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Stem cells of the islets of langerhans and their use in treating diabetes
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一种干细胞、糖尿病的技术，应用在胰腺细胞、非胚胎多能干细胞、基因治疗等方向，能够解决不确定等问题
Inactive Publication Date: 2005-03-02
THE GENERAL HOSPITAL CORP
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[0004] The origin of islet cells, whether they are embryonic or mature mammalian cells, remains uncertain despite much research
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Embodiment 1
[0279] Example 1 Isolation of Nestin-Positive Stem Cells from Mouse Pancreas
[0280] Mouse islets were isolated from 2-3 month old Sprague-Dawley mice using collagenase lysis as described by Lacy and Kostianovsky. Human islets were obtained from the Diabetes Institute, Miami, FL using collagenase lysis. Islets were cultured at 37°C for 96 hours in 12-well plates (Falcon 3043 plates, Becton Dickinson, Lincoln Park, NJ) on which the plates were coated with concanavalin A. The medium was RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, 100 μg / ml streptomycin, 100 units / ml penicillin, 0.25 μg / ml amphotericin B (GIBCO BRL, Life Science Technology , Gaithersburg, MD), and 71.5 mM beta-mercaptoethanol (Sigma, St. Louis, MO).
[0281] After 96 hours, fibroblasts and other non-islet cells adhered to the surface of the concanavalin A-coated culture wells while islets were floating (not attached to the surface). At this point, the medium...
Embodiment 2
[0283] Example 2 Differentiation of pancreatic stem cells to form islets
[0284] Mouse islets were first cultured on concanavalin A-coated 12-well plates in RPMI medium containing 10% fetal bovine serum, and the islets were cultured for three days without adding culture factors except fetal bovine serum. After this period, in which the islets did not adhere, the islets were transferred to a new plate without concanavalin A.
[0285] Stem cells were then stimulated to proliferate to form a monolayer from the stem cells by exposing them to bFGF-2 (20 ng / ml) and EGF (20 ng / ml) for 24 days. After 24 days, the monolayer reached abundance and surrounding the islets was a population of cells that were picked and subcloned into new 12-well plates and cultured again in medium containing bFGF and EGF.
[0286] Subclones rapidly asexually multiply into the monolayer and expand outward from the center. Cells reached abundance by day 6 and overlapped cell waves by day 12. On the 17th...
Embodiment 3
[0287] Example 3 Isolation and culture of human or mouse islets
[0288] Isolation and culture of human islets. Human islet tissue was obtained from the Islet Allocation Program of the Cell Transplantation Center, Diabetes Institute, University of Miami School of Medicine, Harvard Medical School, Boston, MA, Juvenile Diabetes Foundation for Islet Transplantation. Thoroughly washed islets were hand-picked , in modified RPMI 1640 medium (11.1 mM glucose) supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 1 mM sodium pyruvate, 100 U per mL penicillin G sodium salt, 100 μg / mL streptomycin sulfate, 0.25 ng / mL Amphotericin B, and 71.5uM β-mercaptoethanol, and Eagle 3043 added to a 12-well tissue culture plate coated with Concanavalin A (ConA). Islets were cultured at 37°C for 96 hours in the presence of 5% air and 5% CO2. Under these conditions, more islets remain in suspension (floating), while fibroblasts and other non-islet cells adhere to the medium, and the medium...
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Abstract
Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin and ABCG2 have been identified as molecular markers for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin and / or ABCG2-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.
Description
[0001] This application claims priority to: U.S. Patent Application 09 / 963,875 filed September 26, 2001, U.S. Patent Application 10 / 120,687 filed April 11, 2002, U.S. Patent Application 10 / 12, filed May 2, 2002 136,891. technical field [0002] The present invention relates to the field of stem cells and their differentiation, in particular to pancreatic islet beta cells and nestin (nestin) positive liver stem cells and their differentiation from stem cells and progenitor cells, as well as pancreatic stem cells, progenitor cells, and differentiated beta cells Or use of nestin-positive hepatic stem cells or progenitor cells in organ transplantation. Background technique [0003] This invention was made with at least partial U.S. Government funding supported by National Institutes of Health DK30457, DK30834, DK55365, DK60125, and thus, the U.S. Government has certain rights in this invention. [0004] The origin of islet cells, whether they are embryonic or mature mammalian c...
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