Preclinical long-term safety of intraspinal transplantation of human dorsal spinal GABA neural progenitor cells

doi:10.1016/j.isci.2023.108306

. 2023 Oct 23;26(11):108306.

doi: 10.1016/j.isci.2023.108306. eCollection 2023 Nov 17.

Preclinical long-term safety of intraspinal transplantation of human dorsal spinal GABA neural progenitor cells

Xiaolong Zheng¹, Zhixian Liu¹, Ziyu He¹, Jia Xu^{2

3}, YaNan Wang², ChenZi Gong², Ruoying Zhang¹, Su-Chun Zhang^{4

5}, Hong Chen^{2

3

6}, Wei Wang^{1

6

7}

Affiliations

¹ Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
² Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
³ Stem Cell Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
⁴ Waisman Center, Department of Neuroscience and Department of Neurology, University of Wisconsin, Madison, WI, USA.
⁵ Program in Neuroscience & Behavioral Disorders, Duke-NUS Medical School, Singapore, Singapore.
⁶ Hubei Key Laboratory of Neural Injury and Functional Reconstruction, Huazhong University of Science and Technology, Wuhan 430030, China.
⁷ Key Laboratory of Neurological Diseases of Chinese Ministry of Education, the School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

PMID: 38026209
PMCID: PMC10661464
DOI: 10.1016/j.isci.2023.108306

Preclinical long-term safety of intraspinal transplantation of human dorsal spinal GABA neural progenitor cells

Xiaolong Zheng et al. iScience. 2023.

. 2023 Oct 23;26(11):108306.

doi: 10.1016/j.isci.2023.108306. eCollection 2023 Nov 17.

Authors

Xiaolong Zheng¹, Zhixian Liu¹, Ziyu He¹, Jia Xu^{2

3}, YaNan Wang², ChenZi Gong², Ruoying Zhang¹, Su-Chun Zhang^{4

5}, Hong Chen^{2

3

6}, Wei Wang^{1

6

7}

Affiliations

¹ Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
² Department of Rehabilitation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
³ Stem Cell Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
⁴ Waisman Center, Department of Neuroscience and Department of Neurology, University of Wisconsin, Madison, WI, USA.
⁵ Program in Neuroscience & Behavioral Disorders, Duke-NUS Medical School, Singapore, Singapore.
⁶ Hubei Key Laboratory of Neural Injury and Functional Reconstruction, Huazhong University of Science and Technology, Wuhan 430030, China.
⁷ Key Laboratory of Neurological Diseases of Chinese Ministry of Education, the School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

PMID: 38026209
PMCID: PMC10661464
DOI: 10.1016/j.isci.2023.108306

Abstract

Human pluripotent stem cell (hPSC)-derived neurons have shown promise in treating spinal cord injury (SCI). We previously showed that hPSC-derived dorsal spinal γ-aminobutyric acid (GABA) neurons can alleviate spasticity and promote locomotion in rats with SCI, but their long-term safety remains elusive. Here, we characterized the long-term fate and safety of human dorsal spinal GABA neural progenitor cells (NPCs) in naive rats over one year. All grafted NPCs had undergone differentiation, yielding mainly neurons and astrocytes. Fully mature human neurons grew many axons and formed numerous synapses with rat neural circuits, together with mature human astrocytes that structurally integrated into the rat spinal cord. The sensorimotor function of rats was not impaired by intraspinal transplantation, even when human neurons were activated or inhibited by designer receptors exclusively activated by designer drugs (DREADDs). These findings represent a significant step toward the clinical translation of human spinal neuron transplantation for treating SCI.

Keywords: Biological sciences; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Figure 1**
Long-term survival, full maturation and extensive axon growth of human dorsal spinal GABA neurons (A) Representative images of horizontal sections of adult naive rat C5-C8 spinal cords receiving either human ESC-derived spinal dI4/dIL_A NPCs with or without BiDREADDs expression or cell culture medium, showing long-term survival of grafted cells 12 months post-transplantation. (B) Images of differentiated human neurons within the graft. Quantifications show the proportions of hNu⁺NeuN⁺ human neurons in dI4 (green) and dI4-Bi-DREADDs grafts (red). (C) Images of human neurons expressing the markers of dI4/dIL_A (PTF1A) and inhibitory interneuron identity (PAX2). Images of hNu were omitted for simplicity. Quantifications show the proportions of PTF1A and PAX2 in dI4 (green) and dI4-Bi-DREADD grafts (red). (D) Images of human neurons expressing the markers of subtypes of inhibitory neurons. Images of hNu were omitted for simplicity. Quantifications show the proportions of PV, CB and n-NOS in dI4 (green) and dI4-Bi-DREADDs grafts (red). (E) Images showing that human neurons released much GABA within the graft. (F–H) Images of the graft showing human neurons extensively expressing NSE (F), hMito (G) and KCC2 (H), suggesting their full maturation. (I and J) Images of the graft showing that human neurons grew large numbers of hMAP-2⁺ dendrites (I) and STEM121⁺Tuj1⁺ axons (J). STEM121 is a human-specific cytoplasmic marker. (K–P) Images of hTau⁺ human axons showing their location, traveling, projection and myelination. Numerous hTau⁺ human axons existed within the rat C5–C8 spinal cords (K). They were densely intermingled within the graft (L), traveling within the WM (M) and projecting into the GM of rat spinal cords where rat INs (N) and MNs (O) reside. Human axons were unwrapped by an MBP⁺ myelin sheath (P). Data are represented as the mean ± SEM. n represents number of rats per group. See also Figure S1. Scale bar, 1 mm in (A) and (K), 50 μm in (B–J) and (L–O), and 10 μm in (P).

**Figure 2**
Human dorsal spinal GABA neurons integrated into rat neural circuits (A) Representative images of horizontal sections of rat C5–C8 spinal cords showing tremendous expression of the presynaptic marker hSyn, indicating the potential formation of human synapses. (B–F) Images showing that human synapses were established and were mainly inhibitory GABAergic. Many hSyn⁺ were colocalized with the inhibitory presynaptic markers GAD65/57 (B) and VGAT (C) and were in close apposition with the postsynaptic marker Homer1 (E) and the inhibitory postsynaptic marker GPHN (F). Quantification of the proportion of GAD65/67 (D). (G) Within the graft, human neurons formed extensive synapses between themselves. (H–M) Out of the graft but within the GM of rat spinal cords, human neurons formed synapses with rat neurons, including MNs (I), INs (H), such as PKCγ⁺ (J), CR⁺ (K), and NK1R⁺ (L) neurons, and with rVGLUT1⁺ afferent sensory terminals (M). (N and O) Images of the graft showing hNu⁺NeuN⁺ human neurons were innervated by rat excitatory synapses as revealed by rVGULT1 and rVGLUT2. (P–S) Images of the graft showing hNu⁺NeuN⁺ human neurons were innervated by rat neural circuits, including the CST from forelimb M1/S1 (P), TH⁺ ceruleospinal tract (Q), 5-HT⁺ raphespinal tract (R), and CGRP⁺ nonmyelinating peptidergic nociceptive afferents (S). Data are represented as the mean ± SEM. n represents number of rats per group. See also Figure S2. Scale bar, 1 mm in (A), 20 μm in (G–I) and (N‒S), and 5 μm in (B–F), (J-M) and in images of magnified white box areas (G–I) and (N‒S).

**Figure 3**
Differentiation and integration of mature human astrocytes into the rat spinal cord (A) Representative images of RECA-1⁺ capillaries and their associated laminin⁺ basement membrane in the graft, GM and WM of rat spinal cords. (B) Quantification of the area, total length, average length, total junctions, total ending points and MFI of RECA-1⁺ capillaries. (C) Quantification of coverage of laminin⁺ basement membrane on RECA-1⁺ capillaries and MFI of laminin. (D and E) Images of grafts showing the differentiation of hNu⁺SOX9⁺ human astrocytes and hNu⁺OLIG2⁺/NG2⁺ human oligodendroglia lineage cells. Quantification of SOX9 and OLOG2/NG2 revealed that the differentiation of human astrocytes was greater than that of oligodendrocytes. (F–J) Images of grafts showing differentiated human astrocytes expressing vimentin (F), GFAP (G), S100β (H), ALDH1L1 (I), and AQP4 (J), suggesting their maturation. (K) Representative images of horizontal sections of rat C5–C8 spinal cords showing tremendous expression of hGFAP in the graft and WM, including the lateral and dorsal columns, indicating extensive differentiation and migration of human astrocytes. (L) Images showing hGFAP⁺ processes of human astrocytes densely surrounding human neurons within the graft and rat MNs and INs outside of the graft. (M and N) Images showing hGFAP⁺ processes of human astrocytes expressing the gap junction proteins Cx43 (M) and GLT-1, which are responsible for glutamate clearance (N). (O and P) Images of the graft showing hGFAP⁺ processes of human astrocytes were aligned with the MAP2⁺ dendrites and NF⁺ axons of human neurons. (Q and R) Images showing hGFAP⁺ processes of human astrocytes wrapping laminin⁺ capillaries (Q) and expressing AQP4 contributing to the structure of BSCB (R). Data are represented as the mean ± SEM. n represents number of rats per group. See also Figure S3. Scale bar, 1 mm in (K), 100 μm in (A), 20 μm in (D-L), 10 μm in (N-R), and 5 μm in (M).

**Figure 4**
Long-term safety of intraspinal transplantation of human dorsal spinal GABA NPCs (A and B) Sensory function of rats in the Medium, dI4 and dI4-Bi-DREADDs groups over 12 months post-transplantation. No significant difference was detected in mechanical allodynia (A) and thermal hyperalgesia (B) in the fore and hind paws of rats. (C–F) Motor function of rats in the Medium, dI4 and dI4-Bi-DREADDs groups over 12 months post-transplantation. Forelimb grooming behavior (C), hindlimb overground locomotion (D), fore- and hindlimb skilled locomotion (E), and gait (F) were unaltered by transplantation. (G) Images of the grafts showing hNu⁺NeuN⁺ human neurons in dI4-Bi-DREADDs but not dI4, or hNu⁻NeuN⁺ rat neurons, still preserved the expression of mCherry and HA, the reporters for hM3Dq and KORD, respectively. (H) Images of the grafts showing hNu⁺NeuN⁺ human neurons in dI4-Bi-DREADDs but not dI4 or hNu⁻NeuN⁺ rat neurons expressing c-FOS upon CNO activation. Quantification of the proportion of c-FOS suggested that human neurons were activated by CNO. (I and J) Sensory function of rats in the Medium, dI4 and dI4-Bi-DREADDs groups at 12 months post-transplantation under BiDREADDs manipulation. No significant difference was detected in mechanical allodynia (I) and thermal hyperalgesia (J) in the fore and hind paws of rats after CNO, SalB or vehicle application. (K) Motor function of rats in the Medium, dI4 and dI4-Bi-DREADDs groups at 12 months post-transplantation under BiDREADDs manipulation. No significant difference was detected in skilled locomotion in the fore and hind paws of rats after CNO, SalB or vehicle application. Data are represented as the mean ± SEM. n represents number of rats per group. See also Figure S4 and Table S1. Scale bar, 20 μm in (G and H).

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References

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[1] Courtine G., Sofroniew M.V. Spinal cord repair: advances in biology and technology. Nat. Med. 2019;25:898–908. doi: 10.1038/s41591-019-0475-6. - DOI - PubMed

[2] Courtine G., Sofroniew M.V. Spinal cord repair: advances in biology and technology. Nat. Med. 2019;25:898–908. doi: 10.1038/s41591-019-0475-6. - DOI - PubMed

[3] Fischer I., Dulin J.N., Lane M.A. Transplanting neural progenitor cells to restore connectivity after spinal cord injury. Nat. Rev. Neurosci. 2020;21:366–383. doi: 10.1038/s41583-020-0314-2. - DOI - PMC - PubMed

[4] Fischer I., Dulin J.N., Lane M.A. Transplanting neural progenitor cells to restore connectivity after spinal cord injury. Nat. Rev. Neurosci. 2020;21:366–383. doi: 10.1038/s41583-020-0314-2. - DOI - PMC - PubMed

[5] Zipser C.M., Cragg J.J., Guest J.D., Fehlings M.G., Jutzeler C.R., Anderson A.J., Curt A. Cell-based and stem-cell-based treatments for spinal cord injury: evidence from clinical trials. Lancet Neurol. 2022;21:659–670. doi: 10.1016/S1474-4422(21)00464-6. - DOI - PubMed

[6] Zipser C.M., Cragg J.J., Guest J.D., Fehlings M.G., Jutzeler C.R., Anderson A.J., Curt A. Cell-based and stem-cell-based treatments for spinal cord injury: evidence from clinical trials. Lancet Neurol. 2022;21:659–670. doi: 10.1016/S1474-4422(21)00464-6. - DOI - PubMed

[7] Ribeiro B.F., da Cruz B.C., de Sousa B.M., Correia P.D., David N., Rocha C., Almeida R.D., Ribeiro da Cunha M., Marques Baptista A.A., Vieira S.I. Cell therapies for spinal cord injury: a review of the clinical trials and cell-type therapeutic potential. Brain. 2023;146:2672–2693. doi: 10.1093/brain/awad047. - DOI - PubMed

[8] Ribeiro B.F., da Cruz B.C., de Sousa B.M., Correia P.D., David N., Rocha C., Almeida R.D., Ribeiro da Cunha M., Marques Baptista A.A., Vieira S.I. Cell therapies for spinal cord injury: a review of the clinical trials and cell-type therapeutic potential. Brain. 2023;146:2672–2693. doi: 10.1093/brain/awad047. - DOI - PubMed

[9] Huang H., Chen L., Moviglia G., Sharma A., Al Zoubi Z.M., He X., Chen D. Advances and prospects of cell therapy for spinal cord injury patients. J. Neurorestoratol. 2022;10:13–30. doi: 10.26599/Jnr.2022.9040007. - DOI

[10] Huang H., Chen L., Moviglia G., Sharma A., Al Zoubi Z.M., He X., Chen D. Advances and prospects of cell therapy for spinal cord injury patients. J. Neurorestoratol. 2022;10:13–30. doi: 10.26599/Jnr.2022.9040007. - DOI

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Preclinical long-term safety of intraspinal transplantation of human dorsal spinal GABA neural progenitor cells

Affiliations

Preclinical long-term safety of intraspinal transplantation of human dorsal spinal GABA neural progenitor cells

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

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Abstract

Conflict of interest statement

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