TWI654983B - Adipose tissue-derived stem cells for veterinary use - Google Patents

Abstract

The invention provides compositions and methods for making and using fat-derived stem cells for treating various medical conditions in non-human mammals. In particular, the present invention provides methods and compositions useful for repairing fractures and treating "dry eye" conditions, acute renal failure, and chronic renal failure in non-human mammals.

Description

Animal Adipose Tissue-Derived Stem Cells Cross-reference to related applications

This is a partial continuation of US Application 12 / 997,067, filed on February 14, 2011. This US application is the national phase of International Application No. PCT / US2009 / 046587, filed on June 8, 2009. The application claims the benefits of US Provisional Application No. 61 / 060,701 filed on June 11, 2008 and US Provisional Application No. 61 / 168,148 filed on April 9, 2009 under 35 USC§ 119 (e), which The content of each is incorporated herein by reference in its entirety.

The present invention provides compositions and methods for making and using fat-derived stem cells for treating various medical conditions in non-human mammals, including medical fractures, "dry eye conditions", chronic renal failure, and acute renal failure. .

Adipose tissue contains stromal vascular components (SVF) from which pluripotent cells have been isolated. These cells have various names: processed lipoaspirate (PLA) cells, adipose tissue-derived mesenchymal stem cells, pluripotent adipose-derived stem (MADS) cells, adipose tissue-derived stem cells, adipose tissue-derived stromal cells ( ADSC, ATSC), adipose tissue-derived adult stem (ADAS) cells, adipose tissue-derived adult matrix (ADAS) cells, and adipose tissue-derived cells (ADC).

ADSCs have similar phenotypes and genetic expressions as those of bone marrow stem cells (BMSCs) Atlas. In addition to its ability to self-renew and grow long-term, ADSC can differentiate into different cell types, including adipocytes, osteoblasts, chondrocytes, liver cells, muscle cells, cardiac muscle cells, neurons and epithelial cells. Therefore, ADSC is not only more and more recognized as true adult stem cells, but also considered to be superior to other types of adult stem cells for future clinical applications. However, due to the incidence of donor bone marrow, only an effective amount can be obtained, but adipose tissue is usually available in large quantities, especially in an increasingly obese society. In addition, adult studies have determined that the number of BMSCs in the bone marrow is approximately 1 in 25,000 to 1 in 100,000, and the average frequency of ADSCs in processed lipoaspirates is approximately 2% of nucleated cells. Therefore, the yield of ADSC from about 1 g of fat is about 5000 cells, while the yield of BMSC is about 100 to 1000 cells per ml of bone marrow.

Previous attempts have been made to use ADSC for therapeutic purposes. See, for example, WO 2005/035742. However, these attempts have mainly focused on the autologous use of ADSCs in animal environments. For animal clinics, autologous treatment programs are too time consuming for both veterinarians and pet owners. There is a need in the industry for compositions and methods for treating non-human mammals (such as pets and farm animals) in a manner that does not cause rejection of the transplanted cells. The invention described herein provides these advantages and also provides other benefits.

All patents, patent applications, references, and other publications disclosed herein are incorporated herein by reference in their entirety.

The invention provides compositions and methods for making and using adipose tissue-derived stem cells (ADSC). In one aspect, the invention provides methods for treating various medical conditions in non-human mammals. In one aspect, the invention provides a method of treating urinary incontinence in a non-human mammal, the method comprising administering to a non-human mammal an effective amount of adipose tissue-derived stem cells (ADSC). Non-human mammals may also have secondary symptoms, where the secondary symptoms are bladder infection or urinary blisters or both bladder infection and urinary blisters.

In another aspect of the invention, the invention provides treatment of inflammation in a non-human mammal Methods for sexually transmitted diseases including administering an effective amount of adipose tissue-derived stem cells (ADSC) to a non-human mammal. In one embodiment, the inflammatory disease is arthritis. In another embodiment, the arthritis is osteoarthritis.

In another aspect of the invention, the invention provides methods for reducing pain in a non-human mammal in need, the methods comprising administering to a non-human mammal an effective amount of adipose tissue-derived stem cells (ADSC).

In another aspect of the invention, the invention provides a method of treating diabetes in a non-human mammal in need, the method comprising administering to a non-human mammal an effective amount of adipose tissue-derived stem cells (ADSC).

In another aspect of the invention, the invention provides a method of treating a medical condition of a non-human mammal in need, the method comprising administering to a non-human mammal an effective amount of adipose tissue-derived stem cells (ADSC) Wherein the medical condition is one or more conditions selected from the group consisting of: urinary incontinence, osteoarthritis, degenerative myelopathy, diabetes, tissue regeneration, wound healing, scarring, soft tissue defects, fecal incontinence, dilation Cardiomyopathy, hip dysplasia, avascular necrosis of the femoral head, ligament injury, tendon injury, spinal cord injury, atherosclerosis-related infarction, arthritis, muscular dystrophy, fracture, "dry eye" symptoms, chronic renal failure And acute renal failure.

The present invention provides treatment or alleviation of a condition discussed in a method of any of the foregoing, wherein ADSC is heterologous to a non-human mammal. In one aspect, the treatment is a xenograft, and rejection or inflammation with ADSC treatment is extremely low. In any of the above methods and herein, a non-human mammal can be any of the following non-limiting examples: dog, cat, horse, rabbit, pig, monkey, baboon, chimpanzee, orangutan, tiger, lion, bear, Cheetah and Llama.

In another aspect, the invention provides a set of non-human ADSCs for treating medical conditions, wherein the ADSCs are heterologous to a recipient of the ADSC, and wherein the medical condition is one or more selected from the group consisting of Symptoms of the following groups: urinary incontinence, bones and joints Inflammation, degenerative myelopathy, diabetes, tissue regeneration, wound healing, scarring, soft tissue defects, fecal incontinence, dilated cardiomyopathy, hip dysgenesis, avascular necrosis of the femoral head, ligament injury, tendon injury, spinal cord injury, arteries Atherosclerosis-related infarction, arthritis, muscular dystrophy, fractures, "dry eye" symptoms, chronic renal failure, and acute renal failure.

In another aspect, the invention provides a set of non-human ADSCs for treating medical conditions, wherein the ADSCs are homologous to a recipient of the ADSC, and wherein the medical condition is one or more selected from the group consisting of Symptoms of the following groups: urinary incontinence, osteoarthritis, degenerative myelopathy, diabetes, tissue regeneration, wound healing, scarring, soft tissue defects, fecal incontinence, dilated cardiomyopathy, hip dysgenesis, femoral head absence Vascular necrosis, ligament injury, tendon injury, spinal cord injury, atherosclerosis-related infarction, arthritis, muscular dystrophy, fractures, "dry eye" conditions, chronic renal failure and acute renal failure. Optionally, ADSCs of any of the above groups may include carboxymethyl cellulose (CMC).

In another aspect, the invention provides a composition comprising a purified non-human ADSC population and CMC for transplantation. In one aspect, the transplant is a xenograft, wherein the xenograft portion causes any significant rejection of the composition.

Figure 1 shows the results of experiments on the production of insulin-producing cells. Human ADSCs were transduced with GFP (control), Pdx1, or Pdx1-PV-16 (PV-16). Phase contrast microscopy showed that Pdx1- and PV-16 transduced cells had insulin-like particles in the cytoplasm and culture medium. Immunofluorescence microscopy showed that insulin staining of Pdx1- and PV-16 transduced cells was positive. Cells were located using DAPI nuclear staining.

Figure 2 shows the results of experiments to verify Pdx1 and insulin performance. Human and rat ADSCs were transduced with GFP (control), Pdx1, or Pdx1-PV-16 (PV-16). The performance of Pdx1 in these cells was examined by western blotting (with β-actin as a control, panel A) and RT-PCR (panel B). Human ADSC was further examined for static insulin production by ELISA (in DMEM with 23 mM glucose, panel C).

Figure 3 shows the results of an experiment to examine the expression of genes in the pancreas. Human and rat ADSCs are not transduced (C) by GFP or Pdx1. RT-PCR was used to examine the performance of Pdx1, insulin, glucagon, and NeuroD in these cells and rat urethral smooth muscle cells (RUSMC) (with b-actin as a control, panel A). The statistical analysis of the results of human and rat cells is presented in Figure B (n = 3) and Figure C (n = 5), respectively. An asterisk indicates a significant difference between Pdx1 transduced cells and untransduced cells (P <0.05).

Figure 4 shows the results of experiments on insulin production in response to glucose concentration. Pdx1 transduced cells were grown in buffers containing the indicated concentrations of glucose. After 1 hour, the amount of insulin in the buffer was evaluated by ELISA. Asterisks indicate a significant difference compared to insulin production at 0 mM glucose (P <0.05).

Figure 5 shows the results of an experiment on changes in blood glucose levels (Figure A) and body weight (Figure B). Thirty rats were randomly divided into three groups. The first group (control) received an intraperitoneal injection of 20 mM citrate buffer. Both the second and third groups received an intraperitoneal injection of 60 mg STZ (in 20 mM citrate buffer) / kg body weight. After 1 week, the second group (saline) was treated with saline, while the third group (IPADSC) was treated with IPADSC. All rats were monitored weekly for weight and fasting blood glucose levels. Asterisks indicate a significant difference between IPADSC-treated and saline-treated rats (P <0.05).

Figure 6 shows the changes in fur appearance and cataract levels in rats.

Figure 7 shows the results of experiments on glucose tolerance. At the end of the 7th week after treatment, rats fasted for 7 hours received an intraperitoneal injection of 1 mg glucose / gram body weight. Blood glucose levels in the samples obtained from the tail vein were then monitored at 2-hour, 30-minute intervals. Asterisks indicate a significant difference between IPADSC-treated and saline-treated rats (P <0.05).

Figure 8 shows the recognition of transplanted cells. At the end of week 7 after treatment, rats were sacrificed and their kidneys were collected for histological examination. The presence of transplanted cells in the subcapsular space was examined using HE staining. Immunofluorescence (IF) staining was used to identify cells expressing insulin. The boxed area in the 20 × photograph is the injection site and is enlarged in each 100 × photograph. 100 × Photo in Chinese and Canadian The area of the frame is further enlarged in each 400 × photograph. Note that IPADSC treats tissue-like structures in the subcapsular space of the kidney. This structure was not seen in saline-treated kidneys. IF photographs were obtained from 3 IPADSC treated kidneys. Note the presence of insulin-positive cells.

Figure 9 illustrates the dose effect. EdU was added to ADSC at 0 μM, 10 μM, 20 μM, and 50 μM. Cell locations are identified by DAPI staining of the nucleus (if color photographs are used, they will be blue). EdU is detected by Alexa 594 (if color photos are used, it will be red). The results showed that approximately 50% of the cells were labeled with EdU (A, × 200), regardless of EdU concentration (B, P> 0.05).

Figure 10 shows the results of the time history study. ADSC was labeled with 10 μM EdU and then split at 1 day, 4 days, 7 days, 14 days, and 21 days. The results showed that the EdU signal in positively labeled cells decreased with time (A, × 100; B, P <0.01). Cell lines with greater magnification (1000 ×) on day 21 are shown in Figure C.

Figure 11 shows the results of labeling DNA in vivo in tissue arrays by using EdU. Neonatal rats were injected intraperitoneally with 50 μg of EdU / g body weight in PBS. After 7 h, the main tissues were collected for preparation of tissue arrays (Table 3), and then HE stained (A) or EdU stained (B). The EdU stained tissue array is shown in Figure C at 20 × magnification. EdU-labeled skin samples are shown in Figure D at 200 × magnification (Figure 6A). Cell locations are identified by DAPI staining of the nucleus (if color photographs are used, they will be blue). EdU is detected by Alexa 594 (if color photos are used, it will be red).

Figure 12 shows the results of tracking the transplanted ADSC. Rat ADSCs were labeled with 10 μM EdU for 12 hours and then autologously injected into the bladder neck (A). After 4 weeks, by targeting EdU (which will be red if color photographs are used), α-smooth muscle actin (SMA, which will be green if color photographs are used), and nuclei (if color photographs are used, then It will be blue) Examine the tissue section. EdU-labeled cells can be observed in the bladder neck (B, x 20). Most EdU labeled cells are localized in connective tissue. A few EdU-labeled cells appeared to have differentiated into smooth muscle cells (C, x200).

Figure 13 shows the results of experiments on higher urination pressure in ADSC treated animals.

Figure 14 shows the results of co-localization of EdU and SMA. If you observe color images, the red signal is EdU, the green signal is ASMA, and the blue signal is DAPI. The boxed area in each image in the image above is shown in the corresponding image in the image below (× 400).

FIG. 15 illustrates elastic fibers in the urethra. Left: control. Right: ADSC transplantation (× 400).

Figure 16 shows the results of experiments on higher urination pressure in ADSC treated animals.

Figure 17 shows an experiment on co-localization of EdU and SMA. If you observe color images, the red signal is EdU, the green signal is ASMA, and the blue signal is DAPI. (a ~ c) EdU positive cells localized in the submucosa. (d ~ h) EdU-positive cells (× 200) localized in the muscle. (A) EdU positive cells (× 400) in the submucosa. (B) EdU-positive cells in muscle (× 400).

Figure 18 shows the results of experiments on the endothelial differentiation of ADSCs in the penis. ADSCs were labeled with BrdU and injected into the corpora cavernosa of rats. After 4 weeks, the tissues were examined by immunofluorescence microscopy. Anti-BrdU and RECA-1 antibodies recognize the injected ADSC (which will be green if using a color photograph) and endothelial cells (which will be red if using a color photograph), respectively. The superimposed image (BrdU / RECA) showed that some ADSCs (which would be yellow if color photographs were used) were also positive for RECA-1 staining. Another superimposed image (combined) using phase contrast images shows ADSC localization to sinusoidal endothelial cells. About 5% of BrdU + cells are RECA +, as determined by counting 10 randomly selected regions in the profile.

Figure 19 shows a comparison of cell morphology and growth rate in DMEM and EGM2. Two rat ADSC lines RADSC-1 and RADSC-2 were inoculated into 100 mm petri dishes at the same density (300,000 cells / dish) and grown in DMEM or EGM2 for 3 days. The cell morphology of RADSC-1 is shown in Figure A. The growth rates of the two cell lines are shown in Figure B.

Figure 20 shows experiments identifying the performance of endothelial markers in cells growing in EGM2 the result of. RADSC-1 and RADSC-2 were grown in DMEM (uninduced) or EGM2 (induced). Endothelial markers CD31, vWF and eNOS were then stained. If color images are observed, green indicates the performance of CD31, vWF or eNOS; blue indicates the nucleus. The original magnification is 200 ×. Matrigel formation (tubes) and LDL uptake of the cells were also analyzed. Red indicates the presence of LDL, which is in a coupled form with the red fluorescent dye DiI. Results for the two cell lines are similar; only RADSC-1 are shown. Human umbilical vein endothelial cells (HUVEC) served as a positive control. The experiment was repeated 3 times.

Figure 21 shows some results illustrating the reversibility and re-inducibility of endothelial differentiation. RADSC-1 and RADSC-2 were grown in DMEM for 6-10 days (DMEM first round); endothelial markers were analyzed for half of the cells. The other half of the cells were switched to EGM2, grown for 6 days (first round of EGM2), and the endothelial markers of half of the cells were analyzed. The other half of the cells were switched to DMEM, grown for 10 days (second round of DMEM), and the endothelial markers of half of the cells were analyzed. Finally, the other half of the cells were switched to EGM2, grown for 6 days (second round of EGM2), and analyzed for endothelial markers. Results for the two cell lines are similar; only RADSC-1 are shown. If color images are observed, green indicates the performance of CD31, vWF or eNOS; and blue indicates the nucleus. The original magnification is 200 ×. Red indicates the presence of LDL, which is in a coupled form with the red fluorescent dye DiI. The experiment was repeated 3 times.

Figure 22 shows the results of an experiment on the identification of endothelial inducers by "subtraction". RADSC-1 cells were grown in fully or partially supplemented EGM2 and then analyzed for LDL uptake. Each part of the supplemented EGM2 is indicated by an omitted factor; for example, "-FGF" means EGM2 without FGF2. The experiment was repeated 3 times.

Figure 23 shows the results of an experiment on the identification of endothelial inducing factors by "addition". RADSC-1 cells were grown in EGM2, EBM2 or EBM2 supplemented with the indicated factors, and then analyzed for LDL uptake. The experiment was repeated 3 times.

Figure 24 shows the results of an experiment on the expression of endothelial markers by FGF2. Make RADSC-1 cells in DMEM, EGM2 or EBM2 supplemented with FGF2 and Vitamin C Grow. Endothelial markers CD31, vWF and eNOS were then stained. If color images are observed, green indicates the performance of CD31, vWF or eNOS; and blue indicates the nucleus. The original magnification is 200 ×. Matrigel formation (tubes) of the cells were also analyzed. The experiment was repeated 3 times.

Figure 25 shows the results of experiments on the effect of FGFR inhibitors on endothelial differentiation. RADSC-1 cells are grown in EGM2 or EBM2 supplemented with FGF2 and vitamin C in the presence or absence of the FGFR inhibitor PD173074. The cells were then analyzed for LDL uptake. If color images are observed, red indicates the presence of LDL, which is in a coupled form with the red fluorescent dye DiI. For the purpose of excluding the involvement of VEGF signaling, RADSC-1 cells are also grown in EBM2 supplemented with VEGF / vitamin C, because PD173074 is known to have a weak inhibitory effect on VEGF receptors. The experiment was repeated 3 times.

Figure 26 shows the results of treating canine fractures with porcine ADSC. Figure A provides X-rays (day 0) of the fractured radius and ulnar bone of a dog 1 week after removal of 2 of 5 screws that have been used to attach a dynamic compression plate to the fractured radius. X-rays have revealed severe decalcification and thinning (reduction of bone mass) of the radius at the fracture site below the plate. On the same day ("Day 0"), 5 × 10 ⁶ pig ADSCs were administered subcutaneously to the inside and outside of the plate in a volume of 0.5 mL, and an additional 1 × 10 ⁶ pig ADSCs were administered intravenously to the animal. . Subsequent X-ray exposures were performed 17 days (Figure B) and 26 days (Figure C) after pig ADSC administration, and the decalcification effect of the radius was reversed and the fracture site healed.

The present invention provides compositions and methods for treating various conditions of non-human mammals and alleviating the discomfort and / or pain associated with various conditions of non-human mammals.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

If the definitions described in this section are inconsistent or inconsistent with those described in patents, published patent applications, and other publications incorporated herein by reference, The references are incorporated into the definitions herein and the definitions described in this section shall prevail.

I. General technology

Unless otherwise indicated, the practice of the present invention will make use of conventional techniques in stem cell biology, cell culture, molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology. Well known. These techniques are fully explained in scientific literature, such as Molecular Cloning: A Laboratory Manual , Third Edition (Sambrook et al., 2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (edited by P. Herdewijn, 2004); Animal Cell Culture (RIFreshney) Editor, 1987); Methods in Enzymology (Academic Press Co., Ltd.); Handbook of Experimental Immunology (edited by DMWeir and CCBlackwell); Gene Transfer Vectors for Mammalian Cells (edited by JMMiller and MPCalos, 1987); Current Protocols in Molecular Biology (FMAusubel et al.) Edit, 1987); PCR: The Polymerase Chain Reaction , (Edited by Mullis et al., 1994); Current Protocols in Immunology (Edited by JEColigan et al., 1991) Short Protocols in Molecular Biology (Wiley and Sons, 1999); Embryonic Stem Cells: A Practical Approach (edited by Notaranni et al., Oxford University Press 2006); Essentials of Stem Cell Biology (edited by R. Lanza, Elsevier Academic Press 2006); Stem Cell Assays (Methods in Molecular Biology) (edited by Mohan C. Vemuri, Humana Press) ;the first (October 2007 August); Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Darwin J.Prockop, Donald G.Phinney, Bruce A.Bunnell edited the first edition (2008, March 7) ); Handbook of Stem Cells (edited by Robert Lanza et al., Academic Press (September 14, 2004); Stem Cell Culture Vol 86: Methods in Cell Biology (edited by Jennie P. Mather, Academic Press, first edition (May 15 (2008)); Practical Hematopoietic Stem Cell Transplantation (edited by Andrew J. Cant et al., Wiley-Blackwell, first edition (January 22, 2007)); Hematopoietic Stem Cell Protocols (edited by Kevin D. Bunting, Humana Press, Second Edition (January 31, 2008)); Bone Marrow and Stem Cell Transplantation (Methods in Molecular Medicine) (Edited by Meral Beksac, Humana Press; First Edition (May 3, 2007)); Stem Cell Therapy and Tissue Engineering for Cardiovascular Repair: From Basic Research to Clinical Applications (edited by Nabil Dib et al., Springer, first edition (November 16, 2005)); Blood And Marrow Stem Cell Transplanta tion: Principles, Practice, And Nursing Insights (Kim Schmit-Pokorny (author) and Susan Ezzone (editor), Jones & Bartlett Publishers; third edition (May 22, 2006)); Hematopoietic Stem Cell Protocols (Christopher A. Edited by Klug and Craig T. Jordan, Humana Press; first edition (December 15, 2001); and Clinical Bone Marrow and Blood Stem Cell Transplantation (edited by Kerry Atkinson et al., Cambridge University Press; third edition (2003) December 8)).

II. Definition

"Adipose-derived stem cells", "Adipose tissue-derived stem cells" and ADSC are used interchangeably herein and refer to pluripotent stromal cells or stem cells derived from adipose tissue and capable of self-renewal. "Fat" means any fatty tissue. Adipose tissue can be brown or white adipose tissue, derived from subcutaneous, omental / visceral, breast, gonadal, or other adipose tissue sites. Preferably, the fat is subcutaneous white adipose tissue. The cells may include primary cell cultures or immortal cell lines. The adipose tissue may be from any non-human mammal having adipose tissue. Adipose tissue can be derived from a non-human mammal (i.e., autologous tissue) or the pure line of the individual to be treated, or from a different species (i.e., a heterologous) of a non-human mammal, or from the same species of a non-human mammal but Non-human mammals (ie, same species) that are not to be treated. These cells display a unique combination of cell surface proteins, which may include (but are not limited to) stem cell markers CD 34 and CD 90, tetra-penetrating protein CD9, CALLA (CD10), amine peptidase N (CD13), integrin 1 ( CD29), hyaluronate receptor (CD44), integrin.α.4 and 5 (CD49d, CD49e), ICAM-1 (CD54), decay acceleration factor (CD55), complementary protective factor (CD59), endothelial factor (CD105), VCAM-1 (CD106), Muc-1,8 (CD146), and ALCAM (CD166) (Gronthos et al., J. Cell Physiol. (2001) October; 189 (1): 54 63).

In one aspect, ADSCs derived from porcine families are usually positive for CD90, CD44, CD29 and usually negative for CD31, CD45, and CD11. For example, see Valina et al., European Heart Journal 28: 2667-77 (2007), which reveals that pig ADSCs cultured against CD90 (97.3 + 0.62%), CD44 (98.27 + 0.38%), and CD29 (98.2 + 0.87%) It was positive for CD31 (0.03 + 0.05%), CD45 (0.45 + 0.41%), and CD11 (0.17 + 0.17%).

The term "potential" or "purified formulation" of the multipotent or pluripotent ADSC as used herein refers to a formulation containing one or more cells that is manipulated to provide a cellular formulation that is substantially free of other components. In some aspects, at least about 60% of the cell preparation by weight or amount is free of other components present when the cells are produced. In various aspects, the cells are at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, by weight or number, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% pure. Purified cell preparations can be obtained, for example, by purification (e.g., extraction) from natural sources, fluorescence-activated cell sorting, or other techniques known to those skilled in the art. Purity can be analyzed by any suitable method, such as fluorescence activated cell sorting (FACS), or by visual inspection.

"Purity" used to describe the purity of stem cells does not mean that only stem cells are present in the composition, but indicates that the stem cells have been manipulated so that they have been removed from their natural tissue environment and that it is related to other cells present in the resulting population.

The term "multipotent" or "pluripotent" as used herein means Potential for ADSCs to differentiate into three germ cells: endoderm (e.g., internal gastric mucosa, gastrointestinal tract, lungs), mesoderm (e.g., muscle, bone, blood, urogenital) or ectoderm (e.g., epidermal tissue and nerves) system). Pluripotent stem cells can produce any fetal or adult cell type. It alone cannot develop into a fetus or an adult animal because it does not have the potential to contribute to extraembryonic tissue (eg, placenta in vivo or germ layer in vitro).

"Non-human mammals" include, but are not limited to, farm animals, sport animals, pets, non-human primates, mice and rats. Farm animals can include, but are not limited to, pigs, cattle, horses, goats, and sheep. Pets include, but are not limited to, dogs, cats, rabbits, and ferrets. Other animals within this definition include, but are not limited to, monkeys, baboons, chimpanzees, orangutans, tigers, lions, bears, cheetahs and llamas. Non-human mammals may also be referred to herein as "individuals."

"Treatment" or "treating" means a method of obtaining beneficial or desired results, including clinical results (which includes an animal clinic or hospital). For the purposes of the present invention, beneficial or desired results include, but are not limited to, alleviating the symptoms associated with the condition, reducing the extent of the symptoms associated with the condition, preventing the deterioration of the symptoms associated with the condition, or delaying the development of the disease or condition . In some aspects, the use of one or more of the cell treatments disclosed herein does not accompany any side effect or is accompanied by fewer side effects than the side effects associated with currently available therapies.

"Receiving treatment" includes initial treatment and / or continuous treatment. As used herein, "treatment" is a method of obtaining beneficial or desired results, and the results preferably include clinical results from treatments in animal clinics or hospitals. For the purposes of the present invention, beneficial or desirable clinical results include, but are not limited to, stabilizing or improving the health-related quality of life of non-human mammals suffering from various medical conditions.

As used herein, "delaying" the development of a disease or condition means delaying, hindering, slowing, preventing, stabilizing, and / or delaying the development of a disease or condition. The length of this delay varies depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, Adequate or significant delay may actually cover prevention because the individual does not have the disease or condition. For example, when compared to not using the method, the method may reduce the probability of disease development and / or reduce the degree of disease in a given period of time. In some aspects, the comparisons are based on clinical studies using a statistically significant number of individuals. Disease development can be detected using standard clinical techniques. Development can also refer to the progression of a disease that was initially undetectable and includes the occurrence, recurrence, and onset.

"Reducing" pain (e.g., pain associated with an inflammatory disease, such as a type of arthritis) or one or more symptoms of pain means reducing one or more of the pain in a non-human mammal treated with the ADSC composition of the present invention The degree to which clinical manifestations are expected.

An "effective amount" (when used in the context of treatment or prevention or in the context of reducing pain or alleviating the symptoms of a particular condition) is an amount sufficient to achieve a beneficial or desired result, including clinical results. An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of ADSC is a cell that quantifies one or more symptoms of a condition treated in a non-human mammal. For example, a reduction in the limpness of a non-human mammal that has been administered ADSC is a symptom of arthritis in a non-human animal and the observation of this reduction in this particular symptom may mean that an effective amount of ADSC has been administered to the non-human mammal. In one aspect, ADSCs are further cultured to induce their differentiation along specific pathways. In another aspect, ADSCs are cultured in a manner that does not cause any differentiation.

As used herein, "requiring it" includes a non-human mammal having a disease or disease or "at risk for that disease or disease". As used herein, a "at risk" non-human mammal is a non-human mammal at risk of developing a disease. Non-human mammals that are "at risk" may or may not have a detectable disease or condition, and may or may not have the detectable disease indicated before the treatment methods described herein. "At risk" means that a non-human mammal has one or more so-called risk factors, which are measurable parameters associated with the development of a disease or condition and are known in the industry. Non-human mammals with one or more of these risk factors are more likely to develop diseases or conditions than those without Individuals of these risk factors. These risk factors include, but are not limited to, age, gender, diet, previous disease history, presence of precursor disease, genetic (ie, hereditary) factors, reproduction protocols and factors, and environmental exposure.

"Pharmaceutically acceptable carrier" means based on the knowledge of those skilled in the art that when combined with an active ingredient allows the ingredient to remain biologically active without causing an unacceptable immune response (e.g., severe allergies or anaphylactic shock ). Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as carboxymethyl cellulose (CMC), phosphate buffered saline, water, emulsions (such as oil / water emulsions), and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate buffered saline or physiological saline (0.9%). An exemplary vehicle for cell infusion is CMC. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences , 18th edition, edited by A. Gennaro, Mack Publishing, Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th edition. Mack Publishing, 2000, each of which is incorporated herein by reference in its entirety, in particular with regard to formulations).

General references to "the composition" or "compositions" include and apply to the compositions of the present invention. The invention also provides pharmaceutical compositions comprising the components described herein.

Unless otherwise indicated, as used herein, the singular forms "a" ("a", "an") and "the" include plural referents. For example, "a" ADSC includes one or more adipose tissue-derived stem cells.

An "about" value or parameter referred to herein includes (and illustrates) various aspects related to the value or parameter itself. For example, references to "about X" include "X".

It should be understood that various aspects of the invention described herein include "including" various aspects, "consisting of" and "consisting essentially of".

III. Adipose-derived stem cells (ADSC) and their isolation

Adipose tissue provides a source of pluripotent stromal cells. Adipose tissue is readily available and abundant in many individuals (eg, non-human mammals). It has been documented in many literatures that adipocyte lines can supplement cell populations. Even after surgical removal by liposuction or other procedures, it is common to see fat cells in the individual reappear after a certain period of time. This indicates that adipose tissue contains stromal stem cells capable of self-renewal.

Methods for general isolation, expansion, and differentiation of human adipose tissue-derived stem cells have been previously described (Zuk et al., Tissue Engineering (2001) 7: 211-228; Burris et al., Mol Endocrinol 1999, 13: 410 7; Erickson et al., Biochemical & Biophysical Research Communications 2002, 290: 763 9; Gronthos et al., J Cell Physiol. 2001 October; 189 (1): 54 63; Halvorsen et al., Metabolism 2001, 50: 407 413; Halvorsen et al. , Tissue Eng. 2001 December; 7 (6): 729 41; Harp et al., Biochem Biophys Res Commun 2001, 281: 907 912; Saladin et al., 1999, Cell Growth & Diff 10: 43 48; Sen et al., Journal of Cellular Biochemistry 2001, 81: 312 319; Zhou et al., Biotechnol Techniq 1999, 13: 513 517). Adipose tissue-derived stem cell line Self-minced human adipose tissue is obtained by collagenase digestion and differential centrifugation according to known techniques (Halvorsen et al., Metabolism 2001, 50: 407 413; Hauner et al., J Clin Invest 1989, 84: 1663 1670; Rodbell et al., J Biol Chem 1966, 241: 130 139). These techniques are also applicable to the isolation, expansion, and differentiation of non-human mammalian adipose tissue-derived stem cells. For example, see Fotuhi P. et al., Europace , 9 (12): 1218-21 (2007); Madonna R. et al., Stem Cell , 26 (1): 202-11 (2008); Huang T. et al., J Spinal Cord Med , 30 Supplement, 1: S35-40 (2007); Hemmrich K. et al., J Surg Res. , 144 (1): 82-8 (2008); Qu CQ et al., In Vitro Cell Dev Biol Anim. , 43 (2): 95-100 (2007); Wang KH et al., Biotechnol. Appl. Biochem. (2008); Williams KJ, Cells Tissues Organs , (2008); and Valina C et al., Eur Heart J . , 28 (21): 2667-77 (2007).

ADSCs can be isolated from a variety of non-human mammals, including (but not limited to) porcine, bovine, canine, equine, and feline. In one aspect, the individual who can isolate ADSC and use it for therapeutic and / or prophylactic purposes is a porcine family. The porcine family is usually raised for the purpose of providing a part for xenotransplantation (eg, a valve of a human heart). Therefore, porcine ADSC can be used for xenotransplantation into other non-human animals. In another aspect, the present invention provides ADSCs obtained from privately owned pets, whose access can be regulated by the Food and Drug Administration (FDA). ADSC from these pets can be used for xenotransplantation, division transplantation, or allogeneic transplantation.

In one aspect, the invention provides an ADSC that is heterologous to a recipient of an ADSC-based treatment. Non-limiting examples of ADSC types that can be used are cattle, horses, sheep and pigs. In another aspect, the present invention provides ADSCs that are species to the recipient of an ADSC-based treatment. In another aspect, the invention provides ADSCs that are isogenic to a recipient of an ADSC-based treatment. In another aspect, the invention provides an ADSC that is autologous to a recipient of an ADSC-based treatment.

ADSCs from non-human mammals can be isolated in any manner known to those skilled in the art. In one aspect of the invention, ADSCs can be isolated according to the following non-limiting methods. First, the separated adipose tissue (ie, adipose tissue or liposuction fat) is washed with PBS containing 1% penicillin and streptomycin, chopped into small pieces, and then the ratio of collagenase solution: fat 5: 1 v / v Mix with a solution containing 0.075% collagenase type IA (Sigma-Aldrich, St. Louis, MO). After incubation for 1 hour at 37 ° C with vigorous shaking, the product was then centrifuged at 220 x g for 10 minutes at room temperature. Three layers are formed: the upper lipid layer, the middle collagenase layer, and the bottom cell pellet. The intermediate layer was collected and filtered with a 200 μm filter, followed by centrifugation. In the second round, the circulating collagenase type IA in the flow-thru was used to digest fresh adipose tissue again, using a ratio higher than the first round (7: 1 by volume). The bottom cell pellet contains stem cells.

Can be stored before further purification, differentiation, administration to an individual, or any other use Or store ADSC or adipose tissue containing ADSC. Although it has been reported that adipose tissue can be stored at room temperature for 24 hours and stored at 4 ° C for 1 to 3 days, viable cells in adipose tissue have declined sharply during storage. Therefore, in one aspect, the present invention provides a method for preserving the viability of ADSC or adipose tissue using adipose tissue preservation solution (ATPS), wherein the ATPS contains the enzyme superoxide dismutase (SOD) as its basic component. In one aspect, the superoxide dismutase is isolated from mammalian red blood cells. Those skilled in the art can easily obtain SOD from commercially available sources such as Sigma-Aldrich (which also sells SOD isolated from bovine RBC or liver).

In a non-limiting aspect, ATPS consists of 200 mg / ml KH ₂ PO ₄ , 200 mg / L KCl, 2.16 g / L Na ₂ HPO4 ^. 7H ₂ O, 8 g / L NaCl, 30,000 units / L SOD and 5 g / L bovine serum albumin (BSA). To preserve non-human mammalian fat-derived stem cells, 1 × 10 ⁶ fat-derived stem cells can be mixed with 1 ml ATPS and stored at 4 ° C. Those skilled in the art will recognize that the concentration of the reagents contained in ATPS can be changed to an appropriate level without substantially affecting the desired properties of ATPS.

It is well known that the matrix vascular component derived from adipose tissue digestion is composed of many types of cells, such as stem cells, endothelial cells, smooth muscle cells, and other terminally differentiated cells. "Panning" (ie, immunoselection methods for enriching specific cell populations from a mixture of different cell types) has been described in various cell culture textbooks and references known to those skilled in the art. This method is based on the ability to select antibodies bound to a cell culture dish. The cell type mixture was cultured on antibody coated plates and allowed to bind for a short period of time. Non-adherent cells (those who do not bind antibodies) can then be gently lysed from the culture dish, allowing the bound cells to be harvested. This method helps to develop other new technologies (such as density-gradient separation) and methods that take advantage of the unique surface-binding properties of specific cell types. For example, the development of magnetic bead technology allows repeated washing of bead-bound cells, which greatly improves potential separation purity. The size and composition of the paramagnetic beads used by the companies are significantly different and further improvements / improvements occur regularly.

Therefore, in one aspect of the invention, a combination of antibodies that can be used to detect the presence of CD34, CD90, and SSEA1 cell markers is used to select fat-derived stem cells. In addition to these labels, other antibodies can be used, such as those disclosed in US Patent Application No. 2006/0147430 published by Sayre et al. After identifying positive cells, such cells can be cultured, further studied, or administered to an individual in one or more of the treatment methods disclosed herein.

IV. Composition

The invention also provides a therapeutic composition for practicing the therapeutic method of the invention. In some embodiments, the ADSC used is heterogeneous to the recipient. The present invention also provides a set of ADSCs that can be used as "universal donors" for the treatment (treatment or prevention) of various non-human mammals, thereby reducing the possibility of transplant rejection.

In one aspect of the present invention, the therapeutic composition includes a pharmaceutically acceptable excipient (vehicle) or vehicle and the ADSC of the present invention in a mixture and has a strength for experiencing cell or tissue loss or Defective individuals are effective for administration, and the ADSC includes cells or tissues derived therefrom, alone or in combination with one or more bioactive agents.

In another aspect, the present invention provides a therapeutic composition for use in a method comprising or based on the ADSC of the present invention and a pharmaceutically acceptable carrier or medium, the ADSC comprising a lineage undefined cell population derived from it, lineage stereotyping Cell population or tissue. It should be understood that the present invention also encompasses therapeutic compositions comprising a biologically active agent that acts on or regulates the ADSC of the invention and / or cells or tissue derived therefrom, and a pharmaceutically acceptable carrier or vehicle.

The preparation of cell or tissue-based therapeutic compositions is well known in the art. These compositions may be formulated in a pharmaceutically acceptable medium. The cells can be in the form of a solution or embedded in a matrix. The preparation of therapeutic compositions with bioactive agents (e.g., growth factors) as active ingredients is well known in the art. Active therapeutic ingredients are often mixed with excipients or vehicles that are pharmaceutically acceptable and compatible with the active ingredients. In addition, if desired, the composition may contain There are small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents that enhance the effectiveness of the active ingredients.

The bioactive agent can be formulated into a therapeutic composition in the form of a neutralized pharmaceutically acceptable salt. Pharmaceutically acceptable salts include acid addition salts (salts formed with the free amine groups of a polypeptide or antibody molecule), and these salts are associated with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid , Tartaric acid, mandelic acid, and the like). Salts formed from free carboxyl groups can also be derived from inorganic bases (e.g. sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide) and organic bases (e.g. isopropylamine, trimethylamine, 2-ethyl Aminoethanol, histidine, procaine, and the like).

ADSC investment

The therapeutic composition of the invention is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered depends on, for example, the individual to be treated and the weakness. However, a suitable dose of the therapeutic composition of the present invention may range from about 0.05 × 10 ⁶ to 100.0 × 10 ⁶ fat-derived stem cells / 10 mm treatment site, preferably about 0.10 × 10 ⁶ to 50.0 × 10 ⁶ Fat-derived stem cells / 10 mm treatment site, and more preferably about 0.5 × 10 ⁶ to 5.0 × 10 ⁶ fat-derived stem cells / 10 mm treatment site. Suitable protocols for initial and subsequent administrations are also variable, but may include initial administrations, followed by one or more intervals as needed or indicated (for example, weekly, monthly, or yearly) by subsequent injections or other administrations With repeated doses.

Those skilled in the art can readily determine the appropriate cell concentration for a particular purpose. Exemplary dosages are in the range of about 0.05 × 10 ⁶ to 100.0 × 10 ⁶ cells per day per treatment site. In a non-limiting example, approximately 5 × 10 ⁶ ADSCs are injected into non-human joints (eg, stiff joints) to treat osteoarthritis. The precise administration schedule of the therapeutic composition depends on the judgement and expected results of the veterinarian, and is therefore, to a certain extent, unique to each individual.

ADSCs or differentiated cells of the invention can be administered by injection into a target site of an individual, preferably via a delivery device, such as a tube, such as a catheter. In one aspect, the tube additionally Contains a needle, such as a syringe, with which cells can be introduced into an individual at a desired location. Specific non-limiting examples of administering cells to an individual may also include administration by subcutaneous injection, intramuscular injection, intra-articular or intravenous injection. If administered intravenously, an injectable liquid suspension of the cells can be prepared and administered by continuous infusion or as a concentrated injection. In another aspect, if the medical condition to be treated is dilated cardiomyopathy, a person skilled in the art (e.g., a veterinarian) may use a catheter-based injection or ADSC can be delivered to surrounding tissues in a manner that can be distributed to surrounding tissues.

Cells can also be embedded in delivery devices (such as syringes) in different forms. For example, the cells can be suspended in a solution contained in such a delivery device. As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent in which the cells of the invention retain viability. The use of these carriers and diluents is well known in the industry. The solution is preferably sterile and fluid to the extent that it can be easily injected. Preferably, by using, for example, parabens, chlorobutanol, phenol, ascorbic acid, sodium thiomersalate and the like, the solution is more stable against microorganisms under the conditions of manufacture and storage and preservation ( Such as bacteria and fungi). The solution of the present invention can be prepared by incorporating the ADSC or differentiated cells described herein into a pharmaceutically acceptable carrier or diluent and other ingredients listed above as necessary, followed by filtration and sterilization.

Cells can be administered systemically (e.g., intravenously) or locally (e.g., directly into a myocardial defect under the guidance of an electrocardiogram, or by direct administration under visualization during a surgical procedure). For such injections, the cells may be present in an injectable liquid suspension formulation or in a biocompatible medium that can be injected in liquid form and becomes semi-solid at the site of the damaged tissue. A conventional intracardiac syringe or a controllable endoscopic delivery device may be used, as long as the needle cavity or hole has a diameter (such as 30 or larger) sufficient for the shear force to not damage the cells being delivered.

Cells can be administered in any manner that allows them to be transplanted into the intended tissue site and to remodel or regenerate a defective area. ADSCs that can be incorporated or embedded include biological phases Matrix that is compatible, compatible with the recipient, and degraded into products that are not harmful to the recipient. These matrices provide support and protection to ADSCs and differentiated cells in vivo.

Natural and / or synthetic biodegradable substrates are examples of such substrates. Natural biodegradable matrices include plasma clots (eg, derived from mammals), collagen, fibronectin, and laminin matrices. Suitable synthetic materials for cell transplantation matrices must be biocompatible to avoid migration and immunological complications; and they should be able to support a wide range of cell growth and differentiated cell functions. It must also be resorbable, allowing for complete natural tissue replacement. The matrix should be configurable in various shapes and should have sufficient strength to prevent collapse after transplantation. Various studies indicate that biodegradable polyester polymers composed of polyglycolic acid meet all of these criteria, such as Vacanti et al., J.Ped. Surg. , 23: 3-9 (1988); Cima et al., Biotechnol. Bioeng. 38: 145 (1991); described by Vacanti et al., Plast. Reconstr. Surg. , 88: 753-9 (1991). Other synthetic biodegradable support matrices include synthetic polymers, such as polyanhydrides, polyorthoesters, and polylactic acid. Other examples of synthetic polymers and methods for incorporating or embedding cells into such matrices are also known in the art. See, for example, U.S. Patent Nos. 4,298,002 and 5,308,701.

Cell and polymer attachment can be enhanced by coating the polymer with compounds such as: basement membrane components, agar, agarose, gelatin, gum arabic, collagen type I, type II, type III , IV and V, fibronectin, laminin, glycosaminoglycans, mixtures thereof and other materials known to those skilled in the field of cell culture. All polymers used in the matrix must meet the mechanical and biochemical parameters necessary to provide adequate support for subsequent cell growth and proliferation.

One of the advantages of the biodegradable polymer matrix is that angiogenesis and other bioactive compounds can be directly incorporated into the support matrix so that it is slowly released as the support matrix degrades in vivo. Since the cell-polymer structure is vascularized and the structure is degraded, ADSCs can differentiate according to their inherent characteristics. Factors including the following can be incorporated into or provided with the matrix: nutrients, growth factors, differentiation or dedifferentiation (i.e., Cells lose differentiation properties and acquire properties such as proliferation and more general functions), secretions, immunomodulators, inflammation inhibitors, regression factors, bioactive agents that enhance or allow lymphatic network or nerve fiber growth, transparent Acids, and drugs known to those skilled in the art and available from suppliers such as Collaborative Research, Sigma Chemical, etc., and with instructions on the composition of the effective amount, such as vascular endothelial growth factor (VEGF), epidermal growth factor ( EGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) and other vascular growth factors. Similarly, polymers containing peptides such as the attachment peptide RGD (Arg-Gly-Asp) can be synthesized for use in matrix formation (see, for example, U.S. Patent Nos. 4,988,621, 4,792,525, 5,965,997, 4,879,237 And No. 4,789,734).

In another example, cells can be transplanted into a gel matrix (eg, Gelfoam from Upjohn), which is polymerized to form a matrix where ADSCs or differentiated cells can grow. Various encapsulation techniques have been developed (e.g., Lacy et al., Science 254: 1782-84 (1991); Sullivan et al., Science 252: 718-712 (1991); WO 91/10470; WO 91/10425; US Patent No. 5,837,234 (U.S. Patent No. 5,011,472; U.S. Patent No. 4,892,538).

PLGA or poly (lactic-co-glycolic acid) is a Food and Drug Administration (FDA) approved copolymer, which can be used in many therapeutic devices due to its biodegradability and biocompatibility. PLGA is synthesized by random ring-opening copolymerization of two different monomers, namely, a cyclic dimer of glycolic acid and lactic acid (1,4-dioxane-2,5-dione). Common catalysts used to make this polymer include tin (II) 2-ethylhexanoate, tin (II) alkoxide or aluminum isopropoxide. During the polymerization, continuous monomer units (of glycolic acid or lactic acid) are linked together in the PLGA by ester bonds, thereby producing a non-linear aliphatic polyester as a product. PLGA has been successfully used as a biodegradable polymer because it undergoes hydrolysis in the body to produce the initial monomers lactic acid and glycolic acid. By-products of various metabolic pathways in the two single systems. Because the body is able to effectively break down these two monomers, there is no relevance to using PLGA for drug delivery or biomaterial applications Systemic toxicity. There is a greater tendency for ADSC mixed with PLGA / carboxymethyl cellulose (CMC) to remain in the injection area compared to ADSC mixed with saline.

V. Genetically modified cells

In addition, ADSC can be engineered to contain genes that express growth factors, hormones, and cytokines. For example, ADSC can be engineered to express beneficial genes such as, but not limited to, VEGF, BDNF, IGF, TGF, NGF, and other neurotrophic and vascular nourishing growth factors. Injecting specially modified ADSC can help some tissues regenerate; for example, BDNF is for nerves. ADSC can also be engineered to express the β-cell-specific gene Pdx-1, which enables ADSC to secrete insulin. These cells can be transplanted into an individual to treat their diabetes. In another aspect, ADSC can be engineered to produce dystrophin and then implanted into an individual to treat muscular dystrophy. In yet another aspect, ADSCs that exhibit at least one genotype or phenotypic characteristic of chondrocytes are genetically modified to express an exogenous gene or to suppress the expression of an endogenous gene and implanted into an animal. The invention provides methods for genetically modifying such cells and populations prior to transplantation. It should be understood that ADSC can be further differentiated along the lineage towards organ-specific cells (eg, chondrocytes) without the help of genetic modification.

A nucleic acid construct comprising a promoter and a sequence of interest can be introduced into a recipient's prokaryotic or eukaryotic cell as a non-replicating DNA (or RNA) molecule, which can be a linear molecule or better a closed covalent circular molecule . Since these molecules cannot replicate autonomously without an origin of replication, gene expression can be performed by means of transient expression of the introduced sequence. Alternatively, permanent expression can be performed by integrating the introduced DNA sequence into the host chromosome.

In one aspect, a vector is used that is capable of integrating the desired gene sequence into the host cell chromosome. Cells that have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers that allow selection of host cells containing the desired nucleic acid sequence. Markers (if required) can provide prototrophy, biocide resistance (eg, resistance to antibiotics or heavy metals such as copper), or the like, of the auxotrophic host. Selectable marker gene sequence can be directly linked to the DNA gene sequence to be expressed or introduced into the same cell by co-transfection in. Preferably, the mark performance can be quantified.

In a preferred aspect, the introduced nucleic acid molecule can be incorporated into a plastid or viral vector capable of autonomous replication in a recipient host. Any of a wide variety of vectors can be used for this purpose. Important factors in selecting a specific plastid or viral vector include: 1) the cheapness of identifying and accepting recipient cells containing the vector from their recipient cells that do not contain the vector; 2) the number of copies of the desired vector in a particular host And 3) whether it is desirable to be able to shuttle the vector between host cells of different species.

Preferred eukaryotic vectors include, for example, acne virus, SV40, retrovirus, adenovirus, adeno-associated virus, lentivirus and various plastid-based commercially available mammalian expression vectors familiar to those skilled in the art.

After the construct-containing vector or nucleic acid molecule has been prepared for expression, the DNA construct can be introduced into an appropriate host cell by any of a variety of suitable methods: transformation, transfection, viral infection, coupling , Protoplast fusion, electroporation, particle grabbing techniques, calcium phosphate-precipitation, direct microinjection, and the like. After the vector is introduced, recipient cells are grown in a selective medium that is selected for the growth of cells containing the vector. The expression of the selected gene molecule results in the production of heterologous proteins.

"Maintaining" the introduced DNA in a cell is understood to mean that as the introduced DNA continues to grow and proliferate, it continues to exist in substantially all of the cells in question. That is, the introduced DNA is not diluted from most cells that have undergone multiple rounds of cell division. Instead, it replicates during cell proliferation and at least one copy of the introduced DNA remains in almost every daughter cell. The introduced DNA can be maintained in the cell in either of two ways. First, it can be integrated directly into the genome of a cell. This occurs at a very low frequency. Second, it can exist in the form of extrachromosomal elements or episomes. In order that episomal bodies are not diluted during cell proliferation, selectable marker genes can be included in the introduced DNA and cells grown under conditions that require the expression of the marker genes. Even where the introduced DNA has been integrated into the genome, a selectable marker gene can be included to prevent DNA from being transferred from the chromosome resection.

Genetically modified cells can express the gene of interest transiently or constitutively. The manifestations can be intracellular or on the cell surface. In one aspect of the present invention, the ADSC of the present invention is genetically modified to transiently express one or more cytokines that are beneficial for treating a specific medical condition or reducing pain. For example, ADSCs that have been genetically modified to exhibit anti-inflammatory interleukins (e.g., IL-2) can be transplanted to sites where inflammation occurs (e.g., arthritic joints). Transplantation then provides the dual benefit to the individual because ADSCs can become chondrocytes in addition to exhibiting beneficial anti-inflammatory cytokines. Transient performance can last for minutes, hours, days, or even weeks. In some embodiments of the invention, the transient manifestation of beneficial cytokines is 1 week, 2 weeks, or 3 weeks. In other embodiments of the invention, the transient manifestation of beneficial cytokines is the length of time necessary for the symptoms to decrease or even disappear. The reduction or disappearance of symptoms can be determined by a person skilled in the art (e.g., a veterinarian) in the treated individual.

Genetically altered cells can then be introduced into an individual under various conditions in which the transgene is to be expressed in vivo by various methods. As a non-limiting example, the transgene may encode for producing extracellular matrix proteins, preferably where the transgene encodes for producing collagen. Cells containing the transgenes for extracellular matrix proteins can then be introduced into animals. Alternatively, the transgene-containing cells can be injected intraperitoneally or into some other suitable organ storage site.

VI. ADSC Library

The present invention also provides ADSC repositories of various species that have been derived from non-human mammals. Non-limiting examples of ADSC types that can be used are cattle, horses, sheep and pigs. The ADSC library allows the storage and / or storage of various isolated ADSCs from non-human mammals. The ADSC library allows those skilled in the art (eg, veterinarians) to easily and quickly treat animals. The ADSC library also allows those skilled in the art to use it to determine the appropriate autologous, allogeneic, xenogeneic, or allogenic ADSC to treat animals. In one aspect, a set of pig-derived ADSCs that can pass the FDA's transplantation (including xenograft) regulations for animal treatment (therapeutic and Prevent both). In another aspect, a set of pig-derived ADSCs derived from non-FDA approved sources is provided for animal treatment (both treatment and prevention). In another aspect, the ADSC library is heterologous to the treatment recipient. In another aspect, the ADSC library serves as a universal donor for recipients of animal therapy.

VIII. Kits with ADSCs from non-human mammals

Also provided are compositions and kits including ADSC derived from any non-human mammal and suitable packaging. In one aspect, the composition comprises 100% (referred to as purity) ADSC. "Purity" does not mean that only stem cells are present in the composition, but indicates that the stem cells have been manipulated such that they have been removed from their natural tissue environment. With regard to other aspects, the composition comprises 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% purity ADSC. In other aspects, the composition comprises ADSCs of 85%, 80%, 75%, or 70% purity. Kits containing ADSCs from non-human mammals can be used for storage and / or shipment. In some aspects, the invention includes a set of: (i) one or more multipotent ADSCs or transplantable ADSCs derived from one or more non-human mammals, and (ii) using the set of treatments requires the treatment Description of conditions in non-human mammals. In various aspects, the invention describes a set of: (i) one or more pluripotent ADSCs or transplantable ADSCs derived from one or more non-human mammals and (ii) using the set for research or Description of drug screening uses. In another aspect, the composition of ADSC is combined with carboxymethyl cellulose (CMC).

Suitable packaging for the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), vessels, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and And so on. Such articles of manufacture may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packages in single or multiple unit doses and may be further sterilized and sealed. The instructions provided in the kits of this invention are usually written instructions for labeling or packaging inserts (e.g., paper included in the kit), but machine-readable instructions (e.g., on magnetic or optical storage discs) The instructions carried) are also acceptable. Instructions related to the use of ADSCs derived from non-human mammals often include information on dosages, dosing regimens, and routes of administration for intended therapeutic or industrial use. The set may further include a description of the selection of an individual suitability or treatment.

Such containers may be unit doses, bulk packages (eg, multi-dose packages), or sub-unit doses. For example, a kit containing ADSCs sufficient to provide effective treatment to an individual for an extended period of time, such as the following, may also be provided: about 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months , 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. The kit may also include multiple unit doses of cells and instructions for use, and be packaged in sufficient quantities for storage in animal clinics (e.g. animal hospitals), pharmacies in animal hospitals and animal clinics and / or animal hospital supply warehouses and use.

In addition, the kits may contain compositions comprising ADSCs that are heterologous, allogeneic, or allogeneic to the recipient of the treatment.

VIII. ADSC Treatment and Administration

ADSC can be administered to treat a variety of mammalian conditions including, but not limited to, urinary incontinence, osteoarthritis, degenerative myelopathy, diabetes, tissue regeneration, wound healing, scarring, soft tissue defects, fecal incontinence, dilation Cardiomyopathy, hip dysplasia, avascular necrosis of the femoral head, ligament injury, tendon injury, spinal cord injury, atherosclerosis-related infarction, arthritis and muscular dystrophy. Another use of ADSC is to suppress the immune response to prevent graft-versus-host (GVH) disease. ADSC can be used for tissue regeneration or the various factors and / or enzymes it can secrete or its effect on the surrounding environment after differentiation.

Adipose-derived stem cells or differentiated cells can be transplanted into a recipient, where the cells will proliferate and differentiate to form new cells and tissues, thereby providing the physiological processes normally provided by the tissue. The term "transplant" as used herein refers to the transfer of cells alone or to cells embedded in a supporting matrix. The cells can be autologous, allogeneic, allogeneic or heterogeneous cells. The term "tissue" as used herein refers to the aggregation of similar specialized cells that collectively perform a specific function. The tissue is expected to cover all types of biological tissue including both hard and soft tissue. Soft tissue refers to tissue that connects, supports, or surrounds other structures and organs of the body. Soft tissues include muscles, tendons (fibrous bands that connect muscles to bone), fibrous tissue, fat, blood vessels, nerves, and synovial tissue (tissue around joints). Hard tissue includes connective tissue (eg, hard forms such as bone tissue or bone) as well as other muscle or bone tissue.

In another aspect of the invention, ADSC is administered with a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable excipients described herein (eg, vehicles, adjuvants, carriers, or diluents) are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carriers or excipients are those who are chemically inert to the therapeutic composition and have no harmful side effects or toxicity under the conditions of use.

The choice of excipients or carriers will be determined to some extent by the particular therapeutic composition and the particular method used to administer the composition. Therefore, there are many suitable formulations of the pharmaceutical composition of the present invention. The formulations described herein are exemplary only and are in no way limiting.

Generally, physiologically acceptable carriers are aqueous pH buffered solutions. Examples of physiologically acceptable carriers include, but are not limited to, saline, solvents, dispersion media, cell culture media, aqueous buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid, low molecular weight ( Less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamate, Asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt formation Counter ions, such as sodium; and / or non-ionic surfactants, such as TWEEN (TM), polyethylene glycol (PEG), and PLURONICS (TM).

A. ADSC treatment for urinary incontinence

ADSC can be used to treat urinary incontinence in various non-human mammals. Urinary incontinence (UI) is a debilitating and incurable condition to date. In one aspect, UI is defined as an unintentional urine leak that affects approximately 20% of female dogs who have had their testicles removed (ovarian hysterectomy), but less than 1% It is seen in intact female dogs and rarely reported in male dogs, regardless of gonad status.

Dogs with UI have some common secondary problems, such as bladder infections and urinary scalding. Bladder infections are caused by bacteria that migrate upward along the urethra and pass through the more relaxed urethral openings into dogs with UI. Urinary dipping of the skin is the consequence of prolonged contact with leaking urine, which is highly corrosive. In female dogs, UI can occur at any time after 1 week after the testicle is removed and is associated with serious management issues that often cause euthanasia in animals. It is believed that UI in female dogs who have had their testicles removed is due to neurological, vascular, or hormonal changes rather than lower urinary tract mechanical damage suffered during surgery.

Currently, canine UI is primarily treated with phenylpropanolamine (PPA). PPA is an alpha-adrenergic agonist and stimulates the secretion of epinephrine, a hormone substance that increases urinary sphincter muscle tone. In 1999, the Federal Food and Drug Administration banned PPA for human use because it increased the risk of hemorrhagic stroke (bleeding in the brain or bleeding into tissues around the brain).

UI stem cell therapy has been successfully proven in human medicine. Stem cells are now isolated from an individual's skeletal muscle and injected into the urethral sphincter, resulting in muscle cell regeneration and enhanced urethral contractility. However, due to the lack of such stem cells and the limited size of muscle biopsies, this procedure requires the persistent culture of isolated stem cells. In addition, these cells must only become muscle cells and thus cannot restore other cell types (such as nerves and blood vessels) of the urethra. In contrast, ADSCs are available in large numbers and can be differentiated into many cell types, including neurons, vascular endothelial cells, and smooth muscle cells, providing advantages over other treatment types.

Therefore, ADSC can be used to treat UI. In one aspect, the ADSC used is described as a universal donor. ADSCs used as universal donors can be stored in animal clinics or animal hospitals for easy and rapid treatment. Individuals who treat UI have an effective amount of ADSC injected into the sphincter area of the urethra. The effective amount is in the range of 1 to 10 million cells per animal or 5 million per 10 kg, depending on the size of the animal. Accurate injections can be done by using a urine tester that allows both visualization and injection. Those skilled in the art (e.g., veterinarians) can work with The volume of the suspension and the number of cells are adjusted proportionally to the weight of the individual.

After the treatment procedure, the owner of the individual should monitor the individual daily to observe the following characteristics: monitoring of signs of reaction at the injection site, such as redness, pain, and fever, monitoring of allergic reactions in animals and signs of urinary blockage, any other in the animal Unusual signs such as persistent nausea, fatigue, drowsiness, decreased appetite, and difficulty urinating. If any of these characteristics are observed, the individual's owner should immediately notify the veterinarian so that the veterinarian can take appropriate measures to bring the individual into better health. Veterinarians can perform regular assessments after treatment, for example, 10 to 15 days, 25 to 30 days, and 55 to 60 days after treatment.

B. ADSC treatment of osteoarthritis (OA)

ADSC can be used to treat osteoarthritis (OA) in various non-human mammals. OA is the most common cause of chronic pain in dogs, with more than 20% or 10 to 12 million dogs suffering from chronic pain in the United States at any time. OA is characterized by articular cartilage degradation, accompanied by matrix loss, fibrillation, and fissure formation, and can lead to complete loss of cartilage surface. Chondrocytes (the only cells in articular cartilage) maintain the autoregulated synthesis and degradation of the extracellular matrix by secreting macromolecular components (collagen, glycosaminoglycan, and hyaluronic acid) and adjusting the turnover rate of the extracellular matrix. In OA, destructive and pro-inflammatory mediators are produced in excess relative to assimilating and repairing substances, leading to the progressive destruction of articular cartilage.

Stem cell therapies are now used in humans, horses, and dogs in clinical settings. In horses, high success rates (approximately 70%) have been achieved by using adipose tissue-derived stem cells (ADSC) to treat tendon injuries, ligament injuries, osteoarthritis (OA), and osteochondral defects. ADSC treatment of OA appears to be equally effective in dogs. Other canine diseases that are potential targets for stem cell therapy include tendon and ligament damage, hip dysplasia, avascular necrosis of the femoral head, dilated cardiomyopathy, spinal cord injury, degenerative myelopathy, urinary incontinence (UI), and muscular atrophy .

Currently, OA is mainly treated with NASID. However, many scientific studies and clinical experiences have shown that NSAID does not completely relieve pain. Compared to medications such as ADSC Cell therapy, such as therapy, does not rely on a single target receptor or pathway for its effect. Cell therapy plays a nourishing role by secreting cytokines and growth factors and by recruiting endogenous cells to the injured site, and it can promote cell differentiation into resident lineages. Mesenchymal stem cells (which include ADSC) are known to communicate with cells in their local environment, suppressing the immune response and inhibiting apoptosis. Recent research has also shown that bone marrow stem cells, which have a regenerative capacity similar to ADSC, can deliver new mitochondria to damaged cells, thereby saving aerobic metabolism. Therefore, ADSC therapy can reduce pain and enhance healing in OA individuals, thereby improving quality of life.

Therefore, ADSC can be used to treat OA. In one aspect, the ADSC used is described as a universal donor. ADSCs used as universal donors can be stored in animal clinics or animal hospitals for rapid treatment. In one aspect, the invention provides treatment for a non-human mammal (eg, a dog) suffering from OA. Although OA can afflict virtually any joint, it is most commonly associated with the hind knee and hip joints. Treatment can be achieved by injecting an ADSC suspension into these joints. Those skilled in the art (e.g., a veterinarian) can skillfully adjust the volume and cell number of a cell suspension according to individual size. As a non-limiting example, a 20 kg dog can accept 5 million ADSCs per joint in 0.5 ml PBS. After treatment, the owner is advised to leash-walk for 30 minutes each day. Non-human mammals are allowed to continue taking any oral medicine that was taken before ADSC treatment. Any intra-articular treatment before ADSC treatment should be discontinued.

After treatment, animal evaluation will include medical records, physical examinations, and lameness examinations, which include joint mobility and pain records during operation. Clinical outcome measures will be based on an animal orthopedic assessment using a numerical rating scale based on a standardized questionnaire. Baseline results for both owner and animal evaluations can be recorded between 2 and 14 days before the dog receives the test or control formulation by intra-articular injection. Follow-up visits can be made to animal clinics 30, 60, and 90 days after intra-articular injection in dogs. At each visit, the owner may be required to complete a numerical rating scale (1 (best) to 5 (worst)) as part of a standard questionnaire adapted from the Cincinnati Orthopaedic Disability Index, which includes Evaluation of the following parameters: Walking, running, jumping, abruptly turning, lying down, standing down, climbing stairs, descending stairs, squatting to urinate or defecate, stiffness in the morning, stiffness at night, difficulty walking on smooth floors, and willingness to play autonomously.

C. ADSC treatment for wound healing

ADSCs can also be administered to non-human mammals for the purpose of accelerating wound healing and slowing scar formation. This has improved cosmetology (i.e., improved scar healing for cosmetic purposes only) and as an improved surgical scar in vivo (e.g., excessive scarring at a surgical anastomosis site can lead to surgical complications such as contracture of the anastomosis site. During surgery or Delayed local injection of ADSC can improve the healing of surgical tissue sites and reduce the use of adjuvants (surgical complications due to excessive scarring).

Therefore, in one aspect of the invention, ADSC is injected subcutaneously at a desired location (eg, along the suture site of a surgical wound). In some aspects, the use of support materials (such as matrigel or microspheres with CMC) can be introduced to further position the ADSC to the treatment site. Introducing ADSC to the wound site causes ADSC to differentiate in a manner that mimics the body's natural response to the wound. For example, ADSC appears to spontaneously differentiate into other cell types (such as fibroblasts and inflammatory cells) or newly formed blood vessels containing natural wound healing devices.

D. Preventive use of ADSC

The ADSC of the present invention is also used for prevention to delay the development of hip dysplasia and avascular necrosis of the femoral head by administering an effective amount of ADSC to the hip or femoral head, respectively. The effective amount is in the range of 1 million to 10 million cells per animal or 5 million cells / 10 kg, depending on the size of the animal, which can be easily determined by those skilled in the art (e.g., veterinarians).

E. Use of ADSC in fracture repair

The ADSC of the present invention is used to promote the repair of bone fracture, and comprises administering an effective amount of ADSC at the fracture site. In some embodiments, the effective amount of ADSC can be from about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC, about 1 × 10 ⁶ ADSC to about 1 × 10 ⁷ ADSC. In other embodiments, ADSC is administered with an effective amount of bone morphogenetic protein (BMP). In a specific embodiment, the BMP is a mixture of human BMP 2, human BMP 4, human BMP 7, or human BMP, and the mixture comprises one or more of the species BMP 2, human BMP 4, and human BMP 7. In particular embodiments, one or more of the BMPs or BMP mixtures are produced recombinantly. A suitable content of BMP is provided in a commercially available demineralized bone matrix product (eg, AlloMatrix® Custom; Wright Medical Technology Co., Arlington, TN) or can be independently determined by those skilled in the art. In a particular aspect of these embodiments, an effective amount of ADSC is also administered intravenously. In some embodiments, the effective amount of ADSC administered intravenously is between about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC , About 1 × 10 ⁶ ADSC to about 1 × 10 ⁷ ADSC. In other aspects of these embodiments, the administered dose of ADSC and BMP is adjusted depending on the size of the animal. In some embodiments, therefore, the effective amount of ADSC administered is from about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg body weight, about 5 × 10 ⁵ ADSC / kg The weight ranges from about 5 × 10 ⁷ ADSC / kg body weight, from about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. In other specific embodiments, the animal treated is a non-human animal and ADSC is a non-human animal. In a specific embodiment, the ADSC is a pig ADSC.

F. Use of ADSC in the treatment of "dry eye" conditions

Non-human mammals can have a "dry eye" condition (e.g., dry keratoconjunctivitis) due to reduced and inadequate production of tear fluid due to dysfunction of the lacrimal gland and / or eyelid gland, or a change in the composition of the produced tear fluid with reduced effectiveness . The causes of dry eye include, inter alia, autoimmune diseases (eg, Sjörgen's syndrome and / or rheumatoid arthritis), age, and trauma.

The ADSC of the present invention is used to promote the recovery of tears in non-human mammals with dry eye, and comprises administering an effective amount of ADSC to the area around the lacrimal glands of the afflicted non-human mammals. In some embodiments, the effective amount of ADSC can be from about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC, about 1 × 10 ⁶ ADSC to about 1 × 10 ⁷ ADSC. In a specific embodiment, 1 × 10 ⁶ ADSCs in 0.2 mL saline are injected into the area around the lacrimal gland. In another specific embodiment, 1 × 10 ⁶ ADSCs stored in 0.2 mL of saline are injected into the area of the eyelid glands of a non-human mammal to be treated. Repeat injections as needed at the discretion of the veterinarian treating the animal. In specific embodiments, the injections are repeated weekly as needed, for example, for several weeks, according to the veterinarian's decision to treat the animal. In a particular aspect of these embodiments, an effective amount of ADSC is also administered intravenously. In some embodiments, the effective amount of ADSC administered intravenously is between about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC , About 1 × 10 ⁶ ADSC to about 1 × 10 ⁷ ADSC. In other aspects of these embodiments, the administered dose of ADSC and BMP is adjusted depending on the size of the animal. In some embodiments, therefore, the effective amount of ADSC administered is from about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg body weight, about 5 × 10 ⁵ ADSC / kg The weight ranges from about 5 × 10 ⁷ ADSC / kg body weight, from about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. In other specific embodiments, the animal treated is a non-human mammal and ADSC is from a non-human mammal. In a specific embodiment, the ADSC is a pig ADSC. In one embodiment, the non-human mammal treated is a cat, and in another embodiment, the non-human mammal treated is a dog.

G. Use of ADSC in the treatment of chronic renal failure

Chronic kidney disease is characterized by a progressive loss of renal function over an extended period (eg, months or years), which is usually identified by an increase in serum creatinine content and / or an increase in protein content in the urine. The development of chronic renal failure is often associated with the presence of diabetes, hypertension, and glomerulonephritis in afflicted non-human mammals.

ADSCs of the present invention are used to treat, for example, to improve the symptoms of chronic renal failure in a non-human mammal with chronic renal failure, wherein the treatment comprises intravenously administering an effective amount of ADSC to the afflicted non-human mammal. In some embodiments, the effective amount of ADSC can be from about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC, about 1 × 10 ⁶ ADSC to about 1 × 10 ⁷ ADSC. In a specific embodiment, afflicted mammals per kilogram of body weight deliver 1 x 10 ⁶ ADSCs intravenously. Repeat administration as needed at the discretion of the veterinarian treating the animal. In specific embodiments, the administration is repeated weekly as needed, for example, for several weeks, according to the veterinarian's decision to treat the animal. In some embodiments, the effective amount of ADSC administered intravenously is between about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC , About 1 × 10 ⁶ ADSC to about 1 × 10 ⁷ ADSC. In other aspects of these embodiments, the administered dose of ADSC and BMP is adjusted depending on the size of the animal. In some embodiments, therefore, the effective amount of ADSC administered is from about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg body weight, about 5 × 10 ⁵ ADSC / kg The weight ranges from about 5 × 10 ⁷ ADSC / kg body weight, from about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. In other specific embodiments, the animal treated is a non-human mammal and ADSC is from a non-human mammal. In a specific embodiment, the ADSC is a pig ADSC. In another embodiment, the non-human mammal treated is a cat, and in another embodiment, the non-human mammal treated is a dog.

H. Use of ADSC in the treatment of acute renal failure

Acute renal failure is characterized by a relatively rapid loss of renal function. There are many causes of acute renal failure, including blood volume caused by any of the causes, exposure to kidney-harmful substances, and urinary tract obstruction. Acute renal failure can be identified by, for example, elevated blood levels of urea nitrogen and creatinine, and by renal inability to afflict non-human mammals that cannot produce a sufficient amount of urine.

ADSCs of the present invention are used to treat, for example, to improve the symptoms of acute renal failure in non-human mammals with acute renal failure, wherein the treatment comprises injecting an effective amount of ADSC into a non-human breastfeeding patient with acute renal failure The kidney cortex of each kidney of the animal. In some embodiments, the effective amount of ADSC can be from about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC, about 1 × 10 ⁶ ADSC to about 1 × 10 ⁷ ADSC. In a specific embodiment, 1 × 10 ⁶ ADSCs are delivered per kilogram of tortured mammal. In a specific embodiment, 1 × 10 ⁶ ADSCs in saline are injected into the renal cortex of each kidney of a tortured non-human mammal. Repeat administration as needed at the discretion of the veterinarian treating the animal. In specific embodiments, the administration is repeated weekly as needed, for example, for several weeks, according to the veterinarian's decision to treat the animal. In some embodiments, the effective amount of ADSC administered is between about 1 × 10 ⁵ ADSC to about 1 × 10 ⁸ ADSC, about 5 × 10 ⁵ ADSC to about 5 × 10 ⁷ ADSC, about 1 × 10 ⁶ ADSC to approximately 1 × 10 ⁷ ADSC. In other aspects of these embodiments, the administered dose of ADSC and BMP is adjusted depending on the size of the animal. In some embodiments, therefore, the effective amount of ADSC administered is from about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg body weight, about 5 × 10 ⁵ ADSC / kg The weight ranges from about 5 × 10 ⁷ ADSC / kg body weight, from about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. In other specific embodiments, the animal treated is a non-human mammal and ADSC is from a non-human mammal. In a specific embodiment, the ADSC is a pig ADSC. In another embodiment, the non-human mammal treated is a cat, and in another embodiment, the non-human mammal treated is a dog. In yet another embodiment, the ADSC is administered before, after, or concurrently with a hydrogel, such as (but not limited to) a polyglucosamine-based hydrogel. In one aspect of this embodiment, ADSC is distributed in a hydrogel (such as, but not limited to, a polyglucosamine-based hydrogel).

Examples

These examples are intended to be illustrative of the invention and should not be construed as limiting the invention in any way, to illustrate and detail various aspects of the invention discussed above.

Example 1 Experimental treatment of canine urinary incontinence using adipose tissue-derived stem cells Materials and methods Animal selection

Female dogs with significant UI symptoms were selected for this study. These symptoms include drops Autonomous control of urine, loss, and / or scald-soaked dermatitis. To ensure that an individual's UI symptoms are caused by a urethral defect, they must demonstrate responsiveness to PPA treatment. Each owner also agreed to enter this experimental study by signing a consent form that instructs (1) the dog will undergo a test injection of ADSC in the hind legs to ensure that there is no immune response, and (2) cancel the dog's PPA Several days (up to 1 week) for UI symptoms to reappear, (3) dogs must show UI symptoms to reappear for at least 3 days before undergoing ADSC treatment, and (4) if the owner is unwilling to cancel and / or Taking care of the dog after ADSC treatment, the dog may need to be hospitalized until symptoms improve.

Pre-treatment check

Prior to treatment, all dogs undergo a physical examination, routine clinical chemistry and hematological assessments, and urinalysis to ensure that the individual as a whole is healthy and free of primary urinary tract infections. In addition, the test injection site in the hind legs will be checked to ensure no inflammation.

Treatment procedure

In this study, the treatment procedure for UI was to inject 1-10 million porcine ADSCs into the sphincter area of the urethra. Urine testers that allow both visualization and injection are used to complete precise injections. The volume of the suspension and the number of cells vary in proportion to the weight of the individual. For example, a 20 kg dog per injection will receive 2 million ADSCs in 0.2 ml PBS. Typically, 5 million cells are used per 10 kg of body weight.

Post-treatment assessment

The owner will perform assessments daily by observing the following characteristics: monitoring of signs of reaction at the injection site, such as redness, pain, and fever; monitoring of allergic reactions in animals and signs of urinary blockage; any other unusual signs in animals, such as Persistent nausea, fatigue, narcolepsy, decreased appetite, and difficulty urinating.

Veterinarians perform assessments 10 to 15 days, 25 to 30 days, and 55 to 60 days after treatment.

Example 2 Treatment of Osteoarthritis with ADSC Treatment procedure

Although OA can afflict virtually any joint, it is most commonly associated with the hind knee and hip joints. Injecting ADSC into these joints is similar to injecting corticosteroids, except that cell suspensions are used instead of steroids. The volume of the cell suspension and the number of cells vary in proportion to the size of the individual. As an example, a 20 kg dog will receive 5 million ADSCs per joint in 0.5 ml PBS. After treatment, the owner is advised to lead the dog for 30 minutes a day. Dogs are allowed to continue taking any oral medicine they took before ADSC treatment. Any intra-articular treatment before ADSC treatment should be discontinued.

Post-treatment assessment

Animal evaluation will include medical records, physical examinations, and lameness examinations, which include joint mobility and pain records during operation. Clinical outcome measures will be based on an animal orthopedic assessment using a numerical rating scale based on a standardized questionnaire. Baseline results for both owner and animal evaluations will be recorded between 2 and 14 days before the dog receives the test or control formulation by intra-articular injection. Follow-up visits to animal clinics will be required 30, 60, and 90 days after intra-articular injection in dogs. At each visit, the owner will be required to complete a numerical rating scale (1 (best) to 5 (worst)) as part of a standard questionnaire adapted from the Cincinnati Orthopaedic Disability Index, which includes Evaluation: walking, running, jumping, sudden turning, lying down, standing up, climbing stairs, descending stairs, squatting to urinate or defecate, stiffness in the morning, stiffness at night, difficulty walking on smooth floors, and voluntary play Will.

Example 3 Treatment of non-human mammals with diabetes using ADSC

Cell lines β Pdx1 (insulin producing cells) in the key to the development of gene regulation and transformation in the form of lines of Pdx1 PV-16 chromatography (e.g., see Tang et al., Laboratory Investigation 86: 829-841 (2006 ); Cao et al., Diabetes 53: 3168-3178 (2004); Tang et al., Laboratory Investigation 86: 83-93 (2006)). This example illustrates the results of ADSCs being transformed into insulin-producing cells after transfection with a lentivirus carrying the Pdx gene or PV-16. These cells can then be injected into the portal vein (as in island cell cell transplantation) to help individuals with diabetes. This treatment requires approximately 1 billion cells.

Non-human ADSC lines were transfected with Lenti-Pdx1, Lenti-PV-16 or Lenti-GFP (GFP, green fluorescent protein), with the latter serving as a negative control. After transfection, cells were cultured in differentiation medium. After 21 days, the morphology of GFP-transfected cells remained unchanged, while the morphology of Pdx1- or PV-16-transfected cells changed significantly. In addition, cells transfected with Pdx1- or PV-16 appeared to secrete granular material. The expression of Pdx1 mRNA by transfected cells was confirmed and measured by RT-PCR. Western blot was used to confirm the performance of Pdx1 protein.

Insulin production from transfected cells was also analyzed by staining the cells with anti-insulin antibodies.

Example 4 Method for separating ADSC using circulating collagenase

Adipose tissue (ie adipose tissue or liposuction fat) isolated from non-human mammals was washed with PBS containing 1% penicillin and streptomycin, chopped into small pieces, and then a 5: 1 v / v collagenase solution : Fat ratio is mixed with a solution (Sigma-Aldrich, St. Louis, MO) containing 0.075% collagenase type IA. After incubation at 37 ° C for 1 hour with vigorous shaking, the product was then centrifuged at 220 x g for 10 minutes at room temperature. Three layers are formed: the upper lipid layer, the middle collagenase layer, and the bottom cell pellet. The intermediate layer was collected and filtered with a 200 μm filter, followed by centrifugation. Use circulating collagenase type IA in the effluent to digest fresh adipose tissue (also reducing the cost of purchasing a large amount of collagenase), using a ratio higher than the first round (7: 1, by volume).

Example 5 Preservation of ADSC with Superoxide Dismutase

This example illustrates a new adipose tissue preservation solution (ATPS) containing superoxide dismutase isolated from mammalian red blood cells to preserve cell viability. ATPS system of _{200 mg / L KH 2 PO 4} , 200 mg / L KCl, 2.16 g / L Na 2 HPO 4. 7H 2 O, 8 g / L NaCl, 30,000 units / L of superoxide dismutase (SOD) and 5 g / L bovine serum albumin (BSA).

Adipose tissue was stored at 4 ° C for 24 hr and 48 hr using ATPS. Adipose-derived stem cells were isolated using the stored adipose tissue according to the procedure described above. The yields of adipose-derived stem cells obtained from ATPS-preserved tissue and adipose tissue freshly harvested from two individuals were compared. Compared to freshly harvested adipose tissue, the yield of ADSC was 85% at 24 hours and 65% at 48 hours. This is much higher than the yield obtained in the absence of ATPS, as reported by Matsumoto et al., Plast. Reconstr. Surg. (2007) 120 (6): 1510-7.

ATPS has also been tested for the preservation of adipose stem cells. 1 × 10 ⁶ non-human fat-derived stem cells were mixed with 1 ml ATPS and stored at 4 ° C. After 48 hours, a cell viability test (trypan blue method) was performed. The results indicate that less than 5% of the cells were damaged after 48 hours of storage with ATPS.

Example 6 Method for separating better ADSC groups

Effective and accurate separation of specific cell types is the defining aspect of many research projects. The use of antibodies for cell isolation is not new. "Panning" (ie, immunoselection methods for enriching specific cell populations from a mixture of different cell types) has been around for a long time. This method is based on the ability to select antibodies bound to a cell culture dish. The cell type mixture was cultured on antibody coated plates and allowed to bind for a short period of time. Non-adherent cells (those who do not bind antibodies) can then be gently lysed from the culture dish, allowing the bound cells to be harvested. This method helps to develop other new technologies (such as density-gradient separation) and methods that take advantage of the unique surface-binding properties of specific cell types. The development of magnetic bead technology allows repeated washing of the bead-bound cells, greatly improving the potential separation purity. The size and composition of the paramagnetic beads used by the companies are significantly different and further improvements / improvements occur regularly.

The matrix vascular component derived from adipose tissue digestion is composed of many types of cells, such as stem cells, endothelial cells, smooth muscle cells, and other terminally differentiated cells. The magnetic cell system provides excellent magnetic bead-labeled cell sorting.

Freshly isolated ADSCs were analyzed for cell surface antigen performance by flow cytometry according to the manufacturer's protocol. Cells were incubated with primary antibodies (Table 1) in 50 μl wash buffer (PBS containing 1% FBS and 0.1% Na ₃ N) for 30 minutes on ice, and then FITC-conjugated secondary antibodies ( Goat anti-IgG). Cells were then washed twice with washing buffer, fixed with 1% paraformaldehyde in PBS, and analyzed by a fluorescence activated cell sorter (FACS Vantage SE; Becton Dickinson). Results were analyzed using FlowJo software (Tree Star Co., Ashland, OR). The cellular antigens analyzed were CD13, CD31, CD34, CD90, CD105, CD133, SSEA-1 and telomerase. The performance contents are shown in Table 2.

Antibody mix

According to the results of flow cytometry, three types of markers were selected as positive cell markers for analysis. Isolated fat-derived stem cells. The antibody mix includes mouse anti-CD34, mouse anti-CD90, and mouse anti-SSEA1 in a 2: 3: 5 ratio.

Positive selection of CD34 (+) / CD90 (+) / SSEA1 (+) by MACS

Non-human adipose-derived stem cells were grown with CD34 (+) / CD90 (+) / SSEA1 (+). Positive cells were selected using pan-mouse antibody magnetic beads.

Proliferation difference: CD34 (+) / CD90 (+) / SSEA1 (+) vs. CD34 (-) / CD90 (-) / SSEA1 (-) cells

Positive cells and negative empty cells selected using the CD34 (+) / CD90 (+) / SSEA1 (+) mixture were cultured and used for cell proliferation analysis by MTT test. The results showed that positive cells grew faster than negative selection cells.

Cytokinin secretion differences: CD34 (+) / CD90 (+) / SSEA1 (+) versus CD34 (-) / CD90 (-) / SSEA1 (-) cells

Cell culture medium using positive and negative selection cells was tested for cytokine production by a cytokine array. The results indicate that positive selection cells secrete more interleukins, such as MCP1, b-NGF, TIMP-1 TNF-a, IL-1b, CINC-1, and the like.

Differences in restoration of erectile function in vivo: CD34 (+) / CD90 (+) / SSEA1 (+) vs. CD34 (-) / CD90 (-) / SSEA1 (-) cells

Positive and negative cultures were selected for ADSC and injected into rat cavernous body after crush injury of cavernous nerve. After four weeks, erectile function was evaluated by nerve stimulation. The results showed that positive selection cells significantly improved erectile function, while negative cells did not improve.

Example 7 Culture of ADSC in Platelet Lysate

In most ADSC standard media, animal serum is the basic component. However, integration of animal proteins into stem cells has been reported and this is a major problem in human cell therapy. A novel method for culturing ADSCs in platelet lysate medium is described herein.

Platelet lysate was obtained from whole blood according to the following procedure. Whole blood was drawn into 4 50 ml sterile plastic tubes containing sodium citrate dihydrate and centrifuged at 350 × g for 10 minutes. Platelet-rich plasma fractions were washed with an equal volume of phosphate buffered saline (PBS) containing 0.38 mg / ml sodium citrate dihydrate. Platelets were then centrifuged at 510 × g for 10 minutes and the pellet was suspended in DMEM to a final concentration of 1 × 10 ⁹ cells / ml to 2 × 10 ⁹ cells / ml. Platelet lysis (and subsequent release of chemokines and growth factors) was obtained by a single cycle of freezing (80 ° C) and thawing (37 ° C). Platelet lysate (12,000 × g, 10 minutes) stored in DMEM was centrifuged at high speed to remove the cell membrane, and the supernatant was extracted and stored at -80 ° C.

To test the ability to maintain and promote cell growth and proliferation, ADSCs were cultured in serum-free media and media containing 10% FBS, 2% platelet lysate, and 4% platelet lysate. The proliferation effect of platelet lysate on ADSC was evaluated by MTT test. The results indicate that 4% platelet lysate has the same effect on supporting cell growth and proliferation as 10% FBS.

Example 8 Using 60 micron PLGA microspheres and carboxymethyl cellulose as a carrier for ADSC

An injectable poly (lactic-co-glycolic acid) (`` PLGA '') solution was prepared by first mixing 1 μg of 60-μm PLGA microspheres with 1 ml of 0.5% carboxymethyl cellulose (CMC) and dissolving in PBS. ). Approximately 5 × 10 ⁶ ADSCs were mixed with 1 ml of this PLGA / CMC mixture. After 30 minutes of incubation on ice, the ADSC / PLGA mixture was injected into the urethra. As a control, ADSC mixed with saline was injected into different groups of animals. After four weeks, the animals were killed and examined for tissue. The results showed that more ADSCs remained in the injection area in the ADSC / PLGA group than in the saline group.

Example 9 Treatment of wounds and other conditions using ADSC

To assess the extent to which ADSCs can spontaneously differentiate into blood vessels. For example, ADSC is injected subcutaneously into the subcutaneous space of healthy rats. Histological studies have shown that ADSCs differentiate into a variety of local cell types, including blood vessels, fats, muscles, connective tissue / fibroblasts, and cells surrounding the hair follicles.

ADSC was also injected subcutaneously along the suture site of the surgical wound. On one side of the suture Subcutaneous injections of ADSC suspended in buffer were received, while the other side received only buffer injections. The results showed that the side of the wound receiving ADSC developed significantly higher density blood vessels. In addition, the degree of scarring on the side of the wound that received ADSC appeared to be weaker compared to the control side. The experiments were repeated with the design changed so that the same wounds were created in the same animal, and the experimental wounds received ADSC, while the control wounds (or the same) did not receive ADSC. These results are highly reproducible.

Other studies have shown that the injected ADSCs preferentially differentiate into blood vessels in the wound environment. ADSC is introduced into the wound environment using an agent called matrigel. The Matrigel system serves as a biocompatible but otherwise inert material for a dense gel that allows oxygen and micronutrients to diffuse, but is dense enough to prevent local tissue cells from growing in. The important system is that it exists in liquid form under artificial cold temperature and semi-solid form under body temperature.

In the experimental cohort, ADSC was suspended in a set volume of Matrigel, and Matrigel was then injected percutaneously into the subcutaneous space of rat dorsal hump. In the same rat, the same volume of ADSC-free matrigel was injected as a control on the opposite side of its dorsal bulge. Matrigel was kept in the wound site for 10 days, and then Matrigel was excised from the wound space, sectioned, and evaluated for histology. The results showed that labeled ADSCs suspended in matrigel differentiated into vascular / endothelial cells. In addition, the edges and centers of matrigel juxtaposed to the injured oral cavity showed higher vessel density. ADSCs are still labeled with nuclear markers, confirming that the visualization of neovascularization corresponds to ADSC rather than to local vascular growth. Control injections of matrigel showed no angiogenesis.

The results described above show that the host tissue environment within the target site affects the cell types caused by ADSC differentiation. The injected ADSCs respond to the wound environment by preferentially differentiating into blood vessels, which is consistent with the natural wound healing response, that is, local tissue hypoxia affects the preferential differentiation of ADSCs into blood vessels. In addition, ADSC appears to spontaneously differentiate into other cell types including natural wound healing devices, such as fibroblasts and inflammatory cells. Specific wound applications include, but are not limited to, prevention of surgical anastomosis, radiation-induced wounds, surgical anastomosis (e.g., colon Ostomy, urethral stoma) and cosmetic surgery scarring / stenosis. ADSC treatment can also be used to prevent the development of pressure ulcers (bedsore ulcers) or to promote the healing of existing pressure ulcers.

Example 10 Other materials and methods ADSC isolation and culture

Isolation of rat ADSC was performed as described in Ning H et al. Differentiation 74: 510-518 (2006).

Lentiviral transduction

Lentiviral constructs containing mouse Pdx1, Pdx1-PV-16 (PV-16, a genetically modified form of Pdx1) or green fluorescent protein (GFP as a control) have been described. See, for example, Tang DQ et al., Lab.Invest. 86: 829-841 (2006) and Tang DQ et al., Lab.Invest. 86: 83-93 (2006). These constructs were transduced into ADSC at a multiplicity of infection (MOI) of 20 overnight. After two days of recovery, the transduced cells were switched to differentiation medium (DMEM with 23 mM glucose), grown to confluence, and split into approximately 5 × 10 ⁵ cells per 10 cm dish at 1: 3. After that, it is split at 1: 3 every 7 to 10 days.

Measurement of insulin secretion

To measure static insulin secretion, human ADSC lines transduced with GFP, Pdx1, or PV-16 were cultured in a differentiation medium (20 mM glucose) with FBS for 3 weeks. Cell culture media was then collected and analyzed using a commercially available ELISA kit (catalog number EZHIASF-14K, Linco Research, St. Charles, MO) for human insulin. To measure insulin secretion in response to glucose challenge, cells grown in 6-well plates (6 × 10 ⁴ cells / well) were washed 3 times with PBS and then in KRB buffer (120 mM NaCl, 2.5 mM CaCl ₂ , 1.1 mM MgCl ₂ , 25 mM NaHCO ₃ and 0.1% BSA) for 1 h. Glucose was then added to final concentrations of 0 mM, 5.6 mM, 16.7 mM, 23 mM and 33 mM. After an additional 1 hour of incubation, KRB buffer was collected and analyzed using a commercially available ELISA kit (catalog number EZRMI-13K, Linco Research, St. Charles, MO) for rat insulin. The experiments were performed in triplicate.

Establishment of a rat model of type 1 diabetes

Streptozotocin (STZ) is known to selectively destroy pancreatic β-cells (Mansford KR et al., Lancet 1: 670-671 (1968)) and is also used to establish a rat model of type 1 diabetes (El-Sakka AI et al. , Int J Impot Res 11: 123-132 (1999). Two-month-old female Sprague-Dawleys were injected by intraperitoneal injection of 60 mg STZ (stored in 20 mM citrate buffer) / kg body weight. (Sprague-Dawley) rats developed hyperglycemia. Control rats were injected with 20 mM citrate buffer. Thereafter, samples obtained from the tail vein were monitored weekly using an Accutrend band (Roche Diagnostics, Indianapolis, IN). Blood glucose content. When blood glucose reaches 300 mg / dl (in about 7 days), STZ-treated rats are subjected to injection of IP-ADSC or saline under the renal capsule.

Cell transplantation under the renal capsule

The rats were anesthetized by exposure to 1% to 3% isoflurane, and a 2 cm incision was made through the skin and muscles of the left abdomen. The wound was rinsed with 1 ml of penicillin / streptomycin in PBS and the kidneys were pulled externally. A small transverse incision was made in the sac, and 2 × 10 ⁶ IP-ADSC stored in 1 ml PBS or only PBS was dispensed under the sac. The kidney is returned to the abdominal cavity and the incision is closed using a surgical clip. The injection site was marked with 7-0 nylon suture for identification.

Glucose tolerance test

The intraperitoneal glucose tolerance test (IPGTT) was performed as described in Animal Models of Diabetes Complications Consortium (www.amdcc.org). Mice were fasted for 7 hours and then intraperitoneally injected with 1 mg of glucose / gram body weight in saline. Blood glucose levels in the samples obtained from the tail vein were monitored at 30 minute intervals for 2 hours.

Immunochemistry and fluorescence microscopy

Cells were seeded on coverslips with 40% to 60% confluence in DMEM in each well of a 6-well plate. The next day, cells were washed with PBS and fixed with ice-cold methanol for 5 minutes. The cells were washed again with PBS and permeabilized with 0.05% triton X-100 for 8 minutes. After another PBS rinse, the cells were incubated with 5% horse serum for 1 hour and then with an anti-insulin antibody (Abcam Ltd., Cambridge, MA, 1: 500) for 1 hour. After washing 3 times with PBS, cells were incubated for 1 hour using Texas red conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). After washing 3 times with PBS, the cells were stained with 4 ', 6-dimethylmethyl-2-phenylindole (DAPI, for nuclear staining, 1 μg / ml, Sigma-Aldrich, St. Louis, MO) 5 minutes. Stained cells were examined using a Nikon Eclipse E600 fluorescence microscope and images were recorded using a Retiga 1300 Q-phase camera.

Histology and immunofluorescence

Tissue samples were fixed in cold 2% formaldehyde and 0.002% saturated picric acid (pH 8.0) in 0.1 M phosphate buffer solution for 4 hours, and then immersed in a buffer solution containing 30% sucrose overnight. Samples were then embedded in OCT compounds (Sakura Finetic USA, Torrance, CA) and stored at -70 ° C until use. A fixed frozen tissue sample was cut at 10 microns, mounted on a SuperFrost-Plus slide (Fisher Scientific, Pittsburgh, PA) and air-dried for 5 minutes. These slides were stained with hematoxylin and eosin (HE staining) for general histological examination. For immunofluorescence, slides were placed in 0.3% H ₂ O ₂ / methanol for 10 minutes, washed twice in PBS for 5 minutes, and stored in PBS / 0.3% Triton X- at room temperature. 3% of horse serum was incubated for 30 minutes. After draining this solution from the tissue sections, the slides were incubated at 4 ° C overnight and incubated with anti-insulin antibodies (Abcam Ltd., Cambridge, MA, 1: 500) for 1 hour. Control tissue sections were prepared in a similar manner, except that no primary antibody was added. After rinsing, the sections were incubated with Texas Red-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA), followed by 4 ', 6-dimethylmethyl-2-phenylindole (DAPI For nuclear staining, 1 μg / ml, Sigma-Aldrich, St. Louis, MO) staining.

Western blot analysis

Lyse cells in a buffer containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, aprotinin (10 μg / ml), leupeptin (10 μg / ml), and PBS . Cell lysates containing 20 μg of protein were electrophoresed in SDS-PAGE and then transferred to a PVDF membrane (Millipore, Bedford, MA). Ponceau S was used to stain the membrane to verify the integrity of the transferred protein and to monitor unbiased transfer of all protein samples. Detection of proteins on the membrane was performed using an ECL kit (Amersham Life Sciences Co., Arlington Heights, IL) using an anti-Pdx1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The images obtained were analyzed using ChemiImager 4000 (Alpha Innotech Corporation, San Leandro, CA) to determine the integrated density value (IDV) of each protein band. Prior to re-probing with anti-b-actin antibody, the membrane was peeled at 62.5 mM Tris-HCl (pH 6.7), 2% SDS, 10 mM 2-mercaptoethanol at 56 ° C for 30 minutes, and then at 1 Wash 4 times in TBST.

RT-PCR analysis

The cells were homogenized in Tri-Reagent RNA extraction solution (Molecular Research Center, Cincinnati, OH). The extracted RNA was further processed with DNase I to remove traces of contaminating DNA. The amount and integrity of RNA were checked by spectrophotometer and agarose gel electrophoresis, respectively. SuperScript reverse transcriptase and its accompanying reagents (Invitrogen, Carlsbad, CA) were used to reverse transcribe RNA into a "complementary DNA (cDNA) library". Briefly, 2.5 μg of RNA was annealed to 0.4 μg of oligo-dT primer in a 12-μl volume. Then 4 μl of 5 × buffer, 2 μl of 0.1 M DTT, 1 μl of 10 mM dNTP, and 1 μl of reverse transcriptase were added to bring the final reaction volume to 20 μl. After incubation at 42 ° C for 1 hour, the mixture was incubated at 70 ° C for 10 minutes to inactivate the reverse transcriptase. Then 8 μl of TE buffer was added to make a 5 × diluted library. A portion of this library was further diluted to various concentrations (up to 100 x dilutions). Then use 1 μl of each dilution in a 10-μl polymerase chain reaction (PCR) to identify Do not optimize the input in the linear range. Except for a 1-μl dilution library, the PCR mix consisted of 10 ng of each primer pair (Table 1) and reagents supplied with Taq polymerase (Invitrogen). PCR was performed in a DNA Engine (MJ Research Co., Watertown, MA) under the calculated temperature control. The cycle program was set to 94 ° C, 5 seconds; 55 ° C, 5 seconds; 72 ° C, 35 cycles of 10 seconds, and then 72 ° C, 1 cycle of 5 minutes. The PCR products were electrophoresed on a 1.5% agarose gel in the presence of ethidium bromide, visualized by UV fluorescence, and recorded by a digital camera connected to a computer.

data analysis

The data was analyzed using Prism 4 (GraphPad Software Co., Ltd., San Diego, CA) and expressed as mean ± standard deviation. A Student-t test was used to compare between two groups (e.g., treated and control). One-way ANOVA analysis of variance was used to compare between 3 or more groups. Differences at P <0.05 were considered significant.

Example 11 Characteristics of human and rat ADSC cell lines

Isolation and characterization of human and rat ADSC have been reported (Lin G et al., Stem cells and development 17: 1053-1063 (2008); Ning H et al., Differentiation 74: 510-518 (2006)). The newly isolated cells are attached to a plastic petri dish allowing the selection of the morphologically homogeneous population of the ADSC line. The attached cells were in the shape of fibroblasts and grew at a rate of doubling about every 3 days. In the third generation, it was mostly negative for endothelial marker CD31 and hematopoietic marker CD34. It also shows extremely low levels of stem cell markers Oct4, SSEA1 and telomerase. However, although there is no obvious stem cell marker, both of these human and rat ADSCs are able to differentiate into endothelial cells and neuron-like cells (Ning H et al. Differentiation 74: 510-518 (2006); and Ning H et al. , FGF2 Promotes Endothelial Differentiation of Adipose Tissue-Derived Stem Cells. J Sex Med (in publication)).

Example 12 Treatment of Type 1 Diabetes with Adipose Tissue-Derived Stem Cells (ADSC) Transduction and expression of Pdx1 gene in ADSC

Two humans and five rat ADSC lines were used as candidates for Pdx1 transduction. In addition, Pdx1-PV-16 (PV-16) was also used to transfect the two human ADSC lines, and Pdx1-PV-16 (PV-16) was a genetically modified form of Pdx1. The transduction using GFP served as a negative control and was used to determine the transduction efficiency, and it was found that the transduction efficiency was higher than 95% (the percentage of cells showing green fluorescence). One week after transduction, Pdx1- and PV-16 transduced cells (rather than GFP transduced cells) exhibited a morphology indicating secretion of insulin particles (Figure 1), which was subsequently confirmed by immunofluorescence staining (Figure 1). RT-PCR and Western blot analysis also confirmed that Pdx1 was expressed in Pdx1- and PV-16 transduced cells (not in GFP transduced cells) (Figure 2). Finally, ELISA analysis showed static production of insulin in Pdx1-transduced cells (Figure 2); thereby producing ADSC of insulin (IPADSC).

Performance of Pdx1-related genes in IPADSC

Pdx1 controls the performance of several key genes during pancreatic development, including the insulin, glycogen, and NeuroD genes. RT-PCR analysis showed that human ADSCs constitutively expressed glucagon and NeuroD (Figure 3). It also exhibits lower levels of insulin, which is upregulated in IPADSC. ADSCs in control rats expressed insulin, glycogen, and NeuroD at lower levels than IPADSC. The specificity of the expression of these 3 genes in the control and IPADSC was confirmed by the absence of such expression in rat urethral smooth muscle cells (Figure 3).

Increased insulin production in response to glucose challenge

As discussed above, Pdx1 transduction resulted in the generation of IPADSC, which released approximately 30 ng / dl of insulin into the culture medium (Figure 2). Quantitative analysis showed that these cells produced increased levels of insulin in response to increasing concentrations of glucose (Figure 4).

Treatment of diabetic rats with IPADSC

Type 1 DM rats are produced by intraperitoneal injection of STZ. One week after STZ injection, the blood glucose content of these rats ranged from 300 mg / dl to 400 mg / dl, while control mice injected with citrate buffer only had normal blood glucose content. Then use Ten of the STZ treated rats were treated with IPADSC, while the other 10 STZ treated rats were treated with saline. Treatment was performed by transplanting approximately 2 million rat IPADSCs or injecting saline under the renal capsule. The fasting blood glucose content and body weight of these rats were then monitored weekly for 7 weeks. As shown in Figure 5A, throughout the entire course, IPADSC-treated rats had lower blood glucose levels than saline-treated rats (P <0.05). The weight of IPADSC-treated rats was also better than that of saline-treated rats, but the difference was not statistically significant (P> 0.05) (Figure 5B). At the end of week 7, the appearance of the fur and the degree of cataract were examined in all rats, their glucose tolerance was tested, and then they were sacrificed for histological evaluation. The results showed that compared to saline-treated rats, IPADSC-treated rats had a healthier (less shaggy) appearance of fur and a lower degree of cataracts (Figure 6). IPADSC treated rats also had higher levels of glucose tolerance (Figure 7). Finally, a histological examination of the transplanted kidney revealed the presence of the transplanted cells, which were stained positive for insulin (Figure 8).

Example 13 Labeling and Tracking Stem Cells with EdU

Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) is a synthetic nucleoside that can be incorporated into the newly synthesized DNA of replicating cells. BrdU-containing cells can then be detected by immunochemistry using BrdU-specific antibodies. The BrdU labeling method has been mainly used to analyze the cell cycle in cultured cells and to visualize proliferating cells in the central nervous system. It is also used to identify stem cells, which are believed to divide slowly or asymmetrically separate chromosomes, thereby allowing BrdU to reside in slowly isolated stem cells or progeny (non-differentiated) stem cells (rather than differentiated daughter cells). In addition, BrdU labeling has been used to track labeled stem cells or non-stem cells in vitro and then transplanted in vivo. In the case of stem cells, this tracking makes it possible to determine whether the transplanted stem cells have differentiated into a particular cell type.

Currently, the selection method for labeling dividing cells is to incorporate thymidine analog 5-bromo-2-deoxyuridine (BrdU) into the DNA of S-phase cells. After the labeled cells were fixed, BrdU was detected using BrdU-specific antibodies. However, BrdU immunochemistry can be problematic because of the need for strong DNA denaturing conditions (such as strong acid and heat) to expose epitopes that are hidden within the DNA. This will Significant variability was introduced within and between experiments. In an effort to overcome these problems, the optional thymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been introduced. The terminal alkyne group of EdU allows detection using fluorescent azide covalently bonded to the alkyne group. This detection method is fast and specific, and does not require DNA denaturation. The purpose was to study the feasibility of labeling ADSCs in vitro with EdU and tracking labeled cells in vivo.

animal

All animal experiments in the study were approved by the Institutional Animal Care and Use Committee of University of California, San Francisco. A total of 12 three-month-old, non-born Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were randomly divided into two groups. One day before delivery, 200 μg of EdU in PBS was injected (i.p.) in the test group and only (i.p.) PBS was injected in the control group. Newborn rats were used to track in vivo labeled EdU. One week after delivery, adipose tissue was collected from adult rats for ADSC isolation, and then ADSC was used to track EdU-labeled cells in vivo.

EdU mark

ADSCs were grown on glass coverslips in DMEM supplemented with 10% calf serum, penicillin and streptomycin. For dose effects, EdU was added to the culture medium at 0 μM, 10 μM, 20 μM, and 50 μM. After 24 hours, the cells were washed with PBS, followed by the addition of conventional media (Figure 9). For time course studies, ADSC was labeled with 10 μM EdU and then split at 1 day, 4 days, 7 days, 14 days, and 21 days (Figure 10).

EdU staining

After methanol fixation, the cells were washed twice with PBS, and then incubated at room temperature in 3% BSA in PBS and then in 0.5% Triton® X-100 in PBS for 20 minutes. Cells were then incubated for 30 minutes at room temperature in the dark using freshly made Click-it reaction mixture (Invitrogen). The cells were contrast stained with DAPI, mounted in a standard mounting medium and imaged with a fluorescence microscope.

EdU mark of organization

Pregnant rats were injected intraperitoneally with 200 μg of EdU in PBS, and newborn rats were used to collect various tissues at 2 hours, 1 week, and 6 weeks after birth. The collected tissues were fixed in cold 2% formaldehyde and 0.002% saturated picric acid (pH 8.0) in 0.1 M phosphate buffer solution for 4 hours, and then immersed in a buffer solution containing 30% sucrose overnight. Samples were then embedded in OCT compounds (Sakura Finetic USA, Torrance, CA) and stored at -70 ° C until use. Thirty different tissues from both the EdU injection group and the PBS injection group (Table 3) were processed for tissue arrays. A fixed frozen tissue sample was cut at 10 microns, mounted on a SuperFrost-Plus slide (Fisher Scientific, Pittsburgh, PA) and air-dried for 5 minutes. EdU staining was performed on the tissues as described above. Hematoxylin and eosin (HE staining) were also used to stain the tissue for general histological examination (Figure 11).

Tracking transplanted ADSC

A total of 1 × 10 ⁶ rat ADSCs were labeled with 10 uM EdU for 12 hours and autologously injected into the bladder neck. Tissues were collected 1 day, 2 days, 1 week, and 4 weeks after transplantation. Tissue samples were fixed in cold 2% formaldehyde and 0.002% saturated picric acid (pH 8.0) in 0.1 M phosphate buffer solution for 4 hours, and then immersed in a buffer solution containing 30% sucrose overnight. Samples were then embedded in OCT compounds (Sakura Finetic USA, Torrance, CA) and stored at -70 ° C until use. A fixed frozen tissue sample was cut at 10 microns, mounted on a SuperFrost-Plus slide (Fisher Scientific, Pittsburgh, PA) and air-dried for 5 minutes. For immunofluorescence, slides were placed in 0.3% H ₂ O ₂ / methanol for 10 minutes, washed twice in PBS for 5 minutes, and stored in PBS / 0.3% Triton X- at room temperature. 3% of horse serum was incubated for 30 minutes. After draining this solution from the tissue sections, the slides were incubated for 1.5 hours at room temperature with an anti-α smooth muscle actin antibody (Abcam Co., Ltd., Cambridge, MA, 1: 500). Control tissue sections were prepared in a similar manner, except that no primary antibody was added. After rinsing, sections were incubated with FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). After washing with PBS, the slides were incubated with freshly prepared Click-it reaction mixture for 30 minutes at room temperature in the absence of light, followed by 4 ', 6-dimethylmethyl-2-phenyl Indole (DAPI, for nuclear staining, 1 μg / ml, Sigma-Aldrich, St. Louis, MO) staining. See Figure 12. Rat ADSC was labeled with 10 μM EdU for 12 hours, and then autologously injected into the bladder neck (A). After 4 weeks, by targeting EdU (if color photos are used, it will be red), α-smooth muscle actin (SMA, if color photos are used, it will be green) It will be blue) Examine the tissue section. EdU-labeled cells can be observed in the bladder neck (B, x 20). Most EdU labeled cells are localized in connective tissue. A few EdU-labeled cells appeared to have differentiated into smooth muscle cells (C, x200).

Example 14 Treatment of Urinary Incontinence with Allogenic ADSC method

In this experiment, 22 Sprague-Dawley rats born at 2 months of age on the 16th day of pregnancy were used. They were randomly divided into a control group (n = 10) and an ADSC transplantation group (n = 12). After delivery, all rats underwent vaginal balloon dilatation and ovariectomy. After 1 week, rats received injections of ADSC or PBS.

Prior to injection, syngeneic rat ADSC was labeled with 10uM EdU for 12 hours. In the treatment cohort, 1 × 10 ⁶ EdU-labeled ADSCs in 400 μl of PBS were injected into the bladder neck and paraurethral tissues. In the control group, 400 μl of PBS was injected into the same area.

Four weeks after the injection, all animals underwent evaluation of bladder function by awake cystometry. If the bladder is filled with frequent low-volume bladder contractions and urine leaks, the bladder manometry results are classified as "abnormal". After bladder manometry, all animals were euthanized. Collect urethra, vagina, pelvic floor tissue and bladder. Immunofluorescence staining was performed on these tissues to locate EdU, smooth muscle actin (SMA), and nucleus (DAPI staining). Chemical staining was also performed to evaluate differences in elastic fibers between treatment groups. Statistical analysis was performed using the Stuart's t-test.

result

Based on the bladder manometry guidelines described above, 8 of 10 rats in the control group had abnormal urinary function, and 4 of 12 (33.3%) rats in the ADSC treatment group had abnormal urinary function . The mean micturition pressure in the ADSC transplantation group was significantly higher than that in the control group (61.7 ± 13.9 cm H ₂ O versus 34 ± 8 cm H ₂ O, respectively) (P <0.05) (Figure 13). Recognize EdU-labeled cells in the submucosa of the bladder neck and urethra. Some of these EdU-positive cells were also positive for SMA, indicating that ADSCs differentiated into smooth muscle cells (Figure 14). There were significantly more elastic fibers in the urethra of rats treated with ADSC (Figure 15).

Example 15 Treatment of urinary incontinence with heterologous ADSC method

In this experiment, 12 Sprague-Dawley rats born at 2 months of age on the 16th day of pregnancy were used. They were randomly divided into a control group (n = 5) and an ADSC transplantation group (n = 7). After delivery, all rats underwent vaginal balloon dilatation and ovariectomy. After 1 week, rats received injections of ADSC or PBS.

ADSCs were isolated from 1 month old male Yorkshire pigs and labeled with 10 uM EdU for 12 hours and then injected. In the treatment cohort, 1 × 10 ⁶ EdU-labeled ADSCs in 400 ul PBS were injected into the bladder neck and paraurethral tissues. In the control group, 400 ul of PBS was injected into the same area.

Four weeks after the injection, all animals underwent evaluation of bladder function by awake cystometry. If the bladder is filled with frequent low-volume bladder contractions and urine leaks, the bladder manometry results are classified as "abnormal". After bladder manometry, all animals were euthanized. Collect urethra, vagina, pelvic floor tissue and bladder. Immunofluorescence staining was performed on these tissues to locate EdU, smooth muscle actin (SMA), and nucleus (DAPI staining). Chemical staining was also performed to evaluate differences in elastic fibers between treatment groups. Statistical analysis was performed using the Stuart's t-test.

result

Based on the bladder manometry guidelines described above, 4 of the 5 rats in the control group had abnormal urinary function, and 1 of 7 (14.2%) rats in the ADSC treatment group had abnormal urinary function. . The mean micturition pressure was higher in the ADSC transplantation group than in the control group (48.7 ± 16.1 cm H ₂ O versus 34.4 ± 7.8 cm H ₂ O, respectively) (P = 0.097) (Figure 16). Recognize EdU-labeled cells in the submucosa of the bladder neck and urethra. Some of these EdU-positive cells were also positive for SMA, suggesting that ADSCs differentiated into smooth muscle cells (Figure 17).

Example 16 Endothelial Differentiation of ADSC Endothelial differentiation in vivo

To test whether ADSC can differentiate into endothelial cells, ADSC was injected into the penis of a rat and the tissue was examined after 4 weeks. ADSC was recognized by BrdU staining, and about 5% of rat endothelial cell antigen (RECA-1) staining was also positive. These cells were localized to sinusoidal endothelial cells, as revealed by superimposed images of fluorescence and phase contrast microscopy (Figure 18).

Morphology and growth characteristics of cells growing in EGM2 medium

ADSCs are grown in DMEM in a conventional manner. Endothelial cells are cultured in conventional manner in EGM2, which is a commercially available endothelial growth medium. When EGM2 is used to replace DMEM in ADSC culture, these cells reach the sink faster than cells kept in DMEM Mergers seem to be tighter (larger cores). Proliferation analysis confirmed that ADSCs grew much faster in EGM2 than in DMEM (Figure 19).

Endothelial properties

Immunochemistry showed that ADSCs grown in EGM2 showed endothelial-specific markers CD31, vWF, and eNOS (Figure 20). Matrigel formation analysis also showed that ADSCs grown in EGM2 can form endothelial-like tube structures. In addition, LDL uptake analysis showed that ADSCs grown in EGM2 have the capacity for LDL uptake. The endothelial specificity of the three analyses was supported by positive results from HUVEC cells.

Reversibility and re-inducibility of endothelial differentiation

Whether ADSC differentiation is reversible is tested by using DMEM instead of EGM2. This resulted in the disappearance of all endothelial properties (Figure 21). Whether endothelial differentiation is re-inducible is tested by reintroducing EGM2 into cells. This leads to the reproduction of all endothelial properties, albeit at reduced levels. These tests determined that the EGM2 medium contained specific factors capable of inducing endothelial differentiation of ADSCs.

Recognition of Endothelial Inducible Factors

EGM2 medium is supplied by the manufacturer in the form of basal medium (EBM2) and individual vials containing supplementary factors. This packaging format allows the importance of each of the supplementary factors associated with ADSC endothelial differentiation to be tested. Specifically, a "subtractive" EGM2 medium was prepared by omitting one supplementary factor at a time. ADSC was then maintained in each of the subtracted EGM2 media for 1 week, and then analyzed for its LDL uptake capacity, which has been shown to be the most reliable endothelial marker. The results showed that the omission of VEGF, EGF, or IGF had almost no effect on growth factors, while the omission of FGF2 greatly reduced the LDL uptake capacity of ADSC (Figure 22). Among non-growth factors, the omission of hydrocortisone or heparin has basically no effect, while the omission of vitamin C greatly reduces the LDL uptake capacity of ADSC. When both FGF2 and vitamin C are omitted, ADSC does not substantially exhibit any LDL uptake capacity. To further confirm the importance of FGF2 and vitamin C, an "additive" medium was prepared by adding FGF2 and / or vitamin C to EBM2, ADSC was maintained in these media for 1 week, and then the LDL uptake capacity of ADSC was analyzed. The results show that (1) EBM2 supplemented with FGF2 and vitamin C is almost as effective as EGM2, (2) EBM2 supplemented with FGF2 is still effective, although the content is reduced, and (3) EBM2 supplemented with vitamin C is still slightly effective, but The content is greatly reduced (Figure 23). It is also worth noting that cells grown in vitamin C-free EGM2 (Figure 22) or FGF2-supplemented EBM2 (Figure 23) exhibit a circular morphology, which can indicate oxidative stress.

Other endothelial properties induced by FGF2

The above experiments identified FGF2 as the only growth factor required to induce the LDL uptake capacity of ADSC. Then test whether FGF2 can induce the expression of other endothelial properties. The results show that this is indeed the case; that is, cells grown in EGF2 supplemented with FGF2 / vitamin C obtained all the endothelial markers tested, as were cells grown in fully supplemented EGM2 (Figure 24).

FGFR1 inhibitor blocks ADSC endothelial differentiation

To further confirm the key role of FGF2 in ADSC endothelial differentiation, ADSC differentiation experiments were performed in the presence or absence of PD173074, a selective inhibitor of FGF receptor (FGFR1). In the absence of PD173074 (solvent only), cells grown in complete EGM2 or FGF2 / vitamin C supplemented EBM2 acquired LDL uptake capacity (Figure 25). On the other hand, in the presence of PD173074, cells grown in either medium were unable to acquire this ability (Figure 25). To ensure that VEGF signaling does not interfere with this test, since PD173074 is known to have a weak inhibitory effect on the VEGF receptor (VEGFR2), it has been shown that ADSCs grown in EBM2 supplemented with VEGF / vitamin C do not acquire LDL uptake.

Example 17-Fracture Repair

In this example, pig ADSC was administered to reverse decalcification in dogs and thinning of fractured long bones and promote fracture repair.

Non-human patients

The patient in this example was a 7-month-old 900 g dog (small poodle) who had suffered severe fractures in both the radius and ulna of his left foreleg.

Pre-treatment check

The fracture has been repaired surgically by attaching a compression plate to the radius using 5 screws. However, X-ray analysis ("Day 0") has demonstrated evidence of insufficient healing of the radius and the presence of decalcification and severe thinning of the bone (reduction of bone mass) in the fracture area and under the plate. Therefore, in preparation for other treatments, two screws were removed and a commercially available demineralized bone matrix product (Allo Matrix® Custom; Wright Medical Technology Ltd., Arlington, TN) containing human bone morphogenetic protein ("BMP") was used ) Perform initial repair. Allow the incision to heal for 1 week.

Treatment procedure

One week after the two screws have been removed (Figure 26, Figure A), 5 × 10 ⁶ pig ADSCs are administered subcutaneously to the inside and outside of the plate in a volume of 0.5 mL, and the animals are administered intravenously to another 1 × 10 ⁶ pigs ADSC.

Post-treatment assessment

X-ray analysis performed on the 17th day (Figure 26, Figure B) and the 26th day (Figure 26, Figure C) showed that the decalcification effect was reversed, the quality was improved and the bone at the fracture site became thicker, thus filling up The void created by the removal of the screw and the fracture healed.

This example demonstrates the surprising and unexpected ability of heterologous ADSCs to help bone repair in non-human animals.

All references cited above are incorporated by reference into this patent.

Other applicable areas of the invention will become apparent from the detailed explanation provided herein. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Claims (22)

A use of a purified adipose tissue-derived stem cell (ADSC) population for the manufacture of a medicament for the treatment of a fracture in an immunoactive non-human mammal, wherein the ADSC is heterologous to the non-human mammal, wherein ADSC is porcine ADSC, and wherein the agent is used to provide ADSC in a range of about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg body weight. As used in claim 1, wherein the non-human mammal is selected from the group consisting of a dog, cat, horse, rabbit, pig, monkey, baboon, chimpanzee, orangutan, tiger, lion, bear, cheetah, and llama. As claimed in claim 1, wherein the medicament is used to provide ADSC in a range of about 5 × 10 ⁶ ADSC / kg body weight to about 5 × 10 ⁷ ADSC / kg body weight. As claimed in claim 1, wherein the medicament is used to provide ADSC in a range of about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. The use according to claim 1, wherein the agent is for administration with a bone morphogenetic protein. The use as claimed in claim 1, wherein the medicament is for administration at the fracture site. The use according to claim 6, wherein the agent is used for the administration of ADSC by intravenous administration. As for the purpose of claim 7, wherein the range of the ADSC administered intravenously is about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg body weight. For the purpose of claim 8, wherein the range of the ADSC administered intravenously is about 5 × 10 ⁶ ADSC / kg body weight to about 5 × 10 ⁷ ADSC / kg body weight. As for the use of claim 9, wherein the range of the ADSC administered intravenously is about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. A purified adipose tissue-derived stem cell ADSC population for use in the manufacture of a condition selected from the group consisting of "dry eye" conditions, chronic renal failure, acute renal failure, and combinations thereof for treating non-human mammal , Wherein the ADSC is heterologous to the non-human mammal, wherein the ADSC is porcine ADSC, and wherein the agent is used to provide about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg of ADSC in the range of body weight. The use of claim 11, wherein the non-human mammal is selected from the group consisting of a dog, cat, horse, rabbit, pig, monkey, baboon, chimpanzee, orangutan, tiger, lion, bear, cheetah, and llama. The use according to claim 11, wherein the medicament is used to provide ADSC in a range of about 5 × 10 ⁶ ADSC / kg body weight to about 5 × 10 ⁷ ADSC / kg body weight. The use according to claim 11, wherein the medicament is used to provide ADSC in a range of about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. The use according to claim 11, wherein the medicament is for intravenous administration. The use according to claim 15, wherein the agent is used for intravenous administration of ADSC in a range of about 1 × 10 ⁵ ADSC / kg body weight to about 1 × 10 ⁸ ADSC / kg body weight. The use according to claim 16, wherein the agent is used for intravenous administration of ADSC ranging from about 5 × 10 ⁶ ADSC / kg body weight to about 5 × 10 ⁷ ADSC / kg body weight. The use according to claim 11, wherein the medicament is used for intravenous administration of ADSC ranging from about 1 × 10 ⁶ ADSC / kg body weight to about 1 × 10 ⁷ ADSC / kg body weight. For the purpose of claim 11, the condition is a "dry eye" condition. The use according to claim 11, wherein the medicament is for administration into a region around the lacrimal gland of the non-human mammal. The use according to claim 11, wherein the condition is chronic renal failure. The use according to claim 11, wherein the condition is acute renal failure.