Human embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord
Introduction
Human embryonic stem cells (hESC) can provide auseful source of cells for basic developmental studies and cell-based therapies. So far, many promising studies have shown the therapeutic potential of differentiated derivatives of ESC ameliorating neurologic disease in animal models. For example, ESC-derived neural progenitor cells (NPC) transplanted into the brains of rats with Parkinson's disease generate functional dopamine neurons [1,2] and retinal pigment epithelium derived from monkey and human ESC appears to preserve visual function in a rat model of macular degeneration [3,4]. Moreover, grafting of ESC into animal models with spinal cord injuries (SCI) has been shown to improve motor function [5., 6., 7., 8.]. However, their potential for tumor and inappropriate tissue formation [9] remains a significant concern for clinical application. Therefore, in order to restrict the breadth of cell-type differentiation and inappropriate tissue formation, precursor cells with a limited potential to form only neural tissue will probably be the preferred source for central nervous system (CNS) transplantation.
ESC can be induced to generate NPC by (i) mimicking the environment that produces neuroectoderm in the embryo by providing appropriate cell–cell interactions and signals through embryoid body formation, and (ii) depriving the ESC of both cell–cell interactions and signals by low-density culture in serum-free medium, thus evoking a default mechanism for NPC differentiation. Some protocols combine aspects of both approaches by promoting cell–cell interactions to facilitate the formation of all three primary germ layers followed by neural lineage-specific selection under defined conditions [10]. To harness the potential of ESC as a tool for scientific exploration and a source for possible cell replacement, in addition to the degree of maturity of the transplanted cells (stem cell, precursor and/or mature cell), the form of cell transplantation (individual, clumps and/or combined with scaffolding biomaterials) should be considered.
Synthetic three-dimensional biodegradablescaffolds seeded with stem/progenitor cells provide one of the most interesting strategies in the field of biomaterials [11., 12., 13., 14.]. A scaffold seeded with NSC for repairing the CNS can provide a platform for the cells, thus enabling repair of large neural defects. Also, the scaffold may induce stem cells to differentiate. For example, hESC- NPC that are cultured in the form of neural-like tubes in a three-dimensional collagen scaffold display an ependymal-like layer and neural cells with typical synapses [15]. Furthermore, artificial scaffolds made of synthetic biodegradable polymers have shown potential in combination with NPC transplantation [16,17]. Interestingly, an artificial nanofiber scaffold selectively induced rapid differentiation of mouse NPC into neurons and not astrocytes [18], and a collagen sponge self-assembled peptide–amphiphile nanaofiber hybrid scaffold enhanced bone formation [19]. Therefore, the culture of hESC-NPC in a collagen scaffold might provide a new approach for the repair of SCI. Previously, we have developed an efficient differentiation of NPC and neuronal cells with typical cellular, molecular and ultrastructural markers from hESC using a defined adherent culture protocol [15]. We now report on the generation of NPC from hESC in a defined adherent culture and examination of whether implantation of xenografted hESC-NPC in the spinal cord with collagen scaffold could improve clinical behavior in adult rats subjected to SCI by midline lateral hemisection.
Section snippets
hESC culture
The hESC lines Royan H5 and Royan H6 were used in these experiments [20]. The cells were passaged and maintained under feeder-free culture conditions as described elsewhere [21]. Briefly, the cells were cultured in hESC medium containing DMEM/F12 medium (21331–020; Gibco Pailsley, Scotland, UK) supplemented with 20% knock-out serum replacement (KOSR; 10828–028; Gibco), 2 mm l-glutamine (25030–024; Gibco), 0.1 mm β-mercaptoethanol (M7522; Sigma, St Louis, MO, USA), 1% nonessential amino acids
Generation of hESC-NPC and differentiation in vitro
By initiation and continuation of induction inhESC, the cells exhibited the first sign of neural differentiation,i.e. the appearance of columnar cells that formed rosettes(Figure 1C–E) andgeneration of neural-like tubes (Figure 1F, G]. The cells expressed Nestin, Sox1 and Pax6 (as shown by immunofluorescence, stages 2 and 3; Figure 2A), which are neuroectodermal markers expressed during neural plate and tube formation [25]. Flow cytometric analyzes revealed that the expression of these markers
Discussion
We have shown that hESC can differentiateefficiently into NPC and neurons using defined media supplemented with a specific combination of growth factors. The differentiation was induced as an adherent monolayer culture that highly expressed molecular features of NPC, including Nestin, Sox1 and Pax6, and produced neural-like tubes. Furthermore, the neural-like tubes exhibited a multipotential characteristic to differentiate into neurons and glials. The transplanted hESC-NPC plus collagen
Acknowledgements
We acknowledge the assistance of Dr Hamid Gourabi and Dr Ahmad Vosugh in supporting this research, and Adeleh Taee, Sepideh Mollamohammadi, Mohammad Pakzad and Reza Moghimi for technical assistance. This project was supported by SBDC of Royan Institute and Industrial Development and Renovation Organization of I. R. Iran.
Disclosure of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Collagen for neural tissue engineering: Materials, strategies, and challenges
2023, Materials Today BioCollagen-based scaffolds: An auspicious tool to support repair, recovery, and regeneration post spinal cord injury
2021, International Journal of PharmaceuticsCitation Excerpt :Correspondingly, cetuximab functionalized linear ordered collagen scaffolds were implanted into SCI lesion areas in dogs (Li et al., 2017b). Comparable results were recorded by Hatami et al. where NPCs were generated from human embryonic stem cells and implanted with collagen scaffold in the midline of a hemi-section spinal cord (Hatami et al., 2009). The grafted scaffold improved motor and sensory functions, the cells were able to differentiate into neurons in-vitro and migrated toward the spinal cord.
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2020, Materials Science and Engineering CCitation Excerpt :Keirstead et al. [106] showed that ESCs have differentiation potential into mature oligodendrocytes and human ESCs can support healing following SCI in rats. Baharvand et al. [107] observed an enhancement in the locomotor function of rats after transplantation of collagen scaffolds containing human ESCs in SCI rats. Another type of cell used in neural tissue applications is pluripotent stem cells [62].
Spinal cord injury
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2019, Experimental NeurologyCitation Excerpt :At high concentrations of collagen, the cells could not migrate and became apoptotic, indicating an optimal concentration of matrix for cell migration and cell–cell contact is required for stem cell survival and differentiation. Commonly used natural polymers include collagen, laminin, fibronectin, fibrin, HA, chitosan, and alginate (Hatami et al., 2009; McCreedy et al., 2014; Mosahebi et al., 2003; Thompson et al., 2018; Zahir et al., 2008). Natural macromolecules may provide beneficial cellular interactions, especially for cell transplantation.
Biomaterials used in stem cell therapy for spinal cord injury
2019, Progress in Materials ScienceCitation Excerpt :Therefore, the selection of biomaterials used in clinical trials is limited, whereas any biomaterial exhibiting no toxic effects can be used in preclinical studies. Some examples of the biomaterials studied in the preclinical stage are summarized in Table 7 [217,290,291,293–327]. Biomaterials such as fibrin scaffolds have been used to reduce scarring at the transplantation site and facilitate the integration of transplanted stem cells or progenitor cells for the treatment of SCI in animal models [290–292].