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Method for obtaining spinal motoneurons and functional cells thereof based on human iPS (induced pluripotent stem) cells

A motor neuron and neuron technology, applied in the field of biomedicine, can solve problems such as toxicity and side effects

Active Publication Date: 2017-05-24
THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of small molecular compounds also has a bottleneck effect, mainly due to its toxic side effects, and is positively correlated with its working concentration

Method used

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  • Method for obtaining spinal motoneurons and functional cells thereof based on human iPS (induced pluripotent stem) cells
  • Method for obtaining spinal motoneurons and functional cells thereof based on human iPS (induced pluripotent stem) cells
  • Method for obtaining spinal motoneurons and functional cells thereof based on human iPS (induced pluripotent stem) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Reprogramming, culture and identification of human iPS cells

[0063] Reprogramming and culture: DMEM complete medium (containing 10% FBS, 100IU / ml penicillin, 100IU / ml streptomycin) was used to primary culture the cells of skin tissue blocks from patient biopsies. After it was overgrown, it was washed once with DMEM, digested and passaged with 0.25% trypsin-EDTA, and 0.2x 10 6 Cells / well density were seeded into 12-well plates and placed at 37°C, 5% CO 2 cultured in a humidified incubator. Two days later, the cells were infected with Sendai virus (hOct3 / 4, hSox2, hKlf4 and hc-Myc) carrying reprogramming factors at a multiplicity of infection (MOI) of 5. After 24 hours, replace the above-mentioned fresh medium, 1ml / well; then perform a full amount of medium change every other day. On the 6th day of culture, dilute vitronectin with DMEM / F12 at a ratio of 1:100, coat a 6-well plate with 1ml / well, and put it in an incubator overnight. On day 7 of culture, the...

Embodiment 2

[0069] Example 2 Directed differentiation of human iPS cells to spinal motoneurons, identification and differentiation efficiency statistics

[0070] Human iPS cells grown in a 6-well plate (Corning, USA) in good condition were taken, and when they reached 70%-85% confluence in 5-6 days, they were digested with 0.5mM EDTA at room temperature for 1 minute, and then pipetted and centrifuged. wall, collected into a 15ml centrifuge tube (Guangzhou Jiete Biological Filtration Products Co., Ltd., China), and centrifuged at 1000 rpm for 1 minute. Use 10ml neural induction differentiation medium (containing 1:1 DMEM / F12:Neurobasal medium, 0.5x N2 supplement, 0.5x B27 supplement, L-glutamine, non-essential amino acids) to resuspend in a 10cm low adsorption culture dish (Guangzhou Jiete Biological Filtration Products Co., Ltd., China), while adding different small molecule compound combinations DMH1+SB431542+CHIR99021 or LDN193189+SB431542+CHIR99021, placed at 37 ° C, 5% CO 2 , in an i...

Embodiment 3

[0085] Application of Example 3 Inducing Rapid Maturation of Spinal Cord Motor Neurons

[0086] Primary astrocyte culture:

[0087] The mice of C57BL / 6 strain within 1 day after birth were taken out, and the brains were quickly taken out after fully disinfected with 75% alcohol. After peeling off fibrous components such as meninges and blood vessels under a dissecting microscope, they were transferred to a 15ml centrifuge tube (Guangzhou Jiete Biological Filtration Products Co., Ltd., China), and digested with 0.25% trypsin-EDTA in a 37°C water bath for 10 minutes. DMEM complete medium (containing 20% ​​FBS) was used to terminate the digestion, and gently pipet several times to make a single cell suspension. Let the sediment stand for 5 minutes, draw the supernatant and inoculate it into a Matrigel (1:100 dilution)-coated T75 culture flask (Corning), and place it at 37°C, 5% CO 2 cultured in a humidified incubator. After 24 hours, shake the culture flask vigorously to remov...

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Abstract

The invention belongs to the field of biomedicine, and relates to a safe and reliable reprogrammed method for obtaining human iPS (induced pluripotent stem) cells, a method for simply, rapidly and efficiently inducing and differentiating iPS cells into spinal motoneurons, and a method for economically and efficiently obtaining functional spinal motoneurons and application of the method. Human fibroblasts are infected with Sendai virus carrying a reprogramming factor by using vitronectin as the sole growth medium, and are cultured for 3 weeks to obtain human iPS cells without exogenous serum interference; the iPS cells are efficiently induced and differentiated into spinal motoneurons by a combination of multiple small molecule compounds; and further, an astrocyte conditioned culture medium promotes maturation culture and is applied in the formation of neuromuscular junctions. The functional spinal motoneurons obtained by efficient induction can be applied to simulation of disease phenotype and discussion of pathogenesis, and provides a theoretical basis for the screening of effective therapeutic drugs in the future, and even in the future can be applied to the cell transplantation therapy of diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a method for obtaining spinal motoneurons, in particular including a method for obtaining human iPS cells through safe and reliable reprogramming, and a method for inducing and differentiating human iPS cells into spinal motor neurons simply, quickly and efficiently , also includes a cost-effective method for obtaining functional spinal motoneurons. Background technique [0002] Spinal cord motor neurons (motorneuron, MN) play a bridge role in the contraction of skeletal muscles controlled by the central nervous system, and control important life functions such as breathing and swallowing. Because of this, spinal motor neurons have become the primary violation of incurable motor neuron diseases (motorneurondisease, MND), such as amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), spinal muscular atrophy (spinal muscular atrophy, SMA), etc. Target. The current lack of sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N5/10C12N5/0793
CPCC12N5/0619C12N5/0696C12N15/86C12N2501/734C12N2506/45C12N2510/00
Inventor 陈万金林翔王柠张奇杰
Owner THE FIRST AFFILIATED HOSPITAL OF FUJIAN MEDICAL UNIV
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