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Methods and compositions for treating diabetes with iPS derived pancreatic beta-like cells

A diabetes and cell technology, applied in the field of stem cells) and its treatment of diseases, can solve problems such as poor insulin secretion and non-existence

Inactive Publication Date: 2013-04-10
Y 马 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, transplantation of these cells in animals suffers from limited growth capacity, low levels of insulin expression, and poor or non-existent insulin secretion

Method used

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  • Methods and compositions for treating diabetes with iPS derived pancreatic beta-like cells
  • Methods and compositions for treating diabetes with iPS derived pancreatic beta-like cells
  • Methods and compositions for treating diabetes with iPS derived pancreatic beta-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Materials and methods

[0077] iPS production and culture

[0078] Transduction from green fluorescent protein (GFP) transgenic mice (C57BL / 6-Tg(UBC-GFP)30Scha / J Stock#004353) with 4 retroviral transcription factors as described by Yamanaka et al. (12, 13) ( Normal fibroblasts from Jackson Laboratory, Bar Harbor, ME). In the medium containing knockout DMEM (KDMEM, Invitrogen, Carlsbad, CA), 15% ES qualified fetal bovine serum (FBS) (PAA, Ontario, Canada), 2mM L-glutamine (Invitrogen), 1x10 -4 M non-essential amino acids (Invitrogen), 1x10 -4 M 2-mercaptoethanol (Sigma, St Louis, MO), 1Xpen / strep (Invitrogen) and 12.5ng / ml LIF (Chemicon, Billerica, MA) in the ES medium of mice embryos treated with mitomycin C Newly generated undifferentiated mouse iPS cells were maintained on a fibroblast feeder layer. Cultures were passaged every 3-4 days using 0.25% trypsin (Invitrogen) at a split ratio of 1:3-1:6. Mouse embryonic fibroblast feeder cells (Millipore, Billerica,...

Embodiment 2

[0105] Reprogramming of normal mouse skin fibroblasts into induced pluripotent stem cells

[0106] Normal fibroblasts from green fluorescent protein (GFP) transgenic mice were transduced with four retroviral transcription factors (10-12) using previously described methods (26). In the current study, 2 iPS subclonal colonies were picked and expanded 20 days after transduction ( Figure 8 ). The mRNA expression profiles of these 2 iPS subclones showed upregulation of endogenous stem cell markers Oct4, Sox2, Klf4, and c-Myc, similar to embryonic stem (ES) cells ( Figure 9 ). Exogenous viral transgenes exhibited minimal expression. Immunofluorescent staining, used to assess surface and intracellular antigens found in these iPS cells, was positive for well-known markers of pluripotency Oct4, Sox2, Nanog, and SSEA-1 ( Figure 10 ). To determine their ability to proliferate and differentiate, approximately 2x10 6 Dissociated iPS cells were injected subcutaneously into the flan...

Embodiment 3

[0108] Selective differentiation of iPS cells into insulin-producing pancreatic β-like cells

[0109] Using a modification of a previously published protocol for ES cells (17), iPS cells were driven through a 3-stage differentiation into insulin-producing β-like cells. The timeline of this differentiation is shown in figure 1 . In stage 1, ES-like iPS cell cultures were dissociated into single cells and placed in suspension culture, where they formed embryoid bodies ( figure 1 , top panel). After 5 days of suspension culture, the embryoid bodies were returned to adherent culture to undergo further differentiation for 9 days. During this time, cells migrated from the attached spheroids ( figure 1 , middle panel). Nine days later, the onset of phase 3 was marked by changing the medium to selective medium containing laminin, insulin, nicotinamide, selenate, transferrin, progesterone, and Knock-Out replacement serum. During the subsequent 20 days, the cells continued to diff...

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Abstract

Diabetes mellitus is characterized by either the inability to produce insulin (Type 1 diabetes) and or as insensitivity to insulin secreted by the body (Type 2 diabetes). In either case, the body is unable to efficiently move blood glucose across cell membranes to be utilized. This leads to a variety of local and systemic detrimental effects. Current treatments for diabetes focus on exogenous insulin administration and dietary control. Provided herein are treatments of diabetes using a cellular therapy to ameliorate symptoms associated with both reduced insulin secretion and insulin sensitivity. Using induced-pluripotent stem (iPS) cells, beta-like (ss-like) cells similar to the endogenous insulin secreting cells were derived.; These ss-like cells secreted insulin in response to glucose, and corrected a hyperglycemic phenotype in a mouse model of Type 2 diabetes via an iPS cell transplant. Within the Type 2 diabetes mouse model, a long term correction of hyperglycemia was achieved as measured by blood glucose and hemoglobin AIc measurements. Reduction of hyperglycemia was also seen in a chemically-induced mouse model for Type 1 diabetes.

Description

[0001] Cross References to Related Applications [0002] This application is related to US Provisional Application 61 / 339,510 filed March 5,2010. field of invention [0003] In general, the present invention relates to stem cells, including induced pluripotent stem cells (iPS), and their use to treat diseases, such as diabetes. Background of the invention [0004] About 3.5 percent of Americans are diagnosed with diabetes, making this a major health care problem. Type 1 diabetes is an autoimmune disease in which insulin-secreting beta cells in pancreatic islets are irreversibly destroyed by autoimmune attack, resulting in a lack of insulin production. Type 2 diabetes occurs when the pancreas produces insufficient amounts of the hormone insulin and / or tissues of the body become resistant to normal or even high levels of insulin. In either case, introduction of exogenous insulin is usually sufficient to improve symptoms. However, insulin dosage is difficult to adjust. In a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P3/10A61K35/12A61K35/36C12N15/63A61K35/39
CPCA61K35/39C12N5/0676C12N2500/38C12N2500/46C12N2501/392C12N2501/58C12N2506/45A61P3/10A61K35/545C12N5/0696
Inventor Y.马M.瓦纳
Owner Y 马
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