion buffer is added to the tube, which is then sealed and incubated at 37°C. The samples were placed on a rocker in the beta for 20 minutes. This first digestion step removed most of the red blood cells. After 20 minutes, the tube was removed and placed in the BSC. The tissue was allowed to settle and the supernatant was then removed. The remaining tissue was then filled with fresh PBS equal to the starting volume. The tube was again placed on a rocker in a 37°C incubator for another 3 days. It was placed for 0 minutes.

[0231] After 30 min, the digestion mixture was pipetted onto a 70 μm cell strainer (BD Falcon). The digestion reaction was carried out by passing the cells through a centrifuge tube and placing them in an equal volume of neutralization buffer (DMEM w / 10% FBS). The cell suspension was then washed by centrifugation at 300 × g for 5 minutes. After centrifugation, the pellet was resuspended in 20 mL of KSFM medium using the trypan blue exclusion method. The cells were resuspended in water and samples were taken for cell count and viability assessment. Then, one million cells were harvested for RNA, washed in PBS, and flash-cooled in liquid nitrogen. Fill the remaining cell suspension to 50 mL with KSFM medium and centrifuge at 300 x g for 5 minutes. After washing, the cells were resuspended in KSFM at a concentration of 15 million cells per mL. The cell pellet was resuspended.

[0232] Five milliliters of the kidney cell suspension was then transferred to a 15 mL conical centrifuge tube (BD Fa Add 5 mL of 30% (w / v) Optiprep® in 1 mL of HCl (container) and invert 6 times. This resulted in a final blend of 15% (w / v) Optiprep®. After inversion, the tube was carefully layered with 1 mL of PBS. Centrifuge the tube at 800 x g for 5 minutes. After centrifugation, remove the tube and place the cell buffer on top of the mixed gradient. Red blood cells, dead cells, and certain small, less granular cells, especially A small number of living cells, including certain EPO-producing cells, certain renal tubular cells, and certain endothelial cells There was also a pellet containing the population. The band was carefully removed using a pipette and another Transfer to a 5 mL conical tube, remove the gradient medium by aspiration, and resuspend in 1 L of KSFM. The pellet was then recovered by recombining the band cells and pelleted cells. Dilute at least 3x the recovered band volume with KSFM and resuspend at 300 x g After washing, resuspend the cells in 20 mL of KSFM. The cells were counted using the trypan blue exclusion method. For RNA samples, one million cells were harvested from the PBS-treated 500-well plate, washed in PBS, and stored in liquid nitrogen. It was flash frozen.

[0233] Density gradient separation was used to enhance the viability and ability of renal cells to exhibit specific biological activities. Pre-culture "cleaning" for: To obtain a clean viable cell population for culture, first A cell suspension was generated as described above under "Kidney Cell Isolation." A total of up to 100 million cells suspended in sterile isotonic buffer as a means of clarifying the initial preparation. The cells were then transferred to an equal volume of 30% iodixanol prepared at room temperature from a stock solution of 60% (w / v) iodixanol. Optiprep® (thus ultimately 15% w / v Optiprep The mixture was thoroughly mixed with the 1:1 ratio of 1:1 (resulting in a solution) and inverted six times to thoroughly mix. 1 mL of PBS buffer was carefully layered on top of the mixed cell suspension. Carefully load the gradient tube into the centrifuge, ensuring proper balance. The gradient tubes were centrifuged continuously at 800 x g for 15 minutes at 25°C. populations (viable and functional collecting duct cells, renal tubular cells, endocrine cells, glomerular cells, and blood containing ductal cells) at 6% and 8% depending on the density of 1.025–1.045 g / mL (w / v) Optiprep®. Other cells and debris were collected at the bottom of the tube. It was in the form of pellets.

[0234] Renal cell culture: Then, 5% (v / v) FBS with antibacterial / antimycotic agent, 2.5 μg of EGF, 25mg BPE, 1X ITS (insulin / transferrin / selenite) A 50:50 mixture of DMEM (high glucose) / KSFM containing sodium media supplement Tissue culture-treated trypsin was cultured at a concentration of 30,000 cells per cm2 in 150 mL of the mixture. Inoculate the combined cell band and pellet into a 1000 ml flask (Nunc T500) or equivalent. The cells were seeded in a humidified 5% CO2 incubator, providing an atmospheric oxygen level of 21%. After 2 days, the medium was replaced and the cells were cultured under a CO2 / nitrogen gas mixture. The cultures were kept in a 2% oxygen environment provided by a humidified incubator (Sanyo). After 24 hours of incubation, the cells were washed with 60 mL of 1X PBS. The cells were washed and then treated with 40 mL of 0.25% (w / v) trypsin / EDTA (Gibco After removal, the cells were resuspended in an equal volume of KSFM containing 10% FBS. The suspension was neutralized, and the cells were then washed by centrifugation at 300 x g for 10 minutes. After washing, the cells were resuspended in 20 mL of KSFM and transferred to a 50 mL conical tube. The number of viable cells was determined using the trypan blue exclusion method, and then samples were collected for analysis. One million cells were harvested for RNA samples, washed in PBS, and immediately placed in liquid nitrogen. The cells were washed again in PBS and collected by centrifugation at 300 x g for 5 minutes. The clarified cell pellet was resuspended in KSFM at a concentration of 37.5 million cells / mL.

[0235] Enrichment of specific bioactive kidney cells using step density gradient separation: Primarily involves renal tubular cells, but also other cell types (collecting duct, glomerular, vascular, and endocrine) Cultured kidney cells, which also contain small subpopulations, were treated with multiple concentrations of iodixanol (Opt These components were separated into subpopulations using a step density gradient made from a smear of 1000 smears of lysed smear ...s. Cultures were placed in a hypoxic environment for up to 24 hours before being harvested and applied to the gradient. In a 15 mL conical tube, four different densities of media were layered on top of each other (maximum (The denser solution was placed at the bottom and the less dense solution was placed on top to form layers.) A gradient was created. Cells were applied to the top of the step gradient and centrifuged to separate the cells by size and The population was separated into bands based on grain size.

[0236] Briefly, KFSM medium was used as the diluent to dilute the 7, 11, 13, and 16% A density of Optiprep® (60% w / v iodixanol) was prepared. For example: For 50 mL of 7% (w / v) Optiprep®, 5.83 ml Add 60% (w / v) iodixanol stock solution of L to 44.17 mL of KSFM medium and react. Mix thoroughly by swirling. Sterile Tygon L / S tubes connected to capillary tubes A peristaltic pump (Master Flex L / S) equipped with 16 tubes was used at 2 mL per minute. Set the flow rate to 1000 and inject 2 mL of each of the four solutions, starting with the 16% solution and then the 13% The 11% and 7% solutions were filled into sterile 15 mL conical tubes. Add 2 mL of cell suspension containing 5 million cultured rodent kidney cells to the top of the step gradient. (The suspension was prepared as described above in "Kidney Cell Culture"). As the pump begins to deliver the gradient solution into the tube, the solution is adjusted to ensure proper interface between each layer of the gradient. Care was taken to allow the cells to flow slowly down the side of the tube at a 45° angle. The resulting step gradient was then centrifuged continuously at 800 x g for 20 minutes. Afterwards, the tubes were carefully removed without disturbing the interface. Five different cell fractions were obtained (Ba 4 strands and 1 pellet) (B1-B4, + pellet) (see Figure 48, left conical tube) Use either a sterile disposable bulb pipette or a 5 mL pipette. Each fraction was collected and characterized phenotypically and functionally (see Example 10). When rodent kidney alveolar suspensions are subjected to step gradient fractionation immediately after isolation, the tubular cells The fraction enriched for collecting duct-derived cells (and containing specific cells derived from the collecting duct) was 1.062–1.0 Separation at a density of 88 g / mL. In contrast, if density gradient separation is performed after in vitro culture, , the fraction enriched for tubular cells (and containing specific cells derived from the collecting duct) is 1 The densities ranged from 0.051 to 1.062 g / mL. Similarly, rodent kidney vesicle suspensions were isolated. If subjected to step gradient fractionation immediately afterwards, EPO-producing cells, glomerular podocytes, and The fraction enriched in vascular cells ("B4") had a density of 1.025-1.035 g / mL. In contrast, when density gradient separation is performed after in vitro culture, EPO-producing cells are isolated. The fraction enriched in alveoli, glomerular podocytes, and vascular cells ("B4") ranged from 1.073 to 1. The important thing is that the cells were cultured in a low-oxygen environment (from about 1 hour to about 24 hours). By exposing the cells to both the "B2" and "B4" fractions for a period of time, This means that cell distribution was enhanced (hypoxia refers to the condition before the harvesting and step gradient procedure). The effect of hypoxia on band distribution (defined as an oxygen level <21% (atmospheric)) Further details are provided in Example 9).

[0237] Wash each band by diluting with 3x volume of KSFM, mix thoroughly, and incubate for 5 min. Centrifuge at 300 x g for 1 min. Resuspend the pellet in 2 mL of KSFM and add Trypan Blue. Viable cells were counted using dye exclusion and a hemacytometer. Million cells were harvested, washed in PBS, and flash-frozen in liquid nitrogen. These cells were cultured in a two-step culture at Charles River Laboratories. Used for transplantation studies into uremic and anemic female rats produced by a 5 / 6 nephrectomy procedure Quantitative real-time PCR was used to investigate the oxygen regulation of erythropoietin and vEGF. The expression of glomerular markers (nephrin, podocin) and vascular markers (P We characterized B4, including its expression of E-cadherin, N-cadherin, and The phenotype of the "B2" fraction was confirmed by the expression of α-glucan and aquaporin-2. See 49b.

[0238] Thus, the use of a step gradient strategy allows for the isolation of rare populations of EPO-producing cells. This not only allows for the enrichment of (B4) but also a relatively enriched population of functional tubular cells (B2). This also allows for a means of generating isolated fractions (see Figures 50 and 51). The strategy also involves the production of EPO by cells and renal tubular cells, which are then transported to the bloodstream by red blood cells, cell debris, and and other potentially unwanted cells, such as large cell aggregates and certain types of immune cells. Allows it to be separated from the mold.

[0239] A step gradient procedure is used to provide good separation of cellular components at specific densities. The preferred approach to fine-tuning the gradient is , 1} Comparison of high density at the bottom of the gradient (e.g., 16-21% Optiprep) to the top of the gradient This involves forming a continuous density gradient from a relatively low density (e.g., 5-10%) to a relatively low density. The gradient can be prepared by any standard density gradient according to standard methods (Axis Shield). Prepare using solutions (ficoll, percol, sucrose, iodixanol) The cells of interest are loaded onto the continuous gradient and centrifuged continuously at 800 x G for 20 minutes. Separation: Cells of similar size and granularity tend to separate together during the gradient. , the relative position in the gradient can be measured, and the specific gravity of the solution at that position is also measured. Therefore, as a result, specific species can be identified based on their ability to cross density gradients under specific conditions. It is possible to extract defined step gradients that focus on isolating specific cell populations. Optimization may be required when isolating cells from non-normal tissue rather than normal tissue, or when isolating cells from different species. For example, the To ensure that the specific B2 and B4 subpopulations are separable from other species, Optimization was performed on cultures of both human kidney cells and rodent B2 and B4 subpopulations. The optimal gradient for the isolation of Optipr HCl was 7%, 11%, 13%, and 16% (w / v). The optimal gradient for isolating canine B2 and B4 subpopulations is 7%, 10 %, 11%, and 16% (w / v) Optiprep. The optimal gradient for isolation of the B1 and B2 subpopulations consisted of 7%, 9%, 11%, and 16% (w / v). Thus, B2 and B3 were isolated from cultured rodent, canine, and human kidney cells. The density range for localizing 4 is provided in Table 8.

[0240] TIFF0007625287000009.tif35155

[0241] Example 9 - Hypoxic incubation before gradient affects band distribution, composition, and gene expression To determine the effect of oxygen conditions on the distribution and composition of prototypes B2 and B4, different Fresh kidney cell preparations from different species were exposed to different oxygen conditions before the gradient step.

[0242] As described above, standard procedures for rat cell isolation and culture initiation were used to generate new rodent strains. A kidney enriched (NKA) cell preparation (RK069) was established. All flasks were cultured for 2-3 days. The medium was changed and then half of the flasks were cultured at 21% (atmospheric) oxygen. Transfer the remaining flasks to an oxygen-controlled incubator set at 21% oxygen. Cells were harvested from each set of conditions using the standard enzymatic harvesting procedure described above. The cells were harvested. A step gradient was prepared according to standard procedures and the cells were then transferred to "normoxia" (oxygen 21 % and "hypoxic" (2% oxygen) cultures were harvested separately and placed next to each other on the same step gradient. Both conditions yielded four bands and one pellet (Figure 71). However, the cell distribution across the gradient was similar in the 21% and 2% oxygen culture batches. Specifically, hypoxia increased the yield of B2, while simultaneously decreasing the yield of B3. Furthermore, in the gradients generated from hypoxic cultured cells, B4 specific The expression of heterologous genes (e.g., erythropoietin) was enhanced (Figure 73).

[0243] Standard procedures for canine cell isolation and culture were followed as described above (Rodent Isolation and Culture Procedures). A canine NKA cell preparation (DK008) was established using a medium similar to that described in Example 1. All flasks were cultured at 4°C for 4 h. The cells were cultured at 21% (atmospheric) oxygen for 2 days, and then a subset of flasks were cultured at low oxygen (2 %) for 24 hours, and a subset of flasks was maintained at 21%. The rats were then harvested and subjected to the same step gradient (Figure 72). Example 1) Canine cells cultured in hypoxia showed a gradient different from canine cells cultured in atmospheric oxygen. The B The yield of 2 increased, and a concomitant decrease in distribution was observed for B3.

[0244] Table 9 TIFF0007625287000010.tif78160

[0245] These data suggest that exposure to hypoxia before the gradient alters the composition of B2 and certain specialized cells (e.g., pericardium). promotes the distribution of thrombin-producing cells (thrombin-producing cells, vascular cells, and glomerular cells) into B4 Thus, hypoxic incubation followed by density-gradient separation as described above has been shown to be an effective method for generating "B2" and "B4" cell populations across different species. It is the law.

[0246] Example 10 - Transplantation of neo-kidney prototypes into a rat model of renal failure and anemia Isolation and propagation of EPO-producing cells from native tissues has been demonstrated using primary cultures of mouse kidney cells. In 2008, when it was discovered that Epo mRNA and protein were expressed It was first described in (Aboushwareb, T, et al. World J Urol, 26: 295-300, 2008). After that, similar cell isolation and culture The culture method was applied to rat, pig, and human kidney tissue from all species examined. The cultures were small, nucleated, and nucleated cells expressing Epo, as described by Aboushwareb et al. Contains subpopulations, but primarily tubular cells, as well as glomerular cells, collecting duct cells, and blood vessels Preliminary experiments showed that heterogeneous cell cultures contained a variety of cell types, including a population of cells. In vivo transplantation of the product into uremic / anemic rats stabilized renal filtration and improved red blood cell integrity. These data suggest that normality was restored and overall survival improved. and that bioactive cellular components(s) exist within heterogeneous primary kidney cultures and do not provide significant therapeutic benefit. These results suggest that the therapeutic effect can be delivered by

[0247] The aim of this study is to demonstrate both specific functional properties in vitro and translational evidence in vivo. To identify bioactive cellular components contained in heterogeneous renal cell cultures based on the The subfraction (B2) concentrated in the renal tubules and the oxygen-regulated EPO-producing cells, Both the spheroid cells and the subfraction containing vascular cells (B4) are involved in filtration, erythropoiesis, and Heterogeneous mixtures (UN) across a range of clinical parameters assessing biological function and organism-level function. FX) were found to be more effective and durable than the FX. and in vivo performance is presented herein.

[0248] To determine the specific effects of specific components of a heterogeneous population of neo-renal cells, B4 (EPO) B1 (enriched for tubular, glomerular, and vascular cells) and B2 (enriched for tubular-producing cells) Enriched for cells, removing EPO-producing cells, glomerular cells, and vascular cells A fraction of the erythrocyte membrane protein (which is a soluble form of erythrocyte membrane protein) was generated and transplanted into a rat model of renal failure and anemia. This is shown in Table 10.

[0249] Tissue procurement and primary cell culture Primary kidney cells were obtained from male Donna-Lewis rats purchased from Hilltop Labs. Cell culture was established using Hypothermasol (BioLife Solutions) ) at 4°C and received within 24 hours of collection. Primary kidney cells were isolated according to the method (Aboushwareb, T, et al. Wor ld J Urol, 26:295-300, 2008), under atmospheric oxygen conditions (oxygen 21%), 5% (v / v) FBS, 2.5 μg of E. coli, in a humidified 5% CO2 atmosphere at 37°C. GF, 25mg BPE, 1X ITS (insulin / transferrin / sodium selenite) thorium medium supplement), containing antibacterial / antifungal agents (all from Invitrogen) A concentration of 30,000 cells per cm² in a 50:50 mixture of high glucose DMEM Cells were cultured at 100°C on tissue-culture treated polystyrene flasks or dishes. Prior to harvesting, for experiments involving exposure to hypoxia, cultures were incubated at 37°C under hypoxic (2%) conditions. For harvesting or serial passage, the cells were transferred to a 0.25% trypsin-EDTA culture medium. The cultured cells were detached. Samples taken for gene expression analysis were flash frozen in liquid nitrogen and then stored at RT. The tubules, blood vessels, collecting ducts, glomeruli, and oxygen-regulated To generate subfractions enriched for specific renal cell types, including cells that produce EPO, To determine the cell density, cells were fractionated after culture using standard density gradient techniques (14, 28, 36). Briefly, iodixanol (Optiprep™) was used to prepare a sterile isotonic buffer solution. A multi-step gradient was prepared with concentrations ranging from 7 to 16% (v / v) of medium. The cells were layered on top of a step gradient and incubated at 800 x g for 20 min at 25°C. The cell fraction (evident as a band in the gradient after centrifugation) The cells were collected in order from top (B1) to bottom (B4). Viability was assessed using the trypan blue dye exclusion method. The rate was assessed and counted manually using a hemacytometer.

[0250] Test Article: Cell prototypes delivered to the kidney are listed below. Doses for subpopulations B2 and B4 is the relative quantitative distribution for the heterogeneous mixture of cells tested in Example 7 above. was selected based on

[0251] UNFX is unfractionated renal cells generated by dissociation and in vitro culture of renal tissue. Contains a heterogeneous mixture. Isolation and culture conditions include primarily tubular cells, but also collecting ducts, glomeruli, and Advantageous for establishing cell lines that also contain smaller subpopulations of somatic, endocrine, vascular, and other cell types Work at.

[0252] B2 is a relatively abundant subpopulation of UNFX and is a robust albumin-binding protein in vitro. It contains proximal tubule cells capable of uptake, certain distal tubule and collecting duct epithelial cells, They also contain trace amounts of other cell types.

[0253] B4 is a minor subpopulation of UNFX and is a hypoxia-responsive EPO-expressing cell. Glomerular podocytes, specialized vascular cells, and lower forward dispersion compared with B2 or UNFX Enriched for cell populations characterized by random and side scatter (size and granularity) can be.

[0254] immunocytochemistry Cells from B2 and B4 were subcultured after the gradient and immunocytochemistry was used to identify distal and proximal The cells were characterized by the expression of peripheral tubule markers. Cells were seeded at a density of 10,000 cells / well onto a 1000-well plate (Falcon 353219). The cells were cultured until they reached 85% confluence, and then diluted with ice-cold acetone / methanol (1:1). The cells were fixed in the mixture at room temperature for 10 minutes. The fixed cells were washed twice in PBS (Gibco). The sections were washed and blocked for 10 minutes in PBS containing 0.5% BSA (Sigma). Locked cells were incubated with N-cadherin IgG1 (BD Biosciences 6109 21) and E-cadherin IgG2a (BD Biosciences 610182 ) were simultaneously co-labeled with primary mouse monoclonal antibodies against IgG (both at 3 μg / mL / 4°C / overnight). Isotype-matched controls (mouse myeloma IgG (Zymed 02-6100) and mouse myeloma IgG2a (Zymed 02-6200)). The primary labeled cells were washed three times in PBS and then incubated at 3 μg / mL at 4°C overnight. Secondary antibody (goat anti-mouse IgG1 Alexa488 (molecular probe A21121) and and goat anti-mouse IgG2a Alexa647 (molecular probe A21241). The cells were also labeled at a concentration of 1 μg / mL and incubated for 30 minutes at room temperature protected from light. The cells were then washed with BD Pathway 855 BioImager (Becton D Imaged using a Electron Microscope (Electron Microscope, ...

[0255] Gene expression Taqman probe and primer sets listed in the ABI catalog, and ABI-Prism 7300 real-time PCR system (Applied Biosystems stems, Foster City, CA) using quantitative real-time PCR ( The expression levels of the target genes were examined by qRT-PCR. 18s rRNA was used as an endogenous control for normalizing low-abundance transcripts. Peptidylprolyl isomerase B (PPIB) was used to detect the relative abundance of target transcripts. To compare the amount of whole kidney tissue, and OriGene Technologies Multiple targets, including Origene kidney cDNA, were purchased from Origene (Rockville, MD). The following TaqMan primer and probe sets were used: Erythropoietin, Rn01481376, purchased from Oster City, CA. _m1;Hypoxia-inducible factor 2 alpha (HIF2α) Rn00576515_m1;Kdr , Rn00564986_m1;E-cadherin, Rn00580109_m1;Cubi Lin, Rn00584200_m1; CYP2R1, Rn01423150_m1; Nef Phosphorus, Rn00575235_m1; Podocin, Rn00709834_m1; Cust om SRY: forward AAGCGCCCCATGAATGC: reverse primer AGCC AACTTGCGCCTCTCT: Probe-TTTATGGTGTGGTCCCGTG- MGB.

[0256] Surgery and in vivo analysis Female Lewis rats were subjected to two-stage 5 / 6 nephrectomy (5 / 6Nx) and The ATP was purchased from River Laboratories (CRL) and h Triangle Institute, International (RTI ), where the rats were housed, monitored, and subjected to various treatment procedures (treatment groups and (See Table 10 for details.) After surgery, uremia was monitored by weekly serological analysis. The welfare of the animals was confirmed before treatment through the respective Institutes of CRL and RTI. ituional Animal Care and Use Committees The animals were administered by the Institutional Animal Control Unit (IACUC) at their respective facilities. Rats were sedated prior to anesthesia with acetaminophen (4-5%) and administered via a nose cone. The cells were maintained under inhaled soflurane (3%) throughout the surgical procedure. , delivered directly into the kidney remnant. Cells (5x106 UNFX or B2; 1x106 B 4), 23G needle (Becton Dickinson, Frankli 10 ml of sterile saline solution was administered via a sterile 1 cc syringe fitted with a syringe (N Lakes NJ). Cells were implanted intraparenchyma in a volume of 0 μL and followed for up to 6 months after implantation. Afterwards, 1 ml of warm sterile saline was added to the intraperitoneal cavity for hydration, and the muscle layer was heated to 4.0 V. The wound was closed using Icryl sutures and the skin was closed using wound clips (both articles (Also available from Ethicon Inc., Somerville, NJ). Oxygen should be administered postoperatively (inhaled). (via nose cone) and monitor the animal until it is awake and conscious. Rats were given 0.5 cc (0.3 mg / ml) buprenorphine x) intraperitoneally immediately after surgery. The following day, rats were given 0.6 ml 1 mg / ml of tramadol was administered (via oral gavage). Rats receiving Epo (R&D Systems) at 100 IU / kg or 500 The animals were administered either 1.5 IU / kg twice weekly via intraperitoneal injection. Serological and Blood was collected weekly via tail vein or orbital bleeding for hematological analysis. Complete serum and hematology parameters were obtained at pretreatment, mid-study (12-14 weeks), and biopsy. While the neuter was performed, BUN, sCREAT, HCT, and RBC# were monitored weekly. At necropsy, kidney(s), heart, liver, and spleen were weighed, frozen, and stored for 1 h. The pith was exposed before fixation. At random, femurs and sternums were collected for histopathological analysis. Body weights were collected weekly.

[0257] Surgery / blood sampling was performed on group 1RK68 rats with the following rat numbers: 66, 36, 59, 46, 63, 34, 32, 79, 54, 43, 67, 52, 76, 51, 78, 4 4, 62, 48, 69, 71, 74, 33, 77, 81, 82, 83, 86 and 87. Surgery / blood sampling was performed on group 2RK69 rats with the following rat numbers: 68, 38, 7 0*, 37, 31, 65, 45, 41, 64, 42, 50, 35, 58, 80, 56, 5 7, 72, 75, 49, 61, 39, 55, 84, 85, 88, 89 and 90. RK70 surgery was performed on rat numbers: 70 and 65.

[0258] Histopathological evaluation of kidneys and bones. Following necropsy, the remnant kidney was weighed and bisected longitudinally. One half was used for preparation of frozen sections. The other half was used for DNA and RNA isolation, and the other half was stored in 10% buffered formalin for 24 hours. The tissue was then transferred to the histopathology laboratory (Premier Laboratories). The tissue was dehydrated and transferred to 70% ethanol (Chories, Colorado). The tissue was embedded in raffin, cut into 5 micron sections, and stained with hematoxylin and eosin (H&E ), Masson's trichrome stain, and periodic acid Schiff (PAS) stain. According to standard laboratory procedures, the left femur was collected and histologically analyzed using the procedure described above. The specimen was submitted for examination and stained with H&E. Glomerular damage was observed as an increase in the mesangial matrix. Segmental mesangial sclerosis with adhesions of the glomerular tufts and / or thickening of Bowman's capsule and Tubular injury was characterized by tubular atrophy, dilation, and / or hyalinosis. It is characterized by lymphocyte accumulation, accumulation of intraluminal protein casts, tubular necrosis, and interstitial fibrosis. Microscopic evaluation of femoral sections was performed using H&E staining. Sections were stained for bone marrow cells. For any increase / decrease in cellularity and myeloid to red blood cell ratio, 200x and and examined at the original magnification of 400x.

[0259] Measurements: Animals were weighed weekly. Biweekly serological and hematological analyses were performed to assess renal function before and after transplantation. Provided in-life assessment of (BUN & CREAT) and erythropoiesis (HCT & RBC) Complete serum and hematology panels were performed at baseline, week 6, week 12, and prior to necropsy. At necropsy, organs (kidneys, liver, spleen, heart, and lungs) were weighed and collected for histological examination. Femoral bone marrow was collected for histological examination and bone marrow smear analysis.

[0260] Table 10. Research Design TIFF0007625287000011.tif198165

[0261] result Generation and compartmental characterization of candidate bioactive cell populations (B2 & B4) In vivo pilot studies have demonstrated that the expression of NF-κB in cultured heterogeneous populations of kidney cells (UNFX) The results clearly suggested that the bioactive cells contained in the urea-containing steroid hormone (ESH) have the ability to enhance kidney function (see Example 7). (See references). Previous studies have shown that the clinical parameters affected by the delivery of UNFX populations Based on this data, a theoretical candidate cell population for influencing renal homeostasis is functional tubular cells. (based on improved creatinine excretion and protein retention), glomerular cells (protein retention), (based on improved retention) and highly specialized oxygen-responsive EPO-producing cells at the corticomedullary junction. cells (based on the rate of erythropoiesis) (Maxwell, PH, et al. Kidney Int, 52: 715-24, 1997, Obara, N. et al. Blood, 111: 5223-32, 2008). renal tubules, globules The presence of EPO-producing cells has been established and expanded from rats and other species. It was confirmed in kidney cultures using the density gradient method (Qi, W, et al. Nephrol ogy (Carlton), 12: 155-9, 2007, Gesek, F. A, et al.. Am J Physiol, 253: F358-65, 1 987, McLaren, J, et al. Hum Exp Toxicol, 14: 916-22, 1995) is an established culture of a heterogeneous UNFX cell population. Adapted to enable reproducible production of different cell subfractions (B1-B4) from the culture medium. The subfractions were differentiated from each other and characterized functionally, molecularly, and phenotypically. Based on the results, two subfractions (B2 & B4) were identified from UNFX and further in vitro and It was selected as a bioactive candidate for in vivo studies.

[0262] Subfraction B2, which represents the predominant component (approximately 20% to 50%) of the UNFX mixture, was isolated from the renal tubules. Based on relative enrichment for cytoplasmic vesicles (the predominant UNFX cell type in established and expanded cultures), Targeted gene expression analysis was performed to identify the major cellular compartments of the kidney, subfractions B2 and B4. The relative contribution of the subfraction B2 was assessed. , podocin+), and EPO-producing cells (Figure 74d-h) were relatively eliminated. Two proximal tubule markers associated with clinically relevant tubular cell function were expressed in B2. Vitamin D hydroxylase (CYP21R) was significantly enriched in ), and cubilin (3.0x concentrated). Distal tubule cells (E-cadherin+) and aggregates The duct cells (D. biflorus agglutinin+, aquaporin-2+) The majority (>75%) was localized to subfraction B1, which is consistent with the Alzheimer's disease in these studies. Furthermore, due to the relative absence of cytoplasmic cubilin+ cells and the presence of other undesirable characteristics, However, the expression of distal tubule markers, E-cadherin, and erythrocytes was not evaluated. Herin was relatively enriched in B2 (1.6x) compared to B4 (Fig. 74c ).

[0263] Quantitative differential gene expression data are presented in tabular form (Fig. 74i). The relative distribution of proximal and distal cellular components was analyzed using the N-cadherin (proximal) and E-cadherin (distal) assays. This was qualitatively confirmed at the protein level through immunofluorescence staining for (Fig. 74j, k). A key function associated with proximal tubule cells is receptor-mediated transport of albumin from the glomerular filtrate. reabsorption, thus reducing proteinuria and contributing to serum protein homeostasis (7 ) Functional assays to capture cubilin / megalin-mediated albumin uptake (45) and confirmed that the B2 subfraction exhibited robust protein transport activity in cubilin-expressing cells. In contrast, cubilin protein expression and albumin expression were significantly increased (Fig. 75a). Incorporation of amine was detected less frequently in the B4 subfraction (Fig. 75a), and gene expression levels were significantly higher. The difference in cell composition between B2 and B4 observed in the bell was confirmed. Receptor-mediated transport of ATP was significantly reduced by hypothermic inhibition of active transport (data not shown). (not shown), and the specificity of uptake was determined by competing with cubilin / megalin-mediated endocytosis. This was confirmed by blocking the receptor with a synthetic inhibitor, receptor-associated protein (RAP) (30). (Figure 75a).

[0264] The B4 subfraction constitutes a minor component of the unfractionated (UNFX) cell population (<10%). It consists of several specialized cell types, all of which have clear theoretical basis based on their in vitro characteristics. The B4 subfraction was then transferred to the hypoxia-responsive EPO-producing cells, They were selected based on their relative enrichment in globular podocytes and cells of vascular origin (Figures 74-76). Flow cytometry analysis of the UNFX population revealed that Epo-expressing cells were located in the renal tubules of the UNFX population. We have previously shown that small cells (low side scatter) with low granularity (low side scatter) are distinct from the normal cells. The B4 subfraction was further characterized as a forward scatter (FSC) in response to hypoxic stimulation (34). , small, low-granularity cells that significantly upregulated Epo expression (Fig. 74g, h, i, Fig. 75b) The B4 subfraction was enriched approximately 15x with respect to the glomerular podocytes (nephrin and and podocin) and approximately 15x approximately 200x for vascular cells (Kdr). x concentrated (Figure 74i).

[0265] Comparative in vivo function of B2 and B4 in rats with progressive renal failure Survival and weight gain. A pilot study involving intrarenal transplantation of UNFX cells demonstrated that the UNFX population survived for 3 years after orthotopic transplantation. Contains physiologically active cells that can stabilize filtration / urine production and restore red blood cell homeostasis for months. These results suggest that the B2 (albumin uptake) and B4 (oxygen-regulated Epo production) cells The cellular composition and functional properties of both make them the bioactive components of the UNFX population. These are logical subfractions candidates for further investigation, each of which may be improved upon. This is a possible cause of the observed in vivo effects of improved filtration and enhanced erythropoiesis. A uremic / anemic rat was produced via a two-stage 5 / 6 nephrectomy to establish a progressive disease state. After standing (5-6 weeks after injury), serum creatinine levels in nephrectomized rats were already normal. The hematocrit was over 250% of the control (0.76 ± 0.02 vs. 0.3 ± 0.00). 8% reduction in IL-1 expression (41.8±0.67 vs. 45.6±0.70) in syngeneic male cells As in the previous study, UNFX cells were delivered and their B2 and B4 subfractions were isolated. were delivered at doses approximating their relative contribution to the UNFX population: 5x106 / rats B2 at 1x106 / rat and B4 at 1x106 / rat. Additional controls were delivery vehicle only (PBS) and and recombinant human Epo protein (rEp) at 100 IU / kg and 500 IU / kg. All untreated healthy controls and sham Nx groups were treated twice weekly with 100 mg of ... The rats survived for the duration of the study (30 weeks after nephrectomy), as predicted by previous studies. As shown, 100% of untreated Nx rats died within 22 weeks of the nephrectomy procedure (Figure 76 a) Between treatment groups, 100% of rats treated with B2 survived 6 months after treatment, while 100% of rats treated with B4 survived 6 months after treatment. The 6-month survival rates for the untreated and UNFX-treated groups were 20% and 0%, respectively. Rats treated with 100 IU / kg rEpo had a 25% survival rate at 6 months after treatment. The effect of B4 (the EPO-producing cell fraction) was similar to that of treatment with B4. Treatment with 500 IU / kg rEPO resulted in accelerated death; 100% of patients died within 14 weeks of starting the treatment regimen, and most suffered from severe anemia at the time of death. Specifically, low-dose (100 IU / kg) rEPO was shown to increase normal blood cholesterol levels over a period of one month. Maintaining HCT and RBC within range, the study subsequently produced anemia in 75% of rats by 3 months. However, high-dose (500 IU / kg) rEPO significantly reduced HCT and RB over a 1-month period. C to levels greater than 125% of healthy controls, subsequently resulting in severe anemia and death. (See Figures 53-55.) Furthermore, recombinant EPO has been shown to have a significant effect on the immune system over 4 weeks of repeated administration. It should be noted that the erythropoiesis of 6 / 8 rats was not supported. Poor survival after Epo treatment is due to a combination of bone marrow depletion and development of antibodies to rEpo This may be due to the previously described immunity to human Epo (Campeau, PM, et al. Mol Ther, 17: 369-72, 2009).

[0266] As shown in Figure 52, EPO-enriched (B4) cells were stimulated with two different doses (high and low) of EPO. The results were compared with those of recombinant EPO at 16 weeks. Specifically, and as shown in Table 11 below, the hematocrit was significantly higher in EPO-enriched cells. The cells restored the blood flow to 95% of normal levels. Overall, there is very little variability in the results when EPO-enriched cells are delivered. It was found to exist.

[0267] Weight gain measured 16 weeks after treatment was also significantly higher in the EPO-concentrate treated group than in the non-EPO treated group. Improved compared to the enriched treatment group (see Figure 58). Healthy control and sham Nx rats showed an average weight gain of 30% throughout the study, whereas untreated Nx rats showed an initial On average, rats lost 5% of their body weight at the time of death (Fig. 76b). In contrast, rats treated with B2 lost 1% of their body weight at the time of death (Fig. 76b). Six months after implantation, the subjects showed an average weight gain of 14.3% (p<0.0001) (Fig. 7b ), suggesting significant organism-level improvements in feeding and general welfare. and vehicle showed significantly higher weight gain than Nx animals (B4: p=0.0239, vehicle Chronic hypertension can be both a cause and an effect of CKD, and may be involved in its pathogenesis. Regardless, persistent systemic hypertension places an excessive strain on the heart, leading to significant left ventricular hypertrophy (LVH). This results in relative cardiac hypertrophy due to vasoconstriction (Foley, RN, et al. Kidney I nt, 47: 186-92, 1995). Hypertension and LVH are common complications of nephrectomy. This has been confirmed in rodent models (Amann, K, et al. Nephro l Dial Transplant, 13: 1958-66, 1998). Honken In this study, heart weight and body weight data were collected at autopsy, and cardiac hypertrophy / LVH was assessed by Lewis To determine the characteristics of two-stage 5 / 6 nephrectomy-induced renal failure in rats and to determine whether either treatment The purpose of this study was to determine whether treatment(s) reduced or eliminated this hypertrophic response. The rats treated with thiamin showed only a 25% increase in relative heart weight 6 months after treatment, whereas the normal rats showed only a 25% increase in relative heart weight 6 months after treatment. Compared with normal animals, heart weight was increased by 50% in Nx rats (P < 0.00). 01) (Figure 76c). Although not as conspicuous as the treatment effect of B2, both B4 and vehicle treatments The treatments resulted in significantly lower heart weights than untreated Nx controls. The data support treatment-dependent enhancement of renal-regulated cardiovascular function.

[0268] Progression of uremia and anemia. As mentioned above, rats that underwent two-stage 5 / 6 nephrectomy showed a significant improvement in renal function when various treatments were delivered. The patient was uremia and anemia 5-6 weeks after the resection. Uremia will be assessed via sCREAT and BUN weekly during the study, and erythropoiesis will be assessed at the end of the HCT and and RBC#.

[0269] If a sufficient number of rats survived from each group to allow robust comparisons, the efficacy between groups was Statistical evaluation of force was performed using all data from 10 to 20 weeks after treatment. One-way analysis Analysis of variance was performed using JMP from SAS Institute Inc (Cary, NC). Using version 7.0, serum chemistry results for samples taken between 10 and 20 weeks were In this time range, significant differences were observed between treatment groups: top panel, BUN ( p<0.0001); (Figure 77d), creatinine (p<0.0001) (Figure 77e) Analysis of variance of measurements of BUN and CREAT levels from 10 to 20 weeks showed that B2 and B4 Nx rats treated with cells showed increased renal filtration compared with Nx alone and Nx+Unfx cells. showed stabilization and low BUN and CREAT levels, demonstrating the general renal protective effect of the treatment. Furthermore, Nx rats treated with B2 and B4 prototypes showed increased blood There were no significant differences between treatment groups over this time period. Observed: RBC (p<0.0029) (Fig. 77a), HCT (p=0.0005) ( Figure 77b). The data show that B2-treated rats showed a higher High mean HCT and RBC counts (Fig. 77a-c) and low mean CREAT and BUN levels The results clearly showed that B2 was a potent bioactive component of UNFX (Fig. 77d-f). and delivery of the B2 subfraction (without other components of UNFX) significantly improved the magnitude and durability of the treatment response. This biological observation was not observed in other groups. In addition, the organization of native-like glomerular, tubular, and nephron structures in B2-treated animals This was supported by scientific evidence. B4 did not significantly affect filtration function, but RBC# Erythropoiesis was stimulated by B4 treatment, as exemplified by the significant enhancement of HCT. Regular administration of recombinant Epo resulted in an initial polycythemia and a reduction in red blood cell count (Fig. 77a-c). It produced a short-term stimulation of sphere formation and subsequently in the majority (7 / 8) of rats that received the drug. The lack of recombinant EPO in this study led to severe anemia and ultimately death. The overall potential for antibody development against the human EPO protein in immunocompetent rats was demonstrated. This could be the case.

[0270] As shown in Figure 57, when subjected to a swimming endurance test 12 weeks after treatment, the nephrectomized mice NX rats swam for a shorter time on average than healthy rats. Cell-treated rats swam for longer periods on average than NX rats, but with greater variability. On the other hand, rats treated with high doses of B4 cells swam for nearly the same amount of time as the control group, and Survival of all treated groups was significantly higher than that of nephrectomized, untreated rats. It is better than the original.

[0271] Table 11. Clinical values ​​as % of healthy controls at 12-14 weeks after treatment. TIFF0007625287000012.tif236168TIFF0007625287000013.tif234145

[0272] Enhanced tubule-associated function in treated versus untreated uremic rats Protein processing and lipid metabolism. Comprehensive clinical chemi...

Claims

1. A pharmaceutical composition comprising: (I) one or more isolated and enriched populations of human kidney cells for treating kidney disease; and (II) a pharma- ceutical acceptable carrier, The one or more isolated and enriched populations of human kidney cells include (i) prepared by fractionating cultured kidney cells from a kidney sample or cells from human kidney tissue; said fractionating comprising a step of density gradient separation; (ii) As a result of the fractionation, (a) a cell population comprising large granular cells of the renal tubule and collecting duct system having a buoyant density of less than 1.045 g / mL; and (b) a cell population containing debris and small cells of low granularity and viability having a buoyant density greater than 1.091 g / mL Lacking Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1, wherein the first one or more isolated and enriched human kidney cell populations contain a higher proportion of tubular cells than cultured kidney cells or human kidney tissue derived from a human kidney sample and have a buoyant density of 1.045 g / mL to 1.052 g / mL.

3. The pharmaceutical composition of claim 1, wherein the first one or more isolated and enriched human kidney cell populations contain a higher percentage of EPO-producing, glomerular, and vascular cells than cultured kidney cells or human kidney tissue derived from a human kidney sample and have a buoyant density of 1.063 g / mL to 1.091 g / mL.

4. The pharmaceutical composition of claim 2, further comprising a second isolated and enriched population of human kidney cells, the second isolated and enriched population of human kidney cells comprising a higher proportion of EPO-producing, glomerular, and vascular cells than cultured kidney cells or human kidney tissue derived from a human kidney sample, and having a buoyant density of 1.063 g / mL to 1.091 g / mL.

5. 5. The pharmaceutical composition of any of claims 1 to 4, wherein the one or more isolated enriched human kidney cell populations are further prepared by subjecting cultured kidney cells from a kidney sample or cells derived from human kidney tissue to hypoxic conditions prior to fractionation.

6. The pharmaceutical composition of claim 5, wherein subjecting the cells to hypoxic conditions comprises subjecting the cells to an oxygen level of less than 5%.

7. The pharmaceutical composition of any of claims 1 to 6, wherein the pharma- ceutically acceptable carrier comprises water, saline, an aqueous buffer solution, or a combination of saline and an aqueous buffer solution.

8. The pharmaceutical composition of claim 7, wherein the one or more isolated and enriched human kidney cell populations are suspended in the pharma- ceutically acceptable carrier.

9. The pharmaceutical composition of claim 7, wherein 10 4 to 10 9 cells of said one or more isolated enriched human kidney cell populations are suspended in said pharma- ceutically acceptable carrier.

Images