Motoneuron-like cells derived from adipose stem cells as well as preparation method and application of motoneuron-like cells

A motor neuron and adipose stem cell technology, applied in the field of stem cells and biomedicine, can solve the problems of motor neuron-like cell curative effect, low maturity and short survival time of motor neuron-like cells.

Active Publication Date: 2015-11-04
SHANGHAI EAST HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, two research groups independently reported improved protocols for converting ESC / iPSCs into motor neurons, using which they were able to induce hADSCs into motor neuron-like cells, but the obtained motor neuron-like cells had low maturity , short survival time
However, those skilled in the art know that the cells induced by differentiation are still far away from being used for clinical treatment, and many cells induced by differentiation, even highly mature differentiated cells, may not be used in the end due to various reasons. Clinical treatment
Therefore, the efficacy of differentiation-induced motor neuron-like cells in relevant SCI models remains to be resolved

Method used

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  • Motoneuron-like cells derived from adipose stem cells as well as preparation method and application of motoneuron-like cells
  • Motoneuron-like cells derived from adipose stem cells as well as preparation method and application of motoneuron-like cells
  • Motoneuron-like cells derived from adipose stem cells as well as preparation method and application of motoneuron-like cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] Example 1. In vitro isolation, expansion and characterization of human ADSCs

[0165] Human adipose tissue obtained by liposuction was processed and inoculated in T-75 flasks in DMEM / F-12 basal medium containing 10% FBS. The medium was changed twice a week, and after 15 days of culture, adherent hADSCs were enriched, and showed typical spindle-like vortex growth by bright-field microscopic images and hematoxylin and eosin (HE) staining, and with large nucleoli ( figure 1 A). High-purity hADSCs were obtained by serial passage. Purified hADSCs stained positively for the mesenchymal stem cell-specific epitopes CD29, CD44, CD105 for most of the isolated cells (>85%), whereas hematopoietic stem cell-specific markers CD45 and CD133 were negative (figure 1 B and C), which is consistent with previous reports.

[0166] By immunocytochemical staining[10], more than 80% of hADSCs also expressed classical embryonic stem cell markers, such as Sox2, Oct4, c-Myc and Nanog( figure ...

Embodiment 2

[0167] Example 2. Differentiation of hADSCs into electrophysiologically active motor neuron-like cells

[0168] It has been reported that SHH and RA are important for the neutralization and ventralization of ESCs or iPSCs, which contribute to the differentiation from pluripotent stem cells into motor neurons. Therefore, the inventors examined whether SHH and RA can make hADSC transdifferentiate into motor neurons ( figure 2 A). Before induction, most hADSCs (>95%) were negative for neuronal markers MAP2 and synapsin1 and motor neuron marker HB9, except for a small amount of faint and diffuse MAP2 staining ( figure 2 B), suggesting that without induction, spontaneous neuronal differentiation is minimal and hADSCs can maintain their stem cell identity even after a series of passages. The inventors observed drastic morphological changes starting as early as 6 hours after treatment. Surprisingly, within 3 days of induction, the cells began to express the motor neuron markers ...

Embodiment 3

[0170] Example 3. The curative effect of transplanted hADSC-preMN in mouse spinal cord injury model

[0171] The inventors further examined whether hADSC-derived motoneurons could exert therapeutic effects in the SCI model in vivo.

[0172] hADSCs were first treated with SHH and RA for one day. After testing, 60% of the obtained cell population expressed the motor neuron progenitor cell marker Oligo2; 40% of the cells expressed the motor neuron progenitor cell marker PAX6; 70% of the cells expressed the motor neuron cell marker HB9; 80% of the cells expressed the neuronal cell marker MAP2. These preconditioned cells were then selected and transplanted into the injured part of the spine and its outer center in spinal cord-injured mice. Three days before transplantation, hADSCs were infected with lentivirus-GFP in order to trace the transplanted cells. Cell transplantation was performed on the 7th day after the establishment of the SCI model. The mice were then maintained in...

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Abstract

The invention provides a method for preparing motoneuron-like cells derived from adipose stem cells. The method comprises the following steps: treating 3-6 generations of high-purity adipose stem cells by using a motoneuron-like cell inducer to obtain motoneuron-like cells; and treating the adipose stem cells by using the motoneuron-like cell inducer, and then treating continuously by using neurotrophic factors, thereby obtaining more mature motoneuron-like cells. By adopting the method provided by the invention, the more mature motoneuron-like cells derived from the adipose stem cells can be obtained in an extremely short time, and the obtained motoneuron-like cells can be used for preventing or treating motoneuron injuries and spinal cord injuries; and the pre-induced motoneuron-like cells obtained by the method provided by the invention can be better used for preventing and treating the motoneuron injuries and spinal cord injuries.

Description

technical field [0001] The invention relates to the fields of stem cells and biomedicine. Specifically, the present invention relates to human-derived adipose stem cell (Human-Adipose Derived Stem Cell, hADSC)-derived motoneuron-like cell (motoneuron-like cell, MNLC) and its preparation method and in the prevention or treatment of motor neuron damage and Application in spinal cord injury. Background technique [0002] Spinal cord injury (SCI) is a devastating condition with sudden loss of function associated with the level of trauma, resulting in paralysis and associated complications. Following the initial trauma, a cascade of secondary events ensues, including ischemia, cell death, hemorrhage, inflammation, edema, and further tissue damage, leading to demyelination, axonal degeneration, and cavitation at the injury site Phenomenon. Traditional treatment includes surgery to decompress and stabilize the injury, prevent secondary complications, and promote functional recov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0793A61K35/12A61P25/00
Inventor 徐俊高山峨朱红文
Owner SHANGHAI EAST HOSPITAL
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