Written by Chen Min

Editor in charge ︱ Wang Sizhen

Editor︱Yang Binwei

The death of specific motor neurons is the most typical and direct pathological feature of motor neuron disease

On August 10, 2022, researcher Lai Liangxue's team from Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Professor Zhang Kun's team and Associate Professor Zou Qingjian's team from Wuyi University's School of Biotechnology and Big Health jointly published an article entitled "Inducible motor neuron" on Cell Proliferation.

The authors firstly transferred two PB vectors (①PB-NIL: conditionally expressing motor neuron transcription factor Ngn2-Isl1-Lhx3; ②PB-BLG overexpressing anti-apoptotic gene Bcl-xL and marker genes Luciferase, EGFP) into hiPSCs , two positive cell clones, NIL-hiPSCs (transfected with PB-NIL only) and NILB-hiPSCs (transfected with PB-NIL+PB-BLG at the same time) were obtained by antibiotic screening (Fig.

Fig.

(Source: Min Chen et al.

Interestingly, extending the culture time beyond 2 weeks, the authors observed the generation of a population of non-neuronal cells among the mature neurons; this population of cells abundantly expressed the neural precursor cell markers SOX2, PAX6, and the proliferation marker Ki67 , while forming a typical neural rosette-like structure

Figure 2 Long-term differentiation of NILB-hiPSCs to generate neural precursor cells

(Image source: Min Chen et al.

The authors directly injected NILB-hiPSCs into the subcutaneous tissue of immunodeficient mice to investigate in vivo differentiation
.

The expression of Ngn2, Isl1 and Lhx3 was induced by intraperitoneal injection of Dox (25 mg/kg, 5 days), and the uninduced group was given the same amount of PBS
.

In vivo imaging results showed that the activity of Luciferase in the uninduced group increased rapidly with time, and undifferentiated hiPSCs proliferated rapidly in the subcutaneous tissue
.

In contrast, the activity of Luciferase in the Dox-induced group decreased rapidly with time, and finally disappeared at the 8th week (Fig.
3A)
.

The above experimental results show that Dox-induced subcutaneous injection of NILB-hiPSCs will not form teratoma, and its safety is high
.

In addition, the authors identified the subcutaneous cell clumps by immunofluorescence, and found that the transplanted NILB-hiPSCs were mainly in the direction of neural differentiation subcutaneously, and abundantly expressed motor neuron-related markers TUJ1, MAP2, HB9 and ChAT (Fig.
3B)
.

In samples from week 4, the authors observed a large number of chamber structures and expressed neural precursor cell markers SOX2 and PAX6, as well as proliferation marker Ki67 (Fig.
3C)
.

To further investigate the subcutaneously differentiated cell types of NILB-hiPSCs, the authors removed the cell clumps at the 4th week of transplantation and cultured them in vitro (Fig.
3D)
.

Primary cells also formed a rosette-like structure and expressed neural precursor cell markers SOX2 and PAX6 (Figure 3E); at the same time, they could differentiate into motor neurons quickly and efficiently without the addition of Dox motor neuron induction medium (Figure 3E).
3F), consistent with the experimental phenomenon observed in vitro
.

Figure 3 In vivo differentiation of NILB-hiPSCs injected subcutaneously

(Image source: Min Chen et al.
, Cell Proliferation, 2022)

In motor neuron disease, motor neurons in the spinal cord of patients die extensively, so the authors injected NILB-hiPSCs into the spinal cord tissue of immunodeficient mice to investigate their survival and differentiation
.

Similar to the subcutaneous injection, cells transplanted into the spinal cord decreased substantially in the first 2 weeks, but the number of cells rebounded by week 4, and then stably survived until week 20 (Fig.
4A and B)
.

The results of immunofluorescence staining showed that NILB-hiPSCs formed synapses and other structures in the mouse spinal cord; and a large number of cells expressed neuronal markers TUJ1, MAP2, HB9 and ChAT (Fig.
4C)
.

More importantly, the transplanted cells integrated into mouse spinal cord tissue and generated strong action potentials and ionic currents (Fig.
4D)
.

It indicated that NILB-hiPSCs could differentiate into functionally mature motor neurons in spinal cord tissue
.

Similar to subcutaneous transplantation, cells injected into the spinal cord also formed chamber-like structures and expressed neural precursor cell markers SOX2 and PAX6 (Fig.
4E), and proliferation marker Ki67 (Fig.
4F), indicating that the transplanted cells were in the spinal cord can continue to multiply
.

Unexpectedly, a small fraction of cells also expressed OLIG2 (Fig.
4G), indicating further differentiation of neural precursor cells into motor neuron precursor cells
.

The above results demonstrate that the transplanted cells can survive in spinal cord tissue for 5 months and differentiate into a mixed cell population of motor neurons, neural precursor cells, and motor neuron precursor cells
.

Figure 4 In vivo differentiation of NILB-hiPSCs injected into spinal cord

(Image source: Min Chen et al.
, Cell Proliferation, 2022)

Conclusion and Discussion, Inspiration and Prospects In summary, this study combined in vitro gene modification of stem cells with in vivo induction and differentiation technology for the first time, and successfully constructed a technical system for efficient and directional induction of motor neurons in vivo
.

In the follow-up, in-depth research will focus on the therapeutic effect of NILB-hiPSCs on MNDs animal models
.

More importantly, hiPSCs can differentiate into various types of somatic cells through transcription factors, if Ngn2-Isl1-Lhx3 is replaced by other cell-specific transcription factors (such as liver-specific transcription factors Foxa3-Hnf1A-Hnf4A, kidney-specific transcription factors Sex transcription factor Six2), can induce various types of functional cells in vivo, and then used for transplantation therapy of corresponding diseases
.

It can be seen that this study not only provides a new strategy for MNDs stem cell therapy, but also has important value and significance for other diseases
.

However, because the transposon vector used in this study may integrate foreign genes into key positions in the genome, it may cause safety concerns such as stem cell carcinogenesis or uncontrollable differentiation process
.

In another recently published article, the team introduced drugs to induce the suicide of pluripotent stem cells to remove undifferentiated cells in the body, which to a certain extent improved the safety of stem cell therapy in vivo [5]
.

In the future, site-specific integration of exogenous genes can also be used to minimize the safety risks brought by transgenes to cell therapy
.

Original link: https://onlinelibrary.
wiley.
com/doi/10.
1111/cpr.
13319

Researcher Lai Liangxue, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Associate Professor Zou Qingjian and Professor Zhang Kun from the School of Biotechnology and Big Health, Wuyi University are the co-corresponding authors of the paper, Dr.
Chen Min from the School of Biotechnology and Big Health, Wuyi University, Master Li Chuan and Wang Xia of the Bio Island Laboratory are the co-first authors of the paper
.

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Recommended high-quality scientific research training courses

[1] Symposium on Single-Cell Sequencing and Spatial Transcriptomics Data Analysis (August 27-28, Tencent Online Conference)

【2】Training course︱R language clinical prediction biomedical statistics special training

[3] Workshop on Metagenomics and Metabolomics R Language Analysis and Visualization (Tencent Conference on August 27)

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References (swipe up and down to read) 

1 Wyatt T J, Rossi S L, Siegenthaler M M, et al.
Human motor neuron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss.
Stem cells international, 2011, 2011.

2 Xu L, Yan J, Chen D, et al.
Human neural stem cell grafts ameliorate motor neuron disease in SOD-1 transgenic rats.
Transplantation, 2006, 82(7): 865-875.

3 Popescu I R, Nicaise C, Liu S, et al.
Neural progenitors derived from human induced pluripotent stem cells survive and differentiate upon transplantation into a rat model of amyotrophic lateral sclerosis.
Stem cells translational medicine, 2013, 2(3): 167-174

4 Itzhak F, Jennifer N D, Michael A L.
Transplanting neural progenitor cells to restore connectivity after spinal cord injury, Nature Review Neuroscience, 2020, 21(7): 366-383

5 Liu Y, Yang Y, Suo Y, et al.
Inducible caspase-9 suicide gene under control of endogenous oct4 to safeguard mouse and human pluripotent stem cell therapy.
Mol Ther Methods Clin Dev, 2022, 24: 332-41

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