TW201211256A - Erythropoietin-expressing adipose cell populations - Google Patents

Abstract

The present invention relates to isolated cell populations derived from adipose tissue that express erythropoietin (EPO).

Description

201211256 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to an isolated cell population derived from adipose tissue exhibiting erythropoietin (EPO). [Prior Art] Chronic kidney disease (CKD) affects more than 19 million people in the United States, usually as a result of metabolic disorders including obesity, diabetes, and hypertension. Examination of the data revealed that the rate of increase was due to the development of renal failure secondary to hypertension and non-insulin-dependent diabetes mellitus (NIDDM) (United States Renal Data System: Costs of CKD and ESRD (US Kidney Data System: CKD and ESRD) The price). Bethesda, MD, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238) - These two diseases are also increasing worldwide. Controlling stage 1-3 CKD patients with progressive lifestyle and pharmacological interventions that target potential disease states, while dialysis and drug regimens are used to control patients in stage 4-5. It usually includes antihypertensives, red blood cell stimulating agents (ESA), iron and vitamin D supplements. Chronic renal failure is common in humans as well as in some livestock. Patients with renal failure not only experience loss of renal function (uremia), but also develop anemia because the bone marrow cannot produce sufficient red blood cells (RBC) via red blood cell production. Red blood cell constancy depends on the production of erythropoietin (EP0) of specialized interstitial fibroblasts resident in the kidney and the ability of the targeted red blood cells in the bone marrow to respond to EP0 and produce more RBCs. Anemia of renal failure is due to a reduction in the production of EPO in the kidney and a negative effect of urinary toxic factors on the effects of EPO in the bone marrow. Currently, clinical methods for treating chronic renal failure include dialysis and kidney transplantation to restore renal filtration and urine production, and systemic delivery of recombinant EPO or EPO analogs to restore erythroid mass. Preclinical studies have examined the in vivo efficacy and safety of EPO-producing cells produced by gene therapy methods. However, new therapeutic paradigms of substantial and sustained increase in renal function are needed to slow the progression of the disease and improve the quality of life of this patient population, while reducing the annual burden on the medical care system. Regenerative medicine technology can provide the next generation of treatment options for CKD. The recognized fat is an endocrine organ with significant metabolic biological activity. Adipose tissue includes adipocytes 'angiogenic cells' outer membrane cells 'fibroblasts 'macrophage' stem cells, precursors of MSC-like biological activity and smooth muscle-like cells (Crisan, et al, Cell Stem Cell 3, 301, 2008; da Silva Meirelles, L et al, J Cell Sci 119, 2204, 2006; Lin, G et al, Stem Cells Dev 17, 1053, 2008; Basu, J et al, Tissue Eng Part C, In Press, 2011). Among them, the application of MSC-like and smooth muscle-like cell populations in tissue engineering and regenerative medicine is currently actively being developed (Basu, J. and Ludlow, JW Trends Biotechnol 28, 526, 2010; Basu, J et al., Tissue Eng Part C Methods, 2011 May 19 [Epub ahead of print). At a higher level, adipose tissue can be divided into white or brown based on whether white or brown fat cells predominate. White adipose cells represent the major lipid storage media in adipose tissue, while brown adipose cells are responsible for mediating lipid metabolism and are therefore enriched accordingly in the mitochondria. Adipose tissue can be found to be widely distributed throughout the body as a unique region-specific depot of 156850.doc 201211256. The main reservoirs of white adipose tissue (WAT) are abdominal subcutaneous tissue adipose tissue and visceral adipose tissue (SAT and VAT). VAT itself can be further subdivided into reservoirs of omentum, mesentery, retroperitoneum, gonads and pericardium (Bjorndal, B et al, J Obes, 2011, Volume 2011, Article ID 490650, 15 . pages; Cook, Α· and Cowan, C. , Adipose (March 31, 2009), StemBook, edited by The Stem Cell Research Community). Fat store

A unique pattern of weaving, transcriptomics, proteomics, and expression profiles and biological functions. For example, the secretome produced by the visceral, subcutaneous, and gonad fat reservoirs is source specific (Roca-Rivada, A et al, JP Rotomicics 74(7): 1068-1079, 2011). Further, significant functional differences between subcutaneous, epididymal and mesenteric fat have been observed by transcriptomics and liposome analysis of transgenic mice with humanized lipoprotein profiles (Caesar, R et al, PLoS One 5 ( 7): ell525, 2010). Finally, the multi-lineage differentiation potential of MSC-like biologically active adipose-derived stromal cells appears to be a source-dependent reservoir (Levi, B et al, Plast Reconstr Surg 126, 822, 2010; Schipper, B.M, et al., Ann Plast

Surg 60, 538, 2008). Despite these systematic observations, the analysis and characterization of transcripts, proteomics, and functional differences between fats associated with individual organs remains to be seen. More specifically, understanding the changes in reproductive potential exhibited by stromal cell displays derived from solid organ-related fats from different sources may significantly affect the development of tissue engineering and regenerative medicine (TE/RM) products that target these organs. SUMMARY OF THE INVENTION The present invention relates to a population of cells derived from adipose tissue 156850.doc 201211256 obtained from any suitable fat source in the body. In one embodiment, the population of cells is an isolated population of adipocytes that exhibit erythropoietin (EPO). Suitable sources of adipose tissue include, but are not limited to, heart fat, liver fat, subcutaneous fat, abdominal or visceral fat, white fat, brown S fat, and kidney fat. In one embodiment, the population of cells expressing EPO can be derived from renal adipose tissue. The population of cells can be derived from renal pedicle adipose tissue and/or renal pelvis adipose tissue. In one other embodiment, the population of cells can be derived from a fatty matrix vascular fraction (SVF). In another embodiment, the population of cells also exhibits a manifestation of VEGF 〇 EP0 and/or VEGF as a manifestation of hypoxia regulation. In one other embodiment, the population of cells exhibits an EP0 $Special Record and/or an EPO polypeptide. In another embodiment, the population of cells exhibits a VEGF transcript and/or a VEGF polypeptide. In one other embodiment, the EPO polypeptide exhibited by the S-derived cell population is characterized by a post-translational modification that is different from the non-fat cell population. The non-fat cell population can be selected from the group consisting of keratinocytes, hepatocytes, and primary kidney cells. In another embodiment, the EPO polypeptide expressed by the population of renal adipose-derived cells is characterized by a post-translational modification that is different from the non-renal adipocyte population. The non-renal adipocyte population can be selected from the group consisting of keratinocytes, hepatocytes, visceral fat stromal cells, and primary kidney cells. In some embodiments, the population of renal adipose-derived cells differentially exhibits biomarkers associated with renal regeneration. The differential performance of the biomarker can be an increased performance. The biomarker can be WT-1. The WT-1 biomarker can be a exemplable WT-1 transcript, including a KTS+ and/or KTS_transcript. The biomarker can be 156850.doc 201211256 WT-1 polypeptide. In another aspect, the invention provides a method of preparing a population of adipose stromal cells that exhibit erythropoietin (EPO). In one embodiment, the method includes the step of digesting adipose tissue. The method can also include the step of consuming the digested adipocyte tissue to provide a stromal vascular component (SVF). The consumption step can be performed after the digestion step. The SVF will comprise a population of adipose stromal cells that exhibit EPO. In one embodiment, the adipose tissue can be renal adipose tissue and the renal adipose stromal cell population exhibiting EP0 is a population of renal adipose-matrix (KiSAS) cells as described herein. In one other aspect, the invention provides an implantable construct for improved renal function to a subject in need thereof. In one embodiment, the construct comprises a biocompatible matrix. In another embodiment, the construct further has a population of adipocytes expressing erythropoietin (EP0) that are deposited onto the surface of the substrate or embedded in the surface of the substrate. In one other embodiment, the population of adipose stromal cells exhibiting EP0 is a population of KiSAS cells as described herein. In yet another aspect, the invention provides a method of treating a kidney disease in a subject in need thereof. In one embodiment, the method comprises the step of administering to the subject a composition comprising a population of adipocytes expressing erythropoietin (EP0). In another embodiment, the population of adipose stromal cells exhibiting EP0 is a population of KiSAS cells as described herein. In one other aspect, the invention provides a method of treating a kidney condition in a subject in need thereof by using a construct. In one embodiment, the method comprises the step of administering a construct to the subject. The construct can have a biocompatible matrix. In another 156850.doc 201211256 embodiment, the construct can also have a population of adipocytes expressing erythropoietin (EPO) deposited onto the surface of the substrate or embedded in the surface of the substrate. In one other embodiment, the population of adipose stromal cells exhibiting EPO is a population of KiSAS cells as described herein. [Embodiment] The present invention relates to an isolated cell population derived from adipose tissue, a method of isolating the cell population, a scaffold or matrix inoculated with the cell (construct), and a method of manufacturing the scaffold or matrix, It is related to a method of treating a patient in need thereof using the cell population or construct. It has been found that a population of cells derived from an adipose tissue matrix vascular component (SVF) exhibits erythropoietin (EPOVEPO expression can be an expression of an EPO transcript and/or an EPO polypeptide. The cell population also exhibits a VEGF transcript and/or a VEGF polypeptide. The performance of EP0 and/or VEGF is indicative of hypoxia regulation. The adipose tissue may be derived from any suitable source in the body including, but not limited to, heart fat, liver fat, subcutaneous fat, visceral fat, white fat, brown fat. In the protocol, the adipose tissue may be renal adipose tissue. The renal adipose-derived cell population may be referred to herein as a renal-derived adipose matrix (KiSAS) cell population. The KiSAS cell population exhibits EP0 and vascular endothelial growth factor (VEGF). The performance occurs in a manner of hypoxia regulation. Moreover, the cell is found to exhibit a renal proliferative transcription factor WT1 specific for renal adipose-derived stromal cells, and it has also been found that the renal meridian-specific fat reservoir can be transcribed from different WT1 transcripts. Variant definition. The present invention demonstrates that adipose tissue is a functionally unique reservoir of cells expressing EP0, Alternative cells for cell populations available in tissue engineering and regenerative therapies of the kidneys 156850.doc 201211256 Sources 1. Definitions Unless otherwise indicated, the technical and scientific terms used herein have the ordinary knowledge in the art to which the present invention pertains. The same meaning that people usually understand.

Principles of Tissue Engineering, 3rd edition. (R Lanza, R Langer, & J Vacanti), 2007 provides general guidance to many of the terms used in this application by those of ordinary skill in the art. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the invention. In fact, the invention is not limited in any way by the methods and materials described. For the purposes of the present invention, the following terms are defined below. As used herein, the term "hypoxic" culture conditions refers to culture conditions in which cells are subjected to a reduction in the available oxygen content in a culture system relative to standard culture conditions in which cells are cultured at atmospheric oxygen levels (about 21%). Non-hypoxic conditions are referred to herein as normal or normoxic culture conditions. As used herein, the term "oxygen tunable" refers to the ability of a cell to modulate gene expression (up or down) based on the amount of oxygen available to the cell. "Hypoxia-induced, refers to up-regulation of gene expression in response to a decrease in oxygen tension (regardless of pre- and initial oxygen tension). The term "construct" refers to deposition into one or more synthetic or naturally occurring biocompatible species. One or more cell populations on or in the surface of a scaffold or substrate made of material 156850. Doc 201211256 body. The one or more cell populations may be coated with a biomaterial made of one or more synthetic or naturally occurring biocompatible polymers, proteins or peptides, deposited onto the biomaterial, embedded in the biomaterial, and joined The biological material is inoculated into the biological material or captured in the biological material. The one or more cell populations can be combined with the biomaterial or scaffold or matrix in vitro or in vivo. Typically, one or more biocompatible materials used to form the scaffold/biomaterial are selected to direct, promote or provide at least one cell population deposited thereon to form a multicellular three dimensional system. The one or more biological materials used to create the construct may also be selected to direct, facilitate or provide for the dispersion and/or integration of cellular components of the construct or construct with the endogenous host tissue, or to direct, facilitate or provide constructs or constructs. The cellular components of the substance survive, migrate, tolerate or function. The term "marker" or "biomarker" generally refers to a molecular marker based on DNA, RNA, proteins, carbohydrates or glycolipids whose expression or presence in a cultured cell population can be by standard methods (or disclosed herein) The method) detects and is consistent with one or more cells in the cultured cell population for a particular cell type. The marker can be a polypeptide or a identifiable physical location on the chromosome, such as a gene, a restriction endonuclease recognition site, or a nucleic acid (e.g., mRNA) encoding a polypeptide expressed by a native cell. The target g may be a region of expression of a gene, referred to as a "gene expression marker", or some segment of DNA having no known coding function. Biomarkers can be detected in a population of cells derived directly from tissue samples. The terms "differentially expressed gene", "differential gene expression," and its synonymous form are used interchangeably to mean that their performance in a first cell or cell population is relative to their performance in a second cell or cell population. Activate to a higher or lower level of the gene. The 156850. Doc -10- 201211256 The term also includes genes which are expressed at higher or lower levels at different stages over time during the generation of the first or second cells of culture. It will also be appreciated that differentially expressed genes can be activated or inhibited at the nucleic acid level or protein level, or can be subjected to alternative splicing to produce different polypeptide products. This difference can be demonstrated by differences in, for example, mRNA level, surface appearance, secretion of the polypeptide, or other distribution. Differential gene expression can include comparing the performance of one or more genes or their gene products, or comparing the ratio of expression between one or more genes or their gene products, or even comparing two different processed products of the same gene in a first cell / The cell population differs from the second cell/cell population. The differential performance includes a temporal or cellular expression pattern of the gene or its expression product, for example, a quantitative and qualitative difference between the first cell population and the second cell population. For the purpose of the present invention, when there is a difference between the expressions of the designated genes in the first cell population and the second cell population, it is considered that "the expression of the differential gene" exists. The terms "inhibition", "stimulation", "low performance" and "reduction", used interchangeably, refer to the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more protein or protein subunits. , or the activity of one or more protein or protein subunits is reduced relative to one or more controls, such as, for example, one or more positive controls and/or negative controls. The term "upregulation" or "overexpression" is used to mean The expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more protein or protein subunits, or the activity of one or more protein or protein subunits is increased relative to one or more controls, such as, for example, For example, one or more positive controls and/or negative controls. 156850. Doc • 11 - 201211256 The term “subject” refers to any individual human subject suitable for treatment ‘including a patient who is experiencing or has experienced one or more symptoms, symptoms or other indicative symptoms of kidney disease, anemia or EPO deficiency. Such subjects include, but are not limited to, those recently diagnosed with kidney disease, anemia, or EPO deficiency, or previously diagnosed with kidney disease, anemia, or EPO deficiency and who are now undergoing recurrence or relapse, or at risk of kidney disease, anemia, or EPO deficiency. Healer, no matter what. The subject may have previously been treated for T-nephropathy, anemia or EPO deficiency, or not treated as such. The term "patient" refers to any individual animal that is desired to be treated, more preferably a mammal (including non-human animals such as, for example, dogs) , cats, horses, rabbits, zoo animals, cattle, pigs, sheep and non-human primates). Most preferably, the patient herein is human. The term "sample" or "patient sample" or "biological sample" generally refers to any biological sample obtained from a subject or patient, body fluid, body tissue, cell line, tissue culture, or other source. The term includes tissue biopsy samples such as, for example, kidney biopsy samples. The term includes cultured cells such as, for example, cultured mammalian kidney cells. Methods for obtaining tissue biopsy samples and cultured cells from mammals are well known in the art. If the term "sample" is used alone, it still means that the "sample" is a "biological sample" or a "patient sample", that is, these terms are used interchangeably. The term "anemia" as used herein refers to a deficiency in the production of functional EPO protein by cells producing EPO in a subject, and/or insufficient release of EPO protein into the systemic circulation, and/or the inability of the red blood cells in the bone marrow to respond to EPO proteins. The number of red blood cells and/or the lack of hemoglobin levels. Subjects with anemia cannot maintain red blood cells 156850. Doc -12- 201211256 Constancy. In general, anemia can be accompanied by a decline or loss of renal function (eg, chronic renal failure), anemia associated with relative EPO deficiency, anemia associated with congestive heart failure, and myelosuppressive therapy such as chemotherapy or antiviral therapy ( For example, AZT) is associated with anemia, anemia associated with non-myeloid carcinoma, and viral infections such as HIV.  Anemia, and chronic diseases such as autoimmune diseases (eg, rheumatoid arthritis), liver disease, and anemia of multiple organ system failure. The term "EPO deficiency" refers to any disease or condition, including anemia, that can be treated with a erythropoietin receptor agonist (eg, recombinant EPO, EPO peptide analog or EPO analog) or red blood cell stimulating agent (ESA). As used herein, the term "kidney disease" refers to a condition associated with acute or chronic renal failure at any stage or extent that causes the kidney to lose its ability to perform hemofiltration functions and to remove excess fluid, electrolytes, and waste from the blood. Nephropathy also includes endocrine dysfunction such as anemia (erythropoietin deficiency) and mineral imbalance (vitamin D deficiency). Kidney disease can be derived from the kidney, or it can be secondary to a variety of diseases including, but not limited to, heart failure, blood pressure, diabetes, autoimmune disease or liver disease. Kidney disease can be a condition of chronic renal failure that develops after acute injury to the kidney. For example, ischemia and/or exposure, damage to the kidneys may lead to acute body failure; emergency fiber loss can not be recovered. .  It may lead to the development of chronic renal failure. The term "treatment" is a therapeutic treatment of lions with occupational kidney disease, anemia, lack of Bp〇, insufficient renal tubular transport, or insufficient renal insufficiency. The goal is to delay, prevent or slow down (reduce) the decision. Ba's illness. Need to be included include kidney disease, anemia, EPO deficiency, insufficient tubule transport or glomerular 156850. Doc -13· 201211256 People who are under-represented, and people who are prone to kidney disease, anemia, EPO deficiency, insufficient renal tubular transport or glomerular filtration, or will prevent kidney disease, anemia, EPO deficiency, renal tubular insufficiency or kidney People with insufficient ball filtration. The term "treatment," as used herein, includes stabilizing and/or improving renal function. Cell populations The present invention provides a population of adipose tissue-derived cells that exhibit erythropoietin (EPO) expression by the performance of EPO$Specialized and/or EPO polypeptides. The cell also displays a VEGF transcript and/or a VEGF polypeptide. EPO and VEGF behave hypoxic. The adipose tissue may be derived from any suitable source in the body including, but not limited to, heart fat, liver fat, subcutaneous fat, visceral fat, white fat, brown fat. In one embodiment, the population of cells described herein can be derived from a source of adipose of the subject in need of treatment. The population of cells can also be derived from a source of adiposes that are not autologous to the subject, including, but not limited to, allogeneic, or homologous (homologous or isogenic) sources. In one embodiment, the adipose tissue source can be renal adipose tissue. A population of cells derived from renal fat may be referred to as a population of renal adipose matrix (KiSAS) cells. The KiSAS cell population can be derived from any adipose tissue associated with the kidney, including but not limited to, adipose tissue associated with the renal pedicle and/or renal pelvis (renal hernia or renal pelvis). In one embodiment, the KiSAS cell population is derived from a renal adipose matrix vascular component (SVF). In one aspect, the adipose-derived cell population exhibits EP0 in a manner that is modulated by hypoxic conditions. The cell population of the invention is characterized by EP0 expression and biological response to oxygen 156850. Doc -14- 201211256 Sexuality such that a decrease in oxygen tension in the culture system leads to induction of EPO performance. In one embodiment, EP0 is cultured under conditions in which the cell population undergoes a reduced oxygen content in the culture system when the cell population is in a cell population that is cultured at a normal atmospheric (~21%) level of available oxygen. Performance is induced. In one embodiment, the cells cultured under lower oxygen conditions exhibit a greater level of EP0 相对 relative to cells cultured under normoxic conditions, generally, when the available oxygen content is reduced (also known as hypoxic culture conditions) Culturing cells means that the reduced oxygen content is reduced relative to the culture of cells at normal atmospheric levels of available oxygen (also known as normal or normoxic culture conditions). In one embodiment, the hypoxic cell culture conditions comprise culturing at about less than 1% oxygen, about less than 2% oxygen, about less than 3% oxygen, about less than 4% oxygen, or less than about 5% oxygen. cell. In another embodiment, the normal or normoxic culture conditions comprise about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17 The cells were cultured with % oxygen, about 18% oxygen, about 19% oxygen 'about 20% oxygen, or about 21% oxygen. In one other embodiment, an induction or increased expression of EP0 is obtained, and the cells can be cultured by using less than about 5% of available oxygen and compared to EP0 expression in cells cultured in the atmosphere (about 21%). Observed at the level. In another embodiment, the induction of EP0 is obtained in a cell culture capable of expressing EP0 by a method comprising: a first culture stage in which the cell culture is cultured in atmospheric oxygen (about 21%) for a period of time, and In the second culture stage, where the oxygen content is reduced, the same cells are cultured under about less than 5% available oxygen. In another embodiment, the EP0 response in response to hypoxic conditions is modulated by HIFla. Those of ordinary skill in the art will appreciate other oxygen manipulations known in the art 156850. Doc -15- 201211256 Culture conditions can be used for the cells described herein. In one aspect, the population of cells described herein differ in their EPO performance compared to other cell populations and/or fat sources known to exhibit EPO. The difference can be at the transcript level, the polypeptide level, and/or the post-translation level. In one embodiment, the KiSAS cell population exhibits an EPO polypeptide at a higher level than the non-fat cell population. Non-fat cell populations include, but are not limited to, liver fat, heart fat, white fat, and brown fat. This is illustrated in Figure 5 PJ (see Example 1). It has been found that the EPO-producing cell population of the present invention exhibits a specific post-translationally modified EPO compared to EPO exhibited by other cell populations. This can be demonstrated by using a variety of techniques. For example, isoelectric point pyrolysis (IEF) has been used to detect different forms of EPO in samples obtained from individuals (Catlin et al, Clinical Chemistry 48; 11, 2057-2059 (2002); Breidbach et al, Clinical Chemistry 49:6, 901-907 (2003)) As described in Example 1, compared to other possible sources of EPO, including keratinocytes, hepatocytes, non-renal fat, and primary kidney cell populations, IEF was found to be derived from renal adipose tissue. EPOs have different migration modes (Figure 4A). Electrophoresis channel 3 corresponds to EPO expressed in the KiSAS cell population, and the migration pattern is different from other electrophoresis channels. For example, if the IEF colloid has a pH gradient with the top of the colloid being the strongest (+) and the bottom of the colloid being the strongest (-), then the KiSAS EPO is more than the EPO from the visceral (non-renal) adipose stromal cell population (electrophoresis 4) Move to a more acidic point. In one embodiment, the KiSAS cell population exhibits a more acidic EP0 (expressed by IEF 156850) compared to EP0 expressed by a population of cells selected from the group consisting of: Doc -16- 201211256 shows: keratinocytes, hepatocytes, visceral (non-renal) adipose stromal cells and primary renal cell populations. Figure 4A also shows that EPO exhibited in visceral adipose stromal cells migrates in different migration modes compared to EPO from keratinocytes, hepatocytes, renal fat and primary kidney cell populations. Electrophoresis channel 4 corresponds to the EPO expressed in the visceral fat cell population.  The shift mode is different from other electrophoresis channels. For example, visceral fat EPO migrates to a lower acid point than EPO (electrophoresis channel 3) from a renal fat-derived cell population or EPO (electrical lane 6-7) from a primary renal cell population. In one embodiment, the visceral fat cell population exhibits less acidic EPO (shown by IEF) compared to EPO expressed by a population of cells selected from the group consisting of: keratinocytes, hepatocytes, renal adipose stromal cells And primary kidney cell populations. In another aspect, the KiSAS cell population exhibits biomarkers associated with regeneration. In one embodiment, the biomarker is a population of WT-KiSAS cells that can exhibit one or more of said biomarkers at a higher level than a population of non-renal adipose stromal cells. The non-renal adipose stromal cell population can be a population of visceral adipose stromal cells. 3. A method of isolating a population of cells expressing EPO It has unexpectedly been found that 'adipose tissue is a source of cells that express EPO. In one aspect, the invention provides a method of isolating and isolating a population of adipocytes expressing erythropoietin (EPO) for therapeutic use, including treatment of kidney disease, anemia, EPO deficiency, renal tubular insufficiency, and glomeruli Insufficient filtration. The autologous renal adipose stromal cell population can be obtained directly from the subject in need of treatment 156,850. Doc -17- 201211256 Yes. Non-autologous cell populations can be derived from appropriate donors. The cells can be separated from the biopsy sample and the cells can be frozen or expanded prior to use. For renal fat, the cell population is isolated from fresh digestion, i.e., mechanically or enzymatically digested fatty kidney tissue or from mammalian kidney cell culture in vitro. Renal adipose tissue can be obtained from the kidney pedicle or the kidney sputum. Ti can be cut from the kidneys. Renal adipose tissue from the kidney sputum can be excised from the medulla of the intact human kidney. The renal pelvis fat can be cut from the medulla. After dissection, adipose tissue samples can be processed according to the following exemplary protocol (see Example 1). Add adipose tissue to PBS/0. 1% gentamicin (Iiwitrogen-Gibco) was thoroughly washed at 37. 0 in DMEM-HG (Invitrogen-Gibco). Digestion with 3% collagenase I (Worthington) and 1% BSA for up to 1 hour. The sample can be centrifuged at 600 g for 20 minutes to aspirate the supernatant of the adipocyte. The remaining stromal vascular components can be resuspended in a-MEM/10% FBS (Invitrogen-Gibco) and placed in a tissue culture incubator for 24-48 hours. The non-adherent cell population can be removed by washing 3 times with PBS. The cell population can be cultured under hypoxic conditions for a different period of time in a 〇r depleted (low oxygen) (2%) incubator. To confirm the integrity of the hypoxic regulatory pathway, the performance of biomarkers such as EPO and/or VEGF of the cell population can be examined. In one aspect, the cell population of the invention is characterized by EPO expression and biological responsiveness to oxygen such that a decrease in oxygen tension in the culture system results in induction of EPO performance. The cell population is oxygen-adjustable. In one embodiment, EP0 is cultured under conditions in which the cell population undergoes a reduced oxygen content in the culture system when the cell population is in a cell population that is cultured at a normal atmospheric (~21%) level of available oxygen. Performance is induced. In one embodiment, 156850 is produced relative to EP0 produced under normal oxygen conditions. Doc -18· 201211256 Cells, cells cultured in lower oxygen conditions exhibit a greater level of EPO. In general, culturing cells at a reduced oxygen content (also known as hypoxic culture conditions) refers to a reduced oxygen content relative to the normal atmospheric content of available oxygen (also known as normal or normoxic culture conditions). ) The cells are reduced when cultured. In one embodiment, .  Hypoxic cell culture conditions include culturing the cells at about less than 1% oxygen, about less than 2% oxygen, about less than 3% oxygen, about less than 4% oxygen, or about less than 5% oxygen. In another embodiment, the normal or normoxic culture conditions comprise about 10% oxygen, about 12% oxygen, about 13[deg.]/helium oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, The cells are cultured at about 17% oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, or about 21% oxygen. In one other embodiment, an induction or increased expression of EP0 is obtained, and the cells can be cultured by using less than about 5% of available oxygen and compared to EP0 expression in cells cultured in the atmosphere (about 21%). Observed at the level. In another embodiment, the induction of EP0 is obtained in a cell culture capable of expressing EP0 by a method comprising: a first culture stage in which the cell culture is cultured in atmospheric oxygen (about 21%) for a period of time, and In the second culture stage, where the oxygen content is reduced, the same cells are cultured under about less than 5% available oxygen. In another embodiment, in response to hypoxia, the conditional EP0 expression is modulated by HIF1 alpha. Those with ordinary knowledge in the field will understand.  Other oxygen processing culture conditions known in the art can be used for the cells described herein. Exemplary techniques for isolating and isolating cell populations of the invention include separating by density gradients based on differential specific gravity of different cell types contained in a population of target cells. The specific gravity of any given cell type can be affected by the degree of fineness in the cell, the volume of water within the cell, and other factors. In one aspect, the invention provides for use across a wide variety of species (including 156850. Doc -19· 201211256 But not limited to, human, canine and rodent) optimal gradient conditions for separating cell populations. In another aspect, the invention provides a method of enriching and/or attenuating cell types using fluorescence activated cell sorting (FACS). In one embodiment, BD FACSAriaTM or equivalent can be utilized to enrich and/or attenuate cell types. In another aspect, the invention provides a method of enriching and/or attenuating cell types using magnetic cell sorting. In one embodiment, the KiSAS cell population can be enriched and/or subtracted using the MiltenyiautoMACS® system or equivalent. In another aspect, the invention provides a method of three-dimensionally culturing a population of adipose derived cells. In one aspect, the invention provides a method of culturing a population of cells via continuous perfusion. In one embodiment, the perfusion conditions include a brief, intermittent or continuous flow of liquid (perfusion). In one embodiment, the medium in which the cells are cultured is circulated or agitated intermittently or continuously in such a manner that the force is transmitted to the cells via the flow. In one embodiment, the cells undergoing transient, intermittent or continuous fluid flow are cultured in such a manner that the cells are present in a three-dimensional structure in or on the material providing the framework and/or space for such three-dimensional structure formation. In one embodiment, the cells are cultured on multi-hole beads and subjected to intermittent or continuous liquid flow using a shaker platform, a rotary platform or a spin-on flask. In another embodiment, the cells are cultured on a three-dimensional scaffold and placed in the device such that the scaffold is stationary and the liquid flows directionally through or across the scaffold. Those of ordinary skill in the art will appreciate that other perfusion culture conditions known in the art can be used with the cells described herein. I56850. Doc -20· 201211256 As described herein, low or low oxygen oxygen conditions can be used to prepare a cell population of the invention. However, the method of the present invention can be used without the steps of forming under low oxygen conditions. In one embodiment, conditions with normal oxygen content can be used. Those of ordinary skill in the art will appreciate that other methods of isolation and culture known in the art can be used with the cells described herein. 4. The matrix or scaffold is as described in Bertram et al., U.S. Patent Application Serial No. 20070276507, the entire disclosure of which is incorporated herein by reference in its entirety herein in its entirety, the entire disclosure of the entire disclosure of . In one embodiment, the matrix or scaffold of the present invention may be three dimensional and shaped to conform to the size and shape of the organ or tissue structure. For example, in the use of polymeric stents for the treatment of kidney disease, anemia, EPO deficiency, insufficient tubular transport, or insufficient glomerular filtration, a three-dimensional (3-D) matrix may be required. It is possible to use a variety of different shapes of 3-D brackets. Of course, polymeric matrices can be shaped into different sizes and shapes to accommodate patients of different sizes. The polymeric matrix can also be shaped in other ways to suit the particular needs of the patient. In another embodiment, the polymeric matrix or scaffold can be a biocompatible porous polymeric scaffold. The scaffold can be formed from a variety of synthetic or naturally occurring materials including, but not limited to, open polylactic acid (OPLA®), cellulose ether, cellulose, cellulose esters, fluorinated polyethylene, phenolic, Poly-4-methylpentene, polyacrylonitrile, polyamide, polyamidoximine, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester Carbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyether oxime, 156850. Doc -21 - 201211256 Polyethylene, Polyfluoroolefin, Polyimide, Polyolefin, Polyoxadiazole, Polyphenylene Ether, Polyphenylene Sulfide, Polypropylene, Polystyrene, Polysulfide, Poly Maple, Polytetra Fluorine, polythiol, polytrimethylene, polyurethane, polyethylene, polyvinylidene fluoride, regenerated cellulose, anthrone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginic acid, Hyaluronic acid, agarose, or a copolymer or physical mixture thereof. The branched structure can be changed from a liquid hydrogel suspension to a soft porous scaffold to a rigid, retaining shape multi-well scaffold. Hydrogels can be formed from a variety of polymeric materials and can be used in a variety of biomedical applications. A hydrogel can be physically described as a three-dimensional network of hydrophilic polymers. Depending on the type of hydrogel, it contains varying percentages of water but is generally insoluble in water. Despite their high water content, the 7jC gel is able to additionally bind large volumes of liquid due to the presence of hydrophilic residues. The hydrogel swells sufficiently without changing its gel structure. The basic physical characteristics of the hydrogel can be specifically altered depending on the particular equipment used to characterize the product and the product. Preferably, the hydrogel is made of a polymer that is biologically inert and physiologically compatible with mammalian tissue, a biologically derived material, a synthetically derived material, or a combination thereof. The hydrogel material preferably does not cause an inflammatory reaction. Examples of other materials that can be used to form the hydrogel include (a) modified alginic acid, (b) polysaccharides gelled by exposure to monovalent cations (eg, gellan gum and carrageenan), and (6) very sticky. Liquid, or thixotropy, a gel that forms a gel over time due to the slow evolution of the structure (eg, hyaluronic acid), and (d) a polymeric hydrogel precursor (eg, polyepoxy _ Polypropylene glycol block copolymers and proteins). U.S. Patent No. 6,224,893 B1 provides a suitable preparation for root 156850. Doc -22· 201211256 A detailed description of various polymers of hydrogels according to the invention and the chemical nature of the polymers. The scaffold or biomaterial can be characterized to enable the cells to attach to the scaffold or biomaterial and interact with the scaffold or biomaterial, and/or can provide a porous space in which the cells can be captured. In one embodiment, the porous scaffold or biomaterial of the present invention allows a population of cells to be added or deposited onto a biomaterial constructed as a porous scaffold (eg, by attaching cells) and/or pores of the scaffold (eg, by Capture cells). In another embodiment, the scaffold or biomaterial allows or promotes cells in the scaffold: cell interactions and/or cells: biomaterial interactions to form a construct as described herein. In one embodiment, the biomaterial used in accordance with the present invention comprises hyaluronic acid (HA) in the form of a hydrogel comprising a HA molecule size of 5. 1 kDA to > 2 χ 1 〇 6 kDa. In another embodiment, the biomaterial used in accordance with the present invention comprises hyaluronic acid in the form of a porous foam comprising a HA molecule size also from 5. 1 kDA to > 2 X 1G6 kDa. In yet another embodiment, the biomaterial used in accordance with the present invention comprises a polylactic acid (PLA) based foam having an open cell structure with a pore size of from about 50 microns to about 300 microns. Those of ordinary skill in the art will appreciate that other types of synthetic or naturally occurring materials known in the art can be used to form the stents described herein. 5. Constructs In one aspect, the invention provides inoculation or deposition of the fat-derived 156850 described herein. Doc -23- 201211256 One or more polymeric scaffolds or matrices of a population of EPO-expressing cells. Such scaffolds that have been seeded with a population of cells may be referred to herein as "constructs." In one aspect, the invention provides an implantable construct for treating a kidney disease, anemia, or an EPO deficiency in a subject in need thereof, the construct having one or more of the cell populations described herein. In one embodiment, the construct is deposited on the surface of the stent by a biocompatible material or a biomaterial, a scaffold or matrix comprising one or more synthetic or naturally occurring biocompatible materials, and by attachment and/or capture. The cell population described herein is embedded in the surface of the scaffold. In certain embodiments, the construct is made of a biomaterial and a cell population, the cells are coated with the biomaterial component, deposited onto the biomaterial component, deposited in the biomaterial component, attached to the biomaterial component, Material components are captured, embedded in, or combined with biological material components. This population of cells can be used in combination with a matrix to form a construct. In one embodiment, the population of cells is in direct contact with the substrate to form a construct. In another embodiment, the deposited cell population or cellular component of the construct is a population of KiSAS cells that exhibit EP0. The population of inoculated KiSAS cells can be an oxygen-adjustable cell that exhibits EP0. In other embodiments, the KiSAS cell characteristic is the expression of one or more biomarkers. The biomarker may be a tubular cell biomarker selected from the group consisting of cubilin; E-mucin; NPHS1 (congenital degenerative nephritis 1, Finnish (kidney vasopressin)); ankle protein; vitamin D3 25 - hydroxylase (CYP2D25), Wnt4 and any combination thereof. In another embodiment, the biomarker can be selected from the group consisting of WT VEGF, EP0, and any combination thereof. The biomarker may be a renal regeneration biomarker involved in the development of known renal organs, 156,850. Doc -24- 201211256 For example, WT1. In one embodiment, the cell-deposited construct is adapted to improve renal function of the subject after implantation or administration to the kidney of the subject. In one embodiment, the population of cells deposited onto or formed with the biomaterial or scaffold is derived from a variety of sources, such as autologous sources. Non-autologous sources are also suitable for use, including but not limited to 'allogeneic or homologous (homologous or isogenic) sources. Those of ordinary skill in the art will appreciate that there are a variety of methods suitable for depositing strontium in other ways to combine cell populations with biological materials to form constructs. 6. Methods of Use In one aspect, the invention contemplates methods of providing a population or construct of adipose-derived cells to a subject in need of such treatment. In one embodiment, the method comprises the step of providing a substrate that will inoculate or deposit a population of cells to form a $__^ 沉积 deposition step comprising culturing a population of cells on the substrate. After the substrate has been sunk to form a construct, the construct can be implanted into the treatment site of the patient. In one aspect, the invention provides methods of nephropathy, anemia, or EPO deficiency in a subject in need of treatment with a cell population as described herein. In one embodiment, the method comprises administering to the subject a composition comprising a population of cells. In another embodiment, the composition further comprises for treating a disease or condition as described herein, deposited into a biological material, deposited onto a biological material, embedded in a biological material, coated with a biological material, or Biomaterial capture to form the implantable constructs described herein 3 156850. Doc -25· 201211256 Cell population. In one embodiment, the cell population is used alone or in combination with other cells or biological materials to stimulate regeneration in an acute or chronic disease state, such as a hydrogel, a porous scaffold, or a natural or synthetic Peptide or protein. In another aspect, renal disease, anemia, or EPO deficiency in a subject is effectively treated by the methods of the present invention and can be observed via various indicator symptoms of erythropoiesis and/or renal function. In one embodiment, the indicator symptoms of red blood cell constancy include, but are not limited to, hematocrit (HCT), hemoglobin (HB), red blood cell mean hemoglobin (MCH), red blood cell count (RBC), number of reticulocytes, reticulocyte percentage , red blood cell mean volume (MCV) and red blood cell distribution width (RDW). In one other embodiment, the indicator symptoms of renal function include, but are not limited to, serum albumin, albumin to globulin ratio (A/G ratio), serum phosphorus, serum sodium, kidney size (ultrasonic measurement), serum calcium , phosphorus to calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level and glomerular filtration rate (GFR). Moreover, multiple indicators of overall health and wellness include, but are not limited to, increased or decreased weight, survival, blood pressure (mean systemic blood pressure, diastolic blood pressure or systolic blood pressure), and physical endurance performance. In another embodiment, the treatment of stability of one or more indicator symptoms of renal function is effectively treated. Stabilization of renal function was demonstrated by observing that the indicated symptoms in the treated subjects treated by the method of the invention were compared to the same indicator symptoms in the subjects not treated by the method of the invention. Alternatively, stabilization of renal function can be achieved by treating the subject in a subject treated by the method of the invention compared to the same subject being treated at 156,850. Doc 26· 201211256 Before the same indicator changes in the symptoms to prove. The change in the symptoms of the first indicator can be an increase or decrease in sputum. In one embodiment, the treatment provided by the invention may comprise a stabilization of the blood urea nitrogen (BUN) level in the subject, wherein the BUN level observed in the subject has a similar disease state but is not subjected to the method of the invention The subject being treated is low. In one other embodiment, the treatment can include stabilization of the serum creatinine level in the subject, wherein the serum creatinine level observed in the subject is lower than the subject having a similar disease state but not treated by the method of the invention. . In another embodiment, the treatment can include stabilization of the hematocrit (HCT) level of the subject d, wherein the HCT level observed in the subject is comparable to having a similar disease state but not treated by the method of the invention. The subject is high. In another embodiment, the treatment can include stabilization of the red blood cell (RBC) level in the subject, wherein the RBC level observed in the subject is greater than the subject having a similar disease state but not treated by the method of the invention high. Those skilled in the art will appreciate that one or more other indicator symptoms described herein or known in the art can be measured to determine an effective treatment for a kidney disease in a subject. In another aspect, the invention relates to providing to a subject in need thereof The method of red blood cell constancy. In one embodiment, the method comprises the steps of: (a) administering a population of cells to a subject; and (b) determining a level of symptoms of the erythropoiesis indicator in the biological sample from the subject relative to an indicator in the control Symptom levels are different, wherein the difference in indicator symptom level (i) indicates that the subject responds to administration step (a), or (ii) indicates the subject's red blood cell constancy. In another embodiment, the method comprises the steps of: (4) administering to the subject a composition comprising a population of cells described herein; and 156850. Doc -27· 201211256 (b) Determining the level of symptoms of the red blood cell production index in the biological sample from the subject is different from the index level of the indicator in the control, wherein the difference in the indicator symptom level 1 indicates that the subject responds to Administration step (4), or (9) indicates the red blood cell constence of the subject. In another embodiment, the method comprises the steps of: (4) providing a biomaterial or a biocompatible polymeric scaffold; (b) depositing a population of cells of the invention onto a biomaterial or scaffold or depositing in the manner described herein Biomaterial or scaffold to form an implantable construct; (c) implanting the construct into the subject; and (6) determining the level of symptoms of the red blood cell production indicator from the biological sample from the subject relative to the index in the control Symptom levels are different, with a difference 1 in the indicator symptom level indicating that the subject responds to administration step (8), or (ϋ) indicating the subject's red blood cell constancy. In another aspect, the invention relates to a method of providing renal function stabilization and red blood cell constancy recovery to a subject in need thereof, the subject having renal dysfunction and anemia and/or EPO deficiency. In one embodiment, the method comprises the step of administering a population of cells or a construct comprising the population of cells. In this embodiment, the treatment of the subject is evidenced by an improvement in at least one indicator of renal function accompanied by at least one indicator of red blood cell production by a pre-treatment indicator symptom of the untreated subject or subject. An indicator of improvement in symptoms. In one aspect, the invention provides a method of treating EPO-derived cell populations by administering a fat, (1) treating kidney disease, anemia, or EPO deficiency; (9) stabilizing renal function, (Qi recovering red blood cell constancy, or (iv) any combination thereof, The beneficial effects of administration therein are better or better than the effect of administering a cell population of non-fat-derived EPO-expressing cell populations, or better or better than the effect of not administering a fat-derived cell population. Doc -28 - 201211256 In another embodiment, the adipocyte population provides a modified level of serum blood urea nitrogen (BUN). In another embodiment, the population of adipocytes provides improved retention of protein in serum. In another embodiment, the population of adipocytes provides improved levels of serum cholesterol and/or triglycerides. In another embodiment, the population of adipocytes.  The body provides an improved level of vitamin D. In one embodiment, the population of adipocytes.  The body provides an improved phosphorus to calcium ratio. In another embodiment, the population of adipocytes provides an improved level of hemoglobin. In a further embodiment, the adipocyte population provides improved levels of serum creatinine. In yet another embodiment, the population of adipocytes provides an improved level of hematocrit. In a further embodiment, the population of fatty cells provides an improved level of red blood cells (RBC#). In one embodiment, the improved level of hematocrit is restored to a 95% normal health level. In a further embodiment, the population of adipocytes provides an improved number of reticulocytes. In other embodiments, the population of adipocytes provides an improved percentage of reticulocytes. In still other embodiments, the population of adipocytes provides a modified level of red blood cell distribution width (RDW). In yet another embodiment, the population of adipocytes provides improved levels of hemoglobin. In yet another embodiment, the population of adipocytes provides red, hematopoietic responses in the bone marrow such that the bone marrow cells are nearly normal, and the bone marrow cells: red blood, the ball ratio is near normal. In one other embodiment, the population of adipocytes provides improved blood pressure. In another aspect, the invention provides a cell population exhibiting EPO by administering a fat, (1) treating kidney disease, anemia or EPO deficiency; (ii) stabilizing renal function, (iii) restoring red blood cell constancy, or (iv) any of A combined method wherein the 156850 described herein is administered. Doc -29- 201211256 The beneficial effects of the adipocyte population are characterized by comparable or improved red blood cell constancy compared to the beneficial effects provided by administration of recombinant EPO (rEPO). In one embodiment, the fat-derived EPO-expressing cell population, when administered to a subject in need thereof, provides improved red blood cell constancy (determined by hematocrit, hemoglobin or RBC#) as compared to administration of the recombinant EPO protein. In one embodiment, the adipocyte population provides improved hematocrit, RBC or hemoglobin as compared to recombinant EPO when administered. In a further embodiment, the single dose or delivery administration of the adipocyte population provides an improvement in red blood cell constancy (determined by an increase in hematocrit, hemoglobin or RBC#) in the treated subject, for a significant period of time. The improvement over the single dose of recombinant EPO protein or delivery provides red blood cell constancy for a sustained period of time. In another embodiment, the recombinant EPO is delivered at a dose of about 100 IU/kg, about 200 IU/kg, about 300 IU/kg, about 400 IU/kg, or about 500 IU/kg. Those of ordinary skill in the art will appreciate that other dosage recombination EP0 known in the art may be suitable. Another embodiment of the invention relates to a fat-derived EP0-expressing cell population described herein, or an implantable construct as described herein, for use in the preparation of a kidney disease, anemia or EPO deficiency for use in a subject in need thereof 'Use of a drug that provides red blood cell constancy in a subject in need, or improves renal function in a subject in need thereof. In yet another aspect, the invention provides a method of treating kidney disease in a subject in need thereof, comprising: administering to a subject a fat-derived cell expressing EP0 §¥11 or a cell population comprising a fat-derived EPO-expressing cell Composition of matter. At some 156850. Doc -30- 201211256 In some embodiments, the method comprises determining that the level of symptoms of renal function indicators in the test sample from the subject is different from the level of the indicator in the control, wherein the difference in the indicator symptom level indicates treatment A decrease in one or more renal functions, a stabilization or improvement in one or more renal functions. .  In certain embodiments, the kidney disease to be treated by the method of the invention is accompanied by red blood cells.  The lack of pheromone (EPO). In certain embodiments, the EPO deficiency is anemia. In some embodiments, the EPO deficiency or anemia occurs secondary to renal failure in the subject. In some other embodiments, the EPO deficiency or anemia occurs secondary to a condition selected from the group consisting of chronic renal failure, primary EPO deficiency, chemotherapy or antiviral therapy, non-medullary cancer, HIV infection. , liver disease, heart failure, rheumatoid arthritis or multiple organ system failure. In certain embodiments, the composition for use in the method further comprises a biomaterial comprising one or more biocompatible synthetic polymers and/or naturally occurring proteins or peptides, wherein the population of adipocytes is packaged in the biomaterial Being, deposited onto or in the biological material, captured by the biological material, suspended in the biological material, embedded in the biological material, and/or otherwise combined with the biological material. In certain embodiments, the population of lipid-cells used in the methods of the invention is derived from mammalian adipose tissue or cultured adipose tissue cells. In other embodiments, the population of adipocytes is derived from a kidney sample of the subject in need of the subject. In one embodiment, the sample is a renal adipose tissue biopsy sample. In other embodiments, the population of adipocytes used in the methods of the invention is derived from a sample of non-autologous renal adipose tissue. In yet another aspect, the invention provides a population of adipocytes of the invention or implantable construct 156850. The doc-31-201211256 construct is useful for the preparation of a medicament useful for treating kidney disease, anemia or EPO deficiency in a subject in need thereof. In another aspect, the invention provides a method for regenerating renal function of an autologous kidney in a subject in need thereof. In one embodiment, the method comprises the step of administering or implanting a population or construct of an adipocyte as described herein to the kidney of the subject. Renal function regenerated in the autologous kidney can be characterized by a number of indicator symptoms including, but not limited to, 'function or capacity in the native kidney', improvement in autologous renal function or ability, And the performance of certain markers in the autologous kidney. In one embodiment, the development or improvement of a function or ability can be observed based on the red blood cell constancy and various indicator symptoms of renal function described above. The population of adipocytes described herein, as well as constructs comprising the population of such adipocytes, can be used to provide regenerative effects to the autologous kidney. Regeneration can be characterized by stabilization of one or more indicator symptoms of the renal function (as described herein) and/or recovery of red blood cell constancy (as described herein). 7. Methods and Routes of Administration The adipocyte population and/or construct of the present invention may be administered alone or in combination with other biologically active components. The therapeutically effective amount of the population of adipocytes can be from the maximum number of cells that are safely received by the subject to the minimum number of cells necessary to treat kidney disease (eg, stabilization of one or more renal functions, reduction in rate of decline, or improvement). Variety. In certain embodiments, the methods of the invention provide a medicament for administering a population of adipocytes as described herein 156850. The amount of doc-32-201211256 is about 10,000 cells/kg, about 20,000 cells/kg, about 30,000 cells/kg, about 40,000 cells/kg, about 50,000 cells/kg, about 100,000 cells/kg, about 200,000 cells/kg, about 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/kg, about l. lxlO6 cells / kg, about 1. 2xl06 cells/kg, about 1. 3xl06 cells/kg, about 1. 4χ106 cells/kg, about 1. 5xl06 cells/kg, about 1. 6xl06 cells/kg, about 1. 7xl06 cells/kg, about 1. 8xl06 cells/kg, about 1. 9xl06 cells / kg, about 2. 1xl06 cells / kg, about 2. 1xl06 cells / kg, about 1. 2xl06 cells / kg, about 2 · 3 χ 106 cells / kg, about 2. 4xl06 cells/kg, about 2. 5xl06 cells / kg, about 2. 6xl06 cells/kg, about 2. 7xl06 cells / kg, about 2. 8xl06 cells/kg, about 2. 9xl06 cells/kg, about 3xl06 cells/kg, about 3. 1xl06 cells/kg, about 3. 2xl06 cells / kg, about 3. 3xl06 cells / kg, about 3. 4xl06 cells/kg, about 3_5xl06 cells/kg, about 3. 6xl06 cells/kg, about 3. 7xl06 cells/kg, about 3. 8xl06 cells/kg, about 3. 9xl06 cells/kg, about 4xl06 cells/kg, about 4. 1xl06 cells/kg, about 4. 2xl06 cells / kg, about 4. 3xl06 cells / kg, about 4. 4xl06 cells / kg, about 4. 5xl06 cells / kg, about 4. 6xl06 cells/kg, about 4. 7xl06 cells/kg, about 4. 8xl06 cells / kg, - about 4. 9 x l06 cells/kg, or about 5 x 106 cells/kg. In another embodiment, the cellular dose administered to the subject can be a single dose or a single dose plus another dose. In other embodiments, the dosage can be provided as a construct as described herein. In other embodiments, the cellular dose administered to the subject can be calculated based on the estimated renal quality or functional renal quality. The therapeutically effective amount of the cell population described herein or a mixture thereof can be suspended in pharmacy 156850. Doc -33· 201211256 In an acceptable carrier or excipient. Such carriers include, but are not limited to, basal medium plus 1% serum albumin, saline, buffered saline, dextrose, water, ssogen, alginate, hyaluronic acid, fibrin glue, polyethylene glycol, polyvinyl alcohol , carboxymethyl cellulose and combinations thereof. The formulation should be suitable for the manner of administration. Accordingly, the present invention provides the use of a population of adipocytes for the manufacture of a medicament for treating kidney disease in a subject. In some embodiments, the medicament may further comprise a recombinant polypeptide, such as a growth factor, a chemokine, or a cytokine. In a further embodiment, the medicament comprises a population of human adipose derived cells. The cells used to make the drug can be isolated, derivatized or enriched using any of the variations provided by the methods described herein. The cell preparation, construct or composition is formulated into a pharmaceutical composition suitable for administration to a human according to a conventional protocol. Typically, for example, a composition for intravenous administration, intra-arterial administration or administration in a kidney capsule is a sterile isotonic buffered aqueous solution. If desired, the composition may also include a local anesthetic to relieve any pain at the site of the injection. Typically, the ingredients are provided separately or together in unit dosage form, for example, as an airtight container such as a refrigerated concentrate in an ampoule labeled with an active dose. When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided; whereby the ingredients can be mixed prior to administration. The pharmaceutically acceptable carrier is determined in part by the particular composition to be administered, and by the particular method used to administer the composition. Therefore, there are many suitable preparations for pharmaceutical compositions (see, for example, Alfonso R Gennaro (eds.), Remington: The 156850. Doc -34· 201211256

Science and Practice of Pharmacy, formerly known as Remington's Pharmaceutical Sciences, 20th Edition, Lippincott, Williams & Wilkins, 2003, incorporated herein by reference. Pharmaceutical compositions are typically formulated to be sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practices (GMP) regulations of the US Food and Drug Administration. An aspect of the invention also provides a pharmaceutical formulation comprising a population of adipocytes of the invention and a pharmaceutically acceptable carrier. In some embodiments, the formulation comprises 1〇4 to 1〇9 kidney fat-derived cells. In one aspect, the invention provides a method of providing a population of adipocytes to a subject in need thereof. In one embodiment, the cell population source can be autologous or allogeneic, homologous (homologous or isogenic), and any combination thereof. In situations where the source is not autologous, the method can include administering an immunosuppressive agent. Suitable immunosuppressive agents include, but are not limited to, azathioprine, cyclophosphamide, mizoribine, cyclosporine, tacrolimus hydrate, chlorambucil, chlorpheniric disodium, auranofin, Alprostadil, erolimus hydrochloride, biosynsorb, morozumab, afaset, pentastatin, daclizumab, sirolimus, mycophenolate mofetil, leflunomide, balsam Monoclonal antibody, Streptococcal DNase alpha, • bindarid, cladribine, pimecrolimus, Ilo interleukin, cililizumab, according to law: benzumab, everolimus, anemox, orjamo Monoclonal antibody (gavilimomab), faramolumab 'clofarabine, rapamycin, siplizumab, chaiqin soup, LDP-03, CD4, SR-4355, SK&F_106615, IDEC- 114, IDEC-13 卜 FTY-720, TSK-204, LF-080299, A-8628, A-802715, GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-lOU, 156850.doc • 35- 201211256 ΜΤ-1345 ' CNI-1493 ' CBP-2011 > J-695 > LJP-920 ' L-732531 ^ ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-lg , ER-49890, ER-38925, I SAtx-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 of ordinary skill in the art will be aware of other suitable immunosuppressive agents. The method of treatment of the invention comprises delivering an isolated population of renal adipose-derived cells to an individual. In one embodiment, the cells are preferably administered directly to the site of interest. In one embodiment, a population of cells of the invention is delivered to an individual in a delivery vehicle. In accordance with the present specification, a variety of means for administering cells to a subject will be apparent to those of ordinary skill in the art. This method involves injecting cells into the subject's target. The cells can be inserted into a delivery device or carrier that facilitates introduction into the subject by injection or implantation. In certain embodiments, the delivery vehicle can comprise a natural material. In certain other embodiments, the delivery vehicle can comprise a synthetic material. In one embodiment, the delivery vehicle provides a structure that mimics or properly conforms to the organ configuration. In other embodiments, the delivery vehicle properties are fluid-like. Such a delivery device can include a tube, such as a catheter, for injecting cells and fluid into the body of an intended subject. In a preferred embodiment, the tube additionally has a needle, such as a syringe, through which the cells of the invention can be inserted into the desired site of the subject. In some embodiments, the population of adipocytes is formulated to be administered to a blood vessel via a catheter (wherein the term "catheter" is meant to include the delivery of a substance to a blood vessel 156850.doc -36· 201211256

Any of a variety of tube systems). Alternatively, the cells can be inserted into or inserted into a biomaterial or scaffold including, but not limited to, textiles such as woven fabrics, braids, braids, meshes and nonwovens, perforated films, sponges, and Bubbles, and beads, such as solid or porous beads, microparticles, nanoparticles, etc. (eg, Cultispher-S • Colloidal Beads - Sigma). Cells can be prepared to be delivered in a variety of different forms. For example, cells can be suspended in a solution or gel. The cells can be mixed with a pharmaceutically acceptable carrier or diluent wherein the cells of the invention remain viable. Pharmaceutically acceptable carriers and diluents include saline 'buffered aqueous solutions' solvents and/or dispersion media. The use of such carriers and diluents is known in the art. The solution is preferably sterile and fluid and will generally be isotonic. Preferably, the solution is stable under the conditions of manufacture and storage and is protected from contamination by microorganisms such as bacteria and fungi by the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Those of ordinary skill in the art will appreciate that delivery vehicles for use in the delivery of their cell populations of the invention can include a combination of the above features. The mode of administration of the isolated renal adipose-derived cell population includes, but is not limited to, systemic, intrarenal (e.g., parenchymal), intravenous or intra-arterial injection, and direct injection into the tissue of the intended active site. Additional modes of administration for use in accordance with the present invention include single or multiple injections via direct-laparotomy, via direct laparoscopy, transabdominal, or transdermal. Additional modes of administration for use in accordance with the present invention also include, for example, retrograde infusion and ureteral renal pelvic infusion. Surgical procedures for administration include one-step protocols such as, but not limited to, partial nephrectomy and construct implantation, partial nephrectomy, partial nephrectomy, retinal peritoneal angiogenesis, conus or cone-to-cylinder multifocal biopsy needle tracking , 156850.doc -37- 201211256 and renal pole-like replacement, and a two-step protocol, including, for example, an organoid-internal bioreactor for replantation. In another embodiment, the cellular compositions are delivered separately to a particular site, or simultaneously or in a timed manner by a particular method, by one or more of the methods described herein. Appropriate cell implantation doses in humans can be determined from available information on cellular activity such as EP0 production, or from dose studies conducted in preclinical studies. From in vitro culture and in vivo animal experiments, the amount of cells can be quantified and used to calculate the appropriate dose of implant material. Additionally, the patient can be monitored to determine if additional implantation or material reduction can be performed accordingly. One or more additional ingredients may be added to the cell population, including selected extracellular matrix components, such as one or more types of collagen or hyaluronic acid, and/or growth factors, platelet enriched blood prizes, and drugs known in the art. . Those of ordinary skill in the art will appreciate various formulations and methods of administration suitable for the cell populations described herein. 8. Kits The present invention also encompasses kits comprising any of the following: polymeric matrix and scaffolds of the invention and related materials, and/or cell culture media and instructions for use. Instructions for use may include instructions such as 'culturing cells or administering a population of cells. In one embodiment, the invention provides a kit comprising a scaffold and a flip book as described herein. In a consistent embodiment, the kit includes an agent for detecting the expression of the marker, and a test strip for the face of the drug. Late trials from a group of 156850.doc •38· 201211256 for the purpose of multiple or multiple biomarkers. Kits can also be used to determine the biotherapeutic efficacy of the cell population, mixture or construct described herein. 9. Reports. Lai _ 旙 厅 厅 _ «, the report or description of the characteristics of the squad. The report may include information regarding any defined characteristics of the cell populations described herein. The method and report of the present invention can further include storing the report in a database. Optionally, the method can further generate a record of the subject in the database and fill the record with the data. In one embodiment, the report is a written report. In another embodiment, the report is an auditory report, and in another embodiment the report is an electronic report. Covers the report to physicians and/or patients. Receiving the report can further include establishing a network connection to the server computer containing the data and reports, and requesting information and reports from the server computer. The methods provided by the present invention may also be fully or partially automated. All patents, patent applications and references mentioned in this specification are hereby incorporated by reference in their entirety. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. EXAMPLES Example 1 - Regulation of the regulation of erythropoiesis factor vascular fM cleavage and developmental factors in renal fat 156850.doc -39- 201211256 by deriving from sources derived from kidney and non-kidney SVF In vitro performance of erythropoietin (ΕΡΟ) and vascular endothelial growth factor (VEGF) in cells (SVF cells) amplified from 10% g of Taiwan bovine serum in α-ΜΕΜ medium, we studied from the fatty matrix vascular component (SVF) Whether isolated cells may affect red blood cell production and angiogenesis in vivo. The expression of sputum and VEGF mRNA isolated from SVF cells was regulated by hypoxia. Although the oxygen-regulated EPO manifestations of liver, kidney and brain-derived cells have been documented, it has been reported herein that EPO expression of adipose SVF cells is comparable to that of primary kidney cells or hepatocytes. The isoelectric focusing revealed that the post-translational processing mode of EPO mRNA was different in SVF cells derived from kidney and non-renal fat, and B-sound showed different functions of kidney and non-renal adipose tissue. SVF cells derived from renal fat also specifically express the transcription factor Wilms' tumor 1 (WT1) involved in the development of renal organs during embryogenesis. In summary, these data are consistent with the notion that renal adipose-derived matrix (KiSAS) cells can be triggered to regenerate the regenerative microenvironment in the kidney. These findings open up the possibility of isolating solid-organ-related fat-derived cell populations for therapeutic applications in organ-specific regenerative drug products, and may represent new therapeutic factors for the treatment of EPO-induced reduction of secondary anemia. source. In this paper, we focus our current research on evaluating functional criteria that distinguish stromal cells derived from kidney and non-kidney-derived fats, which are established through analysis of kidney-related regeneration and development markers: erythropoietin (EPO), VEGF and WT1. We demonstrate for the first time that renal adipose tissue represents a reservoir that specifically expresses EPO, and that stromal cell populations derived from kidney and non-kidney-derived fats express EPO and VEGF in a hypoxic manner. Moreover, we show that key nephrosity The expression of the transcription factor WT1 is specific for stromal cells derived from the kidney, and the tick-specific fat reservoir in the kidney can be defined by a differential WT1 transcriptional splice variant. In summary, these data expand the functionally unique, location-specific fat reservoirs from systemic to organ-level and establish the use of kidney-derived fat as tissue engineering and regenerative therapy for the kidney. The basis for replacing cell sources. Materials and Methods Isolation of renal and non-renal fat-derived cells. Human non-renal fat was obtained subcutaneously or via visceral liposuction (Zen-Bio, www.zen-bio.com). Renal fat from the kidney pedicle is passed from the human kidney donated by the National Disease Research Institute (NDRI) according to all NIH regulations governing the use of human tissue for research purposes.

Man) cut it. Renal fat from the kidney sputum is obtained by cutting the intact human kidney and removing the renal pelvis fat from the medulla. Canine kidney used to isolate canine kidney fat was obtained from the University of North Carolina at Chapel Hill (a gift from Dr. Timothy Nichols). Rat kidneys used to isolate rat visceral and organ related fats were derived from male Lewis rats obtained from Charles River Labs. Regardless of species or tissue source, all fat samples were treated as follows: Adipose tissue was thoroughly washed with PBS/0.1% gentamicin, (Invitr〇gen_Gibco), and 0.3% in DMEM-HG (Invitrogen-Gibco) at 37 °C Collagenase I (Worthington), 1% BSA was digested for up to 1 hour. The sample was centrifuged at 600 g for 20 minutes and the supernatant of the adipocyte was aspirated. The remaining vascular components were resuspended in α-ΜΕΜ/10% FBS (Invitrogen-Gibco) and placed in a tissue culture incubator for 24-48 hours. The non-adherent cell population can be removed by washing 3 times with PBS. For experiments involving hypoxia induction, cells were maintained in the 156850.doc •41 · 201211256 〇2-enriched (2%) incubator for the indicated period of time. VEGF jnR^A was used as a control to confirm the integrity of the hypoxic regulatory pathway. Non-adipocytes • Primary renal kidney cells from rat or human kidney are isolated as previously described (Basu et al, Cell Transplant 2011 Mar 24. [Epub ahead of print]; Presnell SC et al., Tissue Eng Part C Methods, Π (3): 261-273, 2011). Human peripheral blood-derived monocytes are isolated as described (Spector, D丄. Cells, a laboratory manual). Cold Spring Harbor Press, 1997> CD34+ GCSF mobilized/non-mobilized peripheral blood mononuclear cells cdnA Available from AllCells LLC. Fetal and adult hepatocyte cDNA and keratinocyte cDNA were purchased from ScienCell Research Laboratories.

TaqMan qRT-PCR • RNA was purified from cell samples using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA was generated from the complete final volume of RNA using the Superscript VILO cDNA Synthesis Kit (Invitrogen-Gibco) according to the manufacturer's instructions. After synthesizing the cDNA, each sample was diluted 1 · 1 直接 and used directly to establish the following qRT-PCR: 10 μΐ master mix (2X ), 1 μΐ primer/probe, 9 μΐ cDNA 〇 below TaqMan primer/probe The group was obtained from Applied Biosystems: rat EPO: Rn01481376_ml, rat VEGF-A: Rn00582935, human EPO: Hs00171267_m, human VEGF-A: Hs00900058_m, human WT-1: Hs01103754_nU. The following primer/probe groups were also obtained: cubulin, NPHSI, E-cadherin, ankle protein, VitD-transferase, and Wnt4. All TaqMan reactions were performed using the preset cycle parameters in the ABI 7300 Instant Thermal Cycler. Analysis of PCR data using comparativeity 156850.doc -42· 201211256

The relative quantification (RQ) method of Ct is carried out. EPO isoelectric point pyrogel analysis • Up to 1 X 106 cells or up to 30 mg total adipose tissue was lysed in protein lysis buffer (50 mM Tris pH 8.0; 150 mM NaCl; 0.5% NP40 and protease inhibitor, Roche). 10 pg of protein lysate was loaded onto an isoelectric point pyrolysis (IEF) gel (Irwitrogen-Gibco) at pH 3-7 and electrophoresed as recommended by the manufacturer. The IEF gel was transferred to nitrocellulose using the iBlot transfer system (Invitrogen-Gibco) with MAB 2871 anti-hEPO monoclonal antibody (R&D Systems) at 1/500 in TBST (Tris-buffered saline, pH 7.0, 0.1 % Tween-20) /2% milk detection overnight. The second antibody was a horse anti-mouse IgG/HRP conjugate (Vector Labs) in 1/60000 in TBST/2% milk. FACs and immuno-fluorescence. Cells (0.5 X 106- 1 X 106 ) were fixed in 2% paraformaldehyde and blocked with Fe receptors to prevent non-specific binding. Cells were permeabilized by incubation in PBS/0.2% Triton X-100/10% horse serum for 30 minutes. The cells were then incubated with a direct conjugated antibody against human WT1 (Santa Cruz) as suggested by the manufacturer. After the last wash (PBS, 0.2% Triton X-100), the Guava EasyCyte micro-performance assay system was used for antigen detection using appropriate fluorescent channels. Obtain at least 5,000-10,000 events from each sample. For immuno-fluorescence analysis, the labeled cell suspension was directly centrifuged to a poly-L-lysine coated slide (Electron Microscopy Sciences) by centrifugation at 1,500 rpm for 5 minutes using a cell centrifugation system (Viescor). on. Cells were counterstained with blocking media (Vector Labs) containing DAPI and visualized using a Leica DMI 4000B fluorescence microscope. Results 156850.doc -43 - 201211256 £P〇 is expressed by a variety of cellular sources. To evaluate the performance of EPO mRNA from fat-derived stromal cells relative to established EPO sources, TaqMan quantitative RT-PCR was performed. Human primary kidney cells were used as calibrators and had a specified rq値 of 1.0. The samples are as follows: H20 as a negative control (1); human primary kidney cells (2); intact fetal liver (3); CD34+ enriched fetal liver cells (4); hepatocytes (5); fourth generation keratinocytes (6) Human skin microvascular endothelial cells (7); human epidermal keratinocytes (8); CDM+, GCSF mobilized peripheral blood-derived monocytes (9); CD34+, normal peripheral blood-derived monocytes (10); (11), the first generation (12), the second generation (13), the fourth generation of fat (fat extract) stromal cells (14); the 0th generation (15), the first generation (16), the second Fat (subcutaneous) stromal cells of generation (17), 3rd generation (18) and 4th generation (19). As shown in Figure 1A, from primary renal cells (cell type 2), fetal liver (cell type 3), adult liver cells (cell type 5), keratinocytes (cell types 8 and 8), and non-mobilized CD34+ PBMNC ( Cell type 10) Strong EPO performance was observed. It was observed that mobilization of CD34+ PBMNC with GCSF resulted in EPO mRNA expression (cell type 9) silence. Observation of multiple stromal cells in non-renal adipose stromal cells such as CD34+ enriched fetal hepatocytes (cell type 4) and passaged fat extracts (cell type 11-14) and subcutaneous (cell type 15-10) adipose stromal cells To a relatively low level of EPO mRNA. The EPO performance of renal and non-renal adipose stromal cells was also evaluated directly relative to renal primary cells. The samples are as follows: primary kidney cells (1 and 2); primary kidney cells exposed to hypoxic conditions (3); third passage fat extracts (4) and subcutaneous (5) stromal cells; first generation fat extracts (6) and subcutaneous (7) stromal cells; first generation of renal sputum-derived renal adipose stromal cells (8); first generation (9) and third generation (10) of pedicle-derived kidney 156850.doc -44 - 201211256 source fat matrix Cells; fetal liver cells were used as a positive control (η); h2 was used as a negative control (12). As shown in Figure 1B, the performance of EPO mRNA from primary kidney cells (cell type 1_3) and non-renal adipose stromal cells (cell types 3-7, 9, 10) was comparable to that observed from primary kidney cells. . The morphological characteristics of renal adipose-derived cells depend on the location of the kidney. We divide the fat from the kidney pedicles derived from the kidneys and the kidneys. Adipocytes derived from the kidney sputum have a unique endothelial morphology compared to adipocytes obtained from the kidney pedicle or from a non-renal source, and this morphology has significantly more fibroblast appearance. The expression of EPO mRNA from renal and non-renal adipose stromal cells is regulated by hypoxia. A key feature of EPO from renal performance is regulation by ambient oxygen (Sytkowski, A.J. Erythropoietin. Blood, brain and beyond. Wiley, 2004, Gleadle, J.M. Nephrology 14, 86, 2006). Based on this standard, experiments were performed to quantify the production of EPO mRNA under conditions of high and low ambient oxygen to determine whether the performance of EPO from adipose cells was physiologically related. The initial study focused on rodents because rats were used to study the small animal models selected by CKD (Ueda et al, Life Sci 84, 853, 2009). · The performance of EPO and VEGF was up-regulated at 4 hours in response to hypoxia, and returned to baseline after 48 hours of normal oxygenation = 3). As shown in Fig. 2A, rat visceral adipose stromal cells responded to hypoxia by up-regulating EPO mRNA expression at 4 hours of treatment. The expression of VEGF mRNA from rat visceral adipose stromal cells was also tightly controlled by hypoxia, up-regulated at 4 hours of treatment, and then down-regulated within 48 hours of normal oxygen return (Fig. 2B). This result is reflected by human KiSAS cells. The EPO in the cells from the two isolates was up-regulated in 8 hours due to hypoxia, and transferred to the baseline containing 156850.doc -45-201211256 for 48 hours of normal oxygen return 0 = 3). As shown in Figures 3A-D, hypoxia-induced upregulation of EPO and VEGF mRNA was observed in adipose stromal cells derived from human kidney pedicles and renal pelvis. The VEGF in the cells from the two isolates was up-regulated in 4 hours due to hypoxia, and returned to baseline within 48 hours of normal oxygenation ("=3). The expression of the EPO protein is comparable between human KiSAS cells and human kidney primary cells. In addition to finding that the expression level of EPO mJRNA in adipose stromal cells is significantly lower than that of primary kidney cells, we also hope to quantify ΕΡ0 from a functional or biotherapeutic perspective by evaluating the performance of EPO proteins between fats from different sources. Therefore, we used the isoelectric point pyrogel (IEF) to study the performance of EPO proteins from adipose-derived stromal cells. The IEF technique has been used to distinguish a number of differently modified ΕΡ0 isoforms with high resolution (Lasne, F. and de Ceaurriz J. Nature 405, 635, 2000). Renal and non-renal adipose stromal cells exhibit different EPO isoforms distinguished by isoelectric focal point gel electrophoresis and western blotting. The samples are as follows: human keratinocytes (1), hepatocytes (2), renal adipose stromal cells (3), non-renal adipose stromal cells (4) and primary renal cells (5), both under normoxic conditions, low Primary kidney cells under oxygen (6), HepG2 cells as a control for impotence (7). The anti-EPO monoclonal antibody was used as a probe for the ink dot for Western analysis. Comparison of electrophoresis lane 5 and sputum showed that the EPO protein expression of primary kidney cell isolates was significantly up-regulated in response to hypoxia. All electrophoresis channels were normalized by the total amount of protein (10 μβ). Figure 4 shows that the EPO protein is directly expressed from human KiSAS cells (electrophoresis 3) and from established EPO cell sources, including keratinocytes (electrophoresis 1), hepatocytes (electrophoresis 2), and primary kidney cells ( The electrophoresis lanes 5, 6) were observed to be comparable to 156850.doc -46 - 201211256 (Bodo et al, FASEB J21, 3346, 2007 'Weidemann, A. and Johnson, RS Kidney Int 75, 682, 2009). EPO is also visceral (non-renal) adipose stromal cells (electrophoresis lane 4), but exhibits a significantly reduced level. Renal and non-renal fat-based cells show different post-translational EP〇 modification patterns' resulting in unique migration on the IEF gel, which can be observed by comparing electrophoresis lane 3 with electrophoresis lane 4. Furthermore, based on isoelectric points, EPO derived from renal or non-renal adipose stromal cells can be further distinguished from EPO expressed by primary kidney cells (comparisons of electrophoresis lanes 3 and 4 with electrophoresis lanes 5 and ό). Finally, the same isoforms expressed by human keratinocytes and hepatocytes (electrophoretic channels 1 and 2) can be distinguished from the same isoforms from all other cell sources. Because dogs are large animal models used to evaluate renal cell therapy selection (Lee et al, Blood Purif 26, 491, 2008), we used EIF gel to examine EPO in canine kidney derived stromal cells and primary kidney cells. The performance of the protein. The samples were as follows: canine primary renal cells under normal oxygen (1) and hypoxia (2), adipose stromal cells derived from renal sputum (3), adipose stromal cells derived from renal pedicle (4), and reconstituted as a positive control. Canine EP0 (5). Anti-EPO monoclonal antibodies were used as blot probes for Westem analysis. All electrophoresis channels were normalized by the total amount of protein (10 gg). Figure 4B demonstrates that canine KiSAS cells derived from kidney sputum fat (Electrical • Lane 3) or Kidney pedicle fat (electrophoresis tract 4 ) exhibit EPO protein levels from canine primary kidney cells (electrophoresis lanes 1 and 2) Observed quite. In addition, in the case of human fat, EPO expressed from stromal cells derived from kidney pedicle or renal pelvis has a unique IEF characteristic that distinguishes between Ep〇 derived from canine fat and EP〇 expressed in canine primary kidney cells. In conclusion, these data confirm that KiSAS cells exhibit an EP prion level comparable to that observed from established EPO cell sources including kidney cells, hepatocytes, and keratinocytes 156850.doc •47– 201211256 and confirm that EPO can be derived from different sources. Identification by isoelectric point spectroscopy (Lasne, F. and de Ceaurriz J. Nature 405, 635, 2000; Bodo et al, FASEB J 21, 3346, 2007; Weidemann, A. and Johnson, RS Kidney Int 75, 682, 2009 ).

Renal adipose tissue is an organ-specific reservoir of EPO. To determine whether the performance of EPO from the adipose stromal cell population reflects the physiologically relevant EPO reservoir in vivo, we examined the EPO protein expression of intact adipose tissue from different sources by IEF. Adipose tissue is a reservoir of cells that can be expressed by EPO electrophoresis and western blotting. White and brown fats are derived from the visceral reservoir. All electrophoresis channels normalize the protein to 1 〇 pg. The table shows the quantitative density analysis of EPO performance, expressed as the band density per unit gel area. As shown in Figure 5, each of the electrophoresis lanes is normalized by the amount of protein loaded. The strong expression of EPO is related to the fat tissue specificity of the kidney. Although EPO can be detected from non-renal organ sources of adipose tissue such as liver and heart, kidney-derived fats are five times more likely to be liver-derived fats, and kidney-derived fats are 2.8 times more abundant than heart-derived fats. Interestingly, the comparison of white and brown fats derived from the visceral fat reservoir showed that EP0 in white fat was five times greater than that of brown fat. The expression of the developmental transcription factor WT1 distinguishes between renal and non-renal adipose stromal cells. We hypothesize that kidney markers other than EPO may be associated with organ-specific performance patterns in fat from different sources. WT1 is a key zinc finger transcription factor' that is extensively involved in organogenesis. WT1 is used to regulate the earliest stages of nephrogenesis as a marker for regeneration (Roberts, SG Curr Opin Genet Dev 15, 542, 2005; Litbarg 156850.doc -48-201211256, et al, Cell Tissue Res. 328, 487, 2007 ; Zhou et al, Nature 454, 109, 2008). We used the specific primers for KTS+ and KTS-transcribed splice variants of WT1 (Hammes et al, Cell 106, 319, 2001) to investigate the performance of WT1 in renal and non-renal adipose stromal cells. As shown in Fig. 6A, the expression of WT1 mRNA was specific to renal adipose stromal cells (electrophoresis lanes 4 and 5), and no expression was observed from visceral fat stromal cells (electrophoresis lanes 2 and 3). Interestingly, the ratio of the two splice variants differs between stromal cells derived from the kidney scorpion fat (electrophoresis channel 4) or the kidney tibia fat (electrophoresis channel 5) derived from the same donor. All electrophoresis channels are normalized to the total amount of cDNA: molecular weight ladders are used for size (1), fat extract stromal cells (electrophoresis channel 2), subcutaneous fat stromal cells (electrophoresis channel 3), renal sputum adipose stromal cells (electrophoresis) Lane 4), renal pedicled stromal cells (electrophoresis tract 5) 〇 WT1 expression was only detected from renal fat. It is noted that the ratio of KTS+/KTS-splice variants differs between the source of renal sputum derived from the same donor and the source of the kidney-derived cells. We evaluated the WT1 RT-PCR analysis by evaluating the expression of WT1 protein in KiSAS cells via FACs and immuno-fluorescence methods. Figure 6B and (: shows that both the renal pedicle and the renal sputum-derived adipose stromal cells have a WT1+ population, from about 45% (kidney pedicle) to 52% (kidney sputum) from the population. 45.6% of the kidney pedicle The adipose stromal cell population is WT1+ 〇52.4% of the renal sputum adipose stromal cell population is WT1+. The expression of WT1 is nuclear and cytoplasmic (Fig. 6D) 'as previously reported (Niksic et al, HumMol Gen 13, 463, 2004). The characterization of 'WT1' is cytoplasmic and nuclear. WT1 (green), DNA (red). Renal adipose-derived cells are characterized by a variety of markers associated with nephrogenesis. By 156850.doc •49· 201211256 by TaqMan QRT- PCR to quantitatively evaluate the performance of other renal markers associated with renal development from renal and non-renal fat-derived cells, we further extended gene expression analysis. Analysis of renal cellulite-derived stromal cells and kidneys derived from the same donor Stromal-derived stromal cells. We found that renal adipose-derived cells express the key progenitor transcription factor WT1. WT1 regulates the performance of GDNF, a secretion that triggers the formation of ureteric buds. Conduction factor (Brodbeck & Englert, Pediatr Nephrol. 2004 Mar; 19(3): 249-55). The performance of WTI-derived cells derived from kidney fat was found to be significantly lower than that observed in primary renal cells. However, at any level there was no The performance of cells derived from non-renal fat was observed. By QRT-PCR, we also found that renal adipose-derived cells express tubular biomarkers, including cubulin, NPHSI, E-cadherin, anthraquinone, VitD hydroxylase and Wnt4°NPHSI are a cell adhesion protein resident in glomerular podocytes (Ruotsalainen et al, Am JPathol 157 (2000): 1905-16). E_fishing adhesion proteins are renal tubular epithelial cells. Marking, the function is to maintain apicobasal polarity (Halbleib & Nelson, Genes Dev. 2006 Dec 1; 20(23): 3199-214). Analysis of E-cadherin and NPHSI expression in non-renal adipocytes, found not to exhibit these Tubulocellular biomarkers.

These studies established a baseline for renal marker expression for the initial attempt to induce tubular phenotype by directed differentiation strategies. To this end, methods for directed differentiation of ancestral populations of embryonic stem cells and adult-derived stem cells toward the renal phenotype have been described. For example, the use of activin-A and retinoic acid is sufficient to induce the expression of multiple early renal development markers in mutated embryoid bodies including WT1, Wnt4 and GDNF (Kim & Dressier, J 156850.doc -50· 201211256

Am Soc Nephrol 16 (2〇05): 3527-34). We observed that the expression of the ankle protein in renal fat-derived cells was up-regulated in response to 1 μ μ of retinoic acid. In addition, the level of glomerular marker ankle protein in non-renal fat-derived cells is increased in response to treatment with bone morphogenetic proteins (BMPs). In this report, we show that kidney-derived fat represents a reservoir that has not been identified to date to produce EPO cells. The downregulation of EPO mRNA observed during normal hypoxia-oxygen conversion was not as tightly regulated as VEGF mRNA showed (Figure 1-3) 'although both VEGF and EPO mRNA were mediated via the same HIF-la/2 (x) The regulatory pathway is responsive to hypoxia (Gleadle, JM Nephrology 14, 86, 2006). This significantly less stringent regulatory control of EPO mRNA may be due to a significantly lower relative performance level of EPO mRNA compared to VEGF mRNA. (Plotkin, MD and Goligorsky, MS Am J Physiol Renal Physiol 291, 902, 2006). The performance of EPO is not evidenced by the human phenomenon produced by cell cultures from observations of renal-derived adipose tissue that is an EPO expression reservoir (figure 5) Interestingly, Figure 5 also shows that although EPO performance from non-renal derived fats is significantly lower than kidney-derived fat, EPO is still a specific marker for distinguishing white and brown adipocytes from visceral-derived fat. As shown in Fig. 4A, the performance of EPO from KiSAS cells is equivalent to that observed in primary kidney cell populations developed from cell therapy for CKD secondary to poor (Aboushwareb, T, etc., World J Uml). 26, 295, 2008) ' implies that kidney-derived fats may represent alternative cell sources for cell carriers for EPO delivery. Changes in isoelectric characteristics of EPO in urine and corresponding serum have previously been documented (Lasne, F et al, Int J Biol) Macromol 41, 354, 2007). Isoelectric isoforms 156850.doc -51 · 201211256 has been used to distinguish between urinary reconstitution and natural EPO in athletes suspected of improper self-medication (Weidemann, A. and Johnson, RS Kidney Int 75) , 682, 2009). Similarly, IEF gel electrophoresis analysis of stromal cell populations derived from kidney and non-kidney reservoirs revealed that this cell type can be functionally distinguished by the unique differences in EPO post-translational modification patterns (Fig. 4A) Moreover, other EPO isoelectric isotypes can be observed in different sources derived from the same organ, such as the difference in EPO manifested by renal sputum or kidney pedicle-derived adipose stromal cells. The IEF signature was confirmed (Fig. 4B). This data led to the belief that the renal sputum and the kidney pedicle represent different fat reservoirs in the kidney. We interpreted the differential EPO of renal and non-renal derived adipose stromal cells. Observations modification patterns enables us WH study may show potential for further differentiate the status of other key renal and non-renal kidney fat reservoir mark. To this end, WT1 is a zinc finger transcription factor that is widely involved in organogenesis. WT1 is used to regulate the earliest stages of nephrogenesis and can be used as a marker for regeneration (Roberts, SG CmrOpin Genet Dev 15, 542, 2005; Litbarg et al, Cell Tissue Res. 328, 487, 2007; Zhou et al, Nature 454, 109 , 2008). The expression of WT1 mRNA was specific for renal adipose stromal cells (Fig. 6A). WT1 mRNA was not detected from visceral-derived fat. The transcriptional regulation of WT1 is complex and has been characterized by a variety of splice variants with distinct biological functions. The KTS+ and KTS-variants of WT1 differ in the presence or absence of three amino acids (KTS) between zinc fingers 3 and 4. Reporting that the ratio of KTS+/KTS-WT1 splice variants is constant across multiple tissues, this balanced operation can trigger severe dysplasia (Hammes, A et al, Cell 106, 319, 2001). It was observed that renal-derived adipose stromal cells (but not non-kidney-derived fat-based 156850.doc-52-201211256 cytoplasmic cells) specifically exhibited EPO expression levels between WT1 and observed adipose stromal cells of renal and non-renal origin. The difference was consistent with the post-translational modification (Fig. 4B). The difference in the ratio of KTS+/KTS-WT1 splice variants in adipose stromal cells from different sources derived from the same organ of the same individual was significant; this difference is in contrast to previous reports (Hammes, A et al, Cell 106, 319, 2001) Conversely, but consistent with the organ and tick specific variability of the EPO IEF features described herein, further confirmation of reservoir-specific functionality in fat is provided. Interestingly, only about 5% of the stromal cell population derived from the renal pelvis or renal pedicle reservoir is WT1+ (Fig. 6B and C), suggesting that there may be two distinct subpopulations of cells in these adipose tissue.

In summary, we show in this report that adipose tissue is a new source of EPO. I have shown that the performance of EPO and VEGF mRNA from fat (kidney and non-kidney sources) is regulated by ambient oxygen, directly comparable to that observed from primary kidney cells or other established EPO sources. Renal and non-renal derived fats have unique functional characteristics, manifested by differences in EPO expression levels and post-translational modifications. These data suggest that kidney and non-renal fat represent fundamentally different fat reservoirs. For this purpose, EPO protein expression of renal and non-renal adipose stromal cells can be distinguished based on the differential migration pattern of IEF gels, which is the result of differences in EPO post-translational modifications between the two cell types. Moreover, KiSAS ^ cells reproduce many additional aspects of the primary renal cell functional phenotype, including the performance of the key progenitor transcription factor WT1. Renal fat may be suitable for the acquisition of tubular function. In this regard, we have observed that established tubular markers in primary cultures of KiSAS cells are induced in response to modulation with known morphogens such as retinoic acid (our unpublished observations). Renal fat may represent a potentially alternative source of alternative cells for the treatment of CKD secondary 156850.doc-53·201211256 chronic anemia, as renal fat can be separated by greater amounts than primary renal cells, as in tubal organs such as Bladder-associated fat is not affected by metastasis in patients with renal cancer (Jenkins, Μ·Α. and Munch, LC Urology 59, 444, 2002; Genheimer, CW, etc., Appl Immunohistochem

Mol Morphol 19, 184, 2011). In summary, the characteristics of renal adipose-derived cells (eg, regulatable EPO and VEGF production, the performance of the key pro-nogenetic transcription factor WT1), suggest that a renal-derived cell population can contribute to therapeutic implantation at the site of injury, Or facilitate the production of a regenerative microenvironment in the diseased kidney. [Simple description of the map]

Figure 1A shows EPO performance of various cell types as determined by TaqMan qRTPCR comparative analysis of human cell and tissue sources. Figure 1B shows that EPO of renal and non-renal derived adipose stromal cells is comparable to primary kidney cells as determined by TaqMan qRT-PCR comparative analysis. Figure 2 shows the modulated expression of EPO (A.) and VEGF (B.) in rat visceral fat-derived cells as determined by TaqMan qRT-PCR analysis. Figure 3 shows the modulated expression of EPO in human kidney pedicle (A) and human renal pelvis (B) adipose stromal cells as determined by TaqMan qRT-PCR analysis, and human kidney pedigree as determined by TaqMan qRT-PCR analysis ( C) and human renal sputum (D) expression of VEGF regulation of adipose stromal cells. Figure 4A shows that EPO of human kidney and non-kidney derived adipose stromal cells is comparable to primary kidney cells, hepatocytes and keratinocytes. Figure 4B shows the performance of EPO in canine kidney-derived lipids 156850.doc -54·201211256 Adipose matrix (KiSAS) cells comparable to canine primary kidney cells. Figure 5 shows the results of EPO protein expression of adipose tissue analyzed by IEF. Figure 6A shows semi-quantitative RT-PCR analysis of WT1 splice variants (KTS+/KTS-) in human kidney and non-kidney derived adipose stromal cells. Figures 6B and C show FACs analysis of WT1+ cell distribution in renal sputum or kidney pedicle-associated adipose stromal cells. Figure 6D shows an immuno-camp analysis of the distribution of WT1+ cells in adipose tissue stromal cells and renal pedicled adipose stromal cells. 156850.doc -55-

Claims (1)

201211256 VII. Scope of Application: 1. An isolated population of fat cells that exhibits erythropoietin (EPO). 2. The cell population of claim 1, wherein the cell population is derived from kidney and adipose tissue. 3. The cell population of claim 1, wherein the cell population is derived from adipose tissue selected from the group consisting of heart fat, liver fat, subcutaneous fat, visceral fat, white fat, brown fat. 4. The cell population of claim 2, wherein the cell population is derived from a renal adipose tissue. 5. The cell population of claim 2, wherein the cell population is derived from a renal adipose tissue. 6. The cell population of any of claims 1-5, which is derived from a fatty matrix vascular component (SVF). 7. The cell population of any one of claims 1-5, which further exhibits vascular endothelial growth factor (VEGF). The cell population of any one of claims 1-5, wherein the EPO expression is a manifestation of hypoxia regulation. 9. The cell population of any of claims 1-5, wherein the VEGF manifestation is a manifestation of hypoxia regulation. 10. The cell population of any of claims 1-5, wherein the EPO transcript is expressed. The cell population of any one of claims 1-5, wherein the VEGF transcript is expressed. 12. The cell population of any of claims 1-5, wherein the EPO polypeptide is expressed. 13. The cell population of any of claims 1-5, wherein the EPO transcript and the EPO polypeptide are expressed. 14. The cell population of claim 1 or 2, wherein the population of cells comprises a post-translationally modified EPO polypeptide that is different from the non-fat cell population. 15. The cell population of claim 14, wherein the non-fat cell population is selected from the group consisting of keratinocytes, hepatocytes, and primary kidney cells. 16. The cell population of claim 2, wherein the population of cells comprises a post-translationally modified EPO polypeptide that is different from the non-renal adipocyte population. The cell population of claim 16, wherein the non-renal cell population is selected from the group consisting of keratinocytes, hepatocytes, visceral fat stromal cells, and primary kidney cells. 18. The cell population of any one of claims 2, 4, and 5, wherein the cell population differentially exhibits a biomarker associated with kidney regeneration. 19. The cell population of claim 18, wherein the differential performance is an increased performance. 20. The cell population of claim 18, wherein the biomarker is WT-1 ° 21. The cell population as described in claim 20, wherein WT-1 156850.doc 201211256 transcription is expressed this. 22. The cell population of claim 21, wherein the WT-1 transcript is WT-1 KTS+. 23. The cell population of claim 21, wherein the \\^-1 transcript is WT-1 KTS. 24. 24. The cell population of claim 21, wherein the WT The -1 transcript is WT-1 KTS+ and WT-1 KTS, 25. The cell population as described in claim 18, wherein the biomarker is a WT-1 polypeptide. 26. A method of preparing a population of adipose stromal cells exhibiting erythropoietin (EPO), the method comprising: a) digesting adipose tissue; and b) consuming digested adipocyte tissue to provide a stromal vascular component (SVF), wherein The SVF includes the population of adipose stromal cells that exhibit EPO. 27. The method of claim 26, wherein the EPO-expressing cell of a stromal cell is a cell, population as described in any one of claims 1 to 25. 28. An implantable construct that provides improved renal function to a subject in need thereof, the implantable construct comprising: a) a biocompatible matrix; and b) deposited onto the surface of the substrate or packaged A population of adipocytes expressing erythropoietin (EPO) buried in the surface of the matrix. The implantable construct of claim 28, wherein the cell population is a cell population as described in any one of claims 1 to 25. 30. Use of a composition for the manufacture of a medicament for the treatment of kidney disease, wherein the composition comprises a population of adipocytes expressing erythropoietin (EPO). The use of the cell of claim 1, wherein the cell population is a cell population as described in any one of claims 1 to 25. 32. Use of an implantable construct for the manufacture of a medicament for treating kidney disease, wherein the implantable construct comprises: a) a biocompatible matrix 'and b) deposited onto the surface of the substrate or embedded A population of adipocytes expressing erythropoietin (EPO) into the surface of the matrix. 33. The use of claim 32, wherein the cell population is a cell population as described in any one of claims 1 to 25. 156850.doc