Method for inducing differentiation of stem cells into islet-like cells

A technology of islet-like cells and induced differentiation, applied in the field of biomedicine, can solve the problems of insufficient cell sources, limited application, severe immune rejection, etc., and achieve the best application prospects, huge social and economic benefits

Inactive Publication Date: 2012-01-11
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows us to use these types of materials from various sources like humans or animals' own body fluids (blood) to create new type of specialized material called pluripotential stroma cells ("PSC"). These PSC are able to differentiate towards specific lineages of connective tissues such as muscle fibers, cartilage, tendon, skin, nerve roots, brain glioma, heart valves, kidneys, pancreas, spleens, etc., which could potentially lead to development of medical treatments related to diseases associated with those organs.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving methods for treating neurology conditions like Alzheimer’s Disease through introductions of new agents called Silicon Receptacle Factor(SiReferring To address these issues, there was developed a technique involving modifying certain parts of somatic cells containing beta globin precursor cells called Islets of Langerhair Tisslecle Homology Group-3 Muscle Retractory Tumor Naïve Breast Cancer Type 2 syndrome, multiple sclerosis associated dystrophesis, autosomal dominant polycysplasia, cerebral deglycemia complex, glioma, retinoid acid reductase 4-5 alpha chloroproteina triphosphatide phospliocyte mitochondria membrane ghost transfer mediator involved in nerve growth factors production process. These techniques could potentially lead to novel therapeutants capable of effectively suppressing inflammatory responses without causing side effects.

Method used

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  • Method for inducing differentiation of stem cells into islet-like cells
  • Method for inducing differentiation of stem cells into islet-like cells
  • Method for inducing differentiation of stem cells into islet-like cells

Examples

Experimental program
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Embodiment 1

[0032] Embodiment 1, the construction of NRSF interference carrier

[0033]Use the network RNA interference fragment design tool to design the interference fragment of the human NRSF gene and the off-target control fragment of the interference, wherein the sequence of the interference fragment is: the sequence of the interference and off-target fragment is: after the two fragments are annealed and phosphorylated by T4 single nucleotide phosphorylase , use T4 ligase to connect to the pSicoR vector after HpaI and Xbal double digestion, after transforming DH5a competent cells, pick the clone and shake the culture to extract the plasmid, use the pSicoR plasmid as a negative control, after XhoI and XbaI double digestion 2% agarose gel electrophoresis, the plasmid that can be digested to produce a 400bp fragment is the plasmid obtained by positive clone (see attached figure 1 ), the clone was sent to a sequencing company for sequencing. The correctly sequenced interference vector p...

Embodiment 2

[0034] Embodiment 2, lentiviral packaging

[0035] Culture 293-FT (purchased from Invitrogen) lentiviral packaging cells, the medium is supplemented with 10% FBS, 0.1mmol / L NEAA, 2mmol / L L-glutamine, 1% penicillin-streptomycin, 500μg / mL G418 High-glucose DMEM medium (product of Sigma Company, Cat#D5648). The lentiviral interference vector pSicoR-GFP-siNRSF and the off-target vector pSicoR-GFP-siControl constructed in step 1 were used with the packaging plasmids pLP1 (purchased from Invitrogen), pLP2 (purchased from Invitrogen) and the envelope protein plasmid pLP / VSVG ( (purchased from Invitrogen Company) was mixed according to the ratio of 5ug: 4.2ug: 2ug: 2.8ug, and then 1.5mL serum-free Opti-MEM was added In medium I (purchased from Invitrogen), mix gently, and dilute 42uL lipofectamine 2000 (purchased from Invitrogen) in 1.5mL of serum-free Opti-MEM in another sterile 5mL tube In medium I, let stand at room temperature for 5 minutes, mix the above two liquids, and incu...

Embodiment 3

[0036] Example 3, Verification of the interference effect of the lentivirus interference vector

[0037] 1. Infection of amniotic fluid stem cells with lentiviral interference vector and off-target vector

[0038] Cultivate amniotic fluid stem cells in cell culture flasks, and infect the cells with viruses when they grow to 80% density. When infecting, add 10ml of virus and polybrene (polybrene) with a final concentration of 8 μg / ml to each well, and shake the culture bottle so that the virus liquid can contact all cells. Medium was changed after overnight culture. The cells infected with lentiviral interference vector and off-target vector were named SiNRSF-hAFSCs and SiControl-hAFSCs, respectively. Cells were then cultured according to normal cell culture methods.

[0039] 2. RT-PCR and real time PCR verification

[0040] Use TRIZOL (invitrogen) reagent to extract the total RNA of siNRSF-hAFSCs and siControl-hAFSCs according to the instructions, and then reverse transcri...

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Abstract

The invention relates to the field of biomedicines, in particular to a method for inducing differentiation of stem cells into islet-like cells in vitro and use of islet-like cells. The invention aims to provide a method for obtaining a large amount of islet-like cells in vitro and the use of the islet-like cells differentiated under induction as seed cells in transplantation of islet cells and preparation of microencapsulated stem cell preparation and as an in-vitro pharmacokinetic model in cell medicine screening. For realizing the aim, the invention adopts a technical scheme that: 1) constructing an NRSF interfering carrier; 2) separating, culturing and amplifying stem cells; 3) inducing stem cells to differentiate into islet-like cells by two stages; and 4) identifying islet-like cells. The construction of the method has a very bright application prospect in regenerative medicine taking gene engineering, cell engineering, tissue engineering and the like as a basis and is expected to create huge social and economic benefit.

Description

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Claims

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Application Information

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Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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