Elsevier

Biomaterials

Volume 33, Issue 29, October 2012, Pages 7039-7046
Biomaterials

Regeneration of peripheral nerves by transplanted sphere of human mesenchymal stem cells derived from embryonic stem cells

https://doi.org/10.1016/j.biomaterials.2012.06.047Get rights and content

Abstract

In cell therapy, the most important factor for therapeutic efficacy is the stable supply of cells with best engraftment efficiency. To meet this requirement, we have developed a culture strategy such as three-dimensional sphere of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) in serum-free medium. To investigate the in vivo therapeutic efficacy of hESC-MSC spheres in nerve injury model, we transected the sciatic nerve in athymic nude mice and created a 2-mm gap. Transplantation of hESC-MSC as sphere repaired the injured nerve significantly better than transplantation of hESC-MSC as suspended single cells in regard to 1) nerve conduction (sphere; 28.81 ± 3.55 vs. single cells; 18.04 ± 2.10, p < 0.05) and 2) susceptibility of nerve stimulation at low voltage (sphere; 0.38 ± 0.08 vs. single cells; 0.66 ± 0.11, p < 0.05) at 8 weeks. Recovery after sphere transplantation was near-complete when compared with the data of normal control (sphere 28.81 ± 3.55 vs normal 32.62 ± 2.85 in nerve conduction : sphere 0.38 ± 0.08 vs normal 0.36 ± 0.67 in susceptibility of nerve stimulation, no significant difference, respectively). Recovery in function of the injured nerve was well corroborated by the histologic evidence of regenerated nerve. In the mechanistic analysis, the supernatant of sphere-forming hESC-MSC contains hepatocyte growth factor and insulin-like growth factor-binding protein-1 significantly more than the supernatant of the single cells of hESC-MSC has, which might be the key factors for the improved engraftment efficiency and greater regeneration of injured peripheral nerve.

Introduction

The peripheral nerves can be recovered from injury through regeneration of axons, leading to the reinnervation of end organs [1]. Despite early diagnosis and modern surgical techniques, the outcome after peripheral nerve injury remains relatively poor. In order to recover the injured peripheral nerve to the pre-injury level, investigators tried several experimental approaches, such as, the administration of an electric field and supplementation with stem cells and neurotrophic factors [2], [3]. Among these techniques, cell transplantation is considered one of the most promising, because several studies demonstrated that peripheral nerve regeneration could be achieved by neural stem cells, bone marrow stromal cells, and umbilical cord-derived mesenchymal stromal cells through neurotrophic factor production and Schwann cell differentiation [4], [5], [6]. However, most adult stem cells have inherent limitations such as the insufficient number of cells and the invasive procedure to obtain them, which limits the applicability of these stem cells to broader clinical fields. We have previously reported the successful derivation of human mesenchymal stem cells (hMSCs) from human embryonic stem cells (hESCs) and demonstrated that hESC-derived MSCs are consistently produced, maintained, and amplified [7]. Furthermore hESC-MSCs were safe in terms of tumorigenesis, effective in repairing the infracted heart, and thus would be useful platform for regenerative medicine [7].

In cell transplantation, another important factor to limit the efficacy is poor engraftment rate after transplantation. Although hESC-MSCs may have superior regenerative capability to adult stem cells, they also are not free from the issue of poor engraftment after transplantation and need additional strategy to augment it for the enhancement of therapeutic efficacy [8], [9], [10]. Therefore, to improve the poor engraftment, we have developed a sphere from hESC-MSCs which restores cell–cell interactions without the need for additional cytokines, serum or matrixes. Previously [7], we demonstrated that the delivery of cells without the disruption of the extracellular matrix led to higher engraftment efficiency and greater therapeutic efficacy. In this study, we tried to test whether hESC-MSCs have capability to regenerate the injured peripheral nerves, to compare therapeutic efficacy between transplantation of hESC-MSCs as sphere versus as single-cell suspension, and finally to decipher the underlying mechanism for the difference in efficacy between sphere versus single-cell suspension of hESC-MSCs. On the basis of the results of the present study that transplanted hESC-MSC spheres can augment the regeneration of the sciatic nerve, we suggest that these spheres constitute candidate stem cells with potential of the widespread applications in regenerative medicine.

Section snippets

Derivation of hESC-MSCs

This study was approved by the institutional review board of Seoul National University Hospital (H-0603-029-170). hESC-MSCs were obtained using a previously reported protocol [7]. In brief, SNUhES3 hES cells (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University Hospital, Seoul, Korea) were detached from feeder cells and incubated in petri dishes without FGF-2 for 14 days to establish the embryonic body (EB). Round EBs were attached to

Functional analysis of hESC-MSC spheres

In two weeks after the injury and treatment with vehicle or cell, no significant differences were observed between groups. At 4 weeks, however, the hESC-MSC sphere group showed a slight augmentation of compound muscle action potentials (CMAPs), compared with the other groups. A period of 4 weeks after injury can be considered as mainly degenerative phase, although slight reinnervation occurred in the denervated muscles. At 8 weeks, augmentation of CMAPs in the hESC-MSC sphere group was

Sequence after peripheral nerve injury

The peripheral nerve regeneration after injury can be divided into the following major events: Wallerian degeneration, axon regeneration/growth, and nerve reinnervation. Wallerian degeneration is a process that starts when a nerve fiber is cut or crushed, in which the part of the axon distal to the injury is separated from the neuron’s cell body and degenerates. This is also known as anterograde or orthograde degeneration [12]. After injury, axonal degeneration that is characterized by

Conclusion

In summary, the injured sciatic nerves in athymic nude mice can be repaired by transplantation of hESC-derived MSCs. The therapeutic efficacy as assessed by several functional tests was better when cells were transplanted as spheres rather than as single-cell suspension. Functional recovery was well corroborated by the consistent histologic findings such as higher engraftment rate as well as greater regeneration of myelinated axons and mature nerve bundles with less replacement fibrosis after

Sources of funding

This study was supported by a grant from the ‘Innovative Research Institute for Cell Therapy’, Seoul National University Hospital (A062260) sponsored by the Ministry of Health, Welfare & Family, Republic of Korea, and this study was supported by a grant of MEST(2010-0020257), and this study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare (A11096211010000301) and the Seoul R&BD Program (305-20110041, SS110011), Seoul, Republic of Korea. The

Disclosure

None.

References (22)

  • E.J. Lee et al.

    Novel embryoid body-based method to derive mesenchymal stem cells from human embryonic stem cells

    Tissue Eng Part A

    (2010)
  • Cited by (37)

    • Endothelin-1 enhances the regenerative capability of human bone marrow-derived mesenchymal stem cells in a sciatic nerve injury mouse model

      2021, Biomaterials
      Citation Excerpt :

      Notably, EDN1 treatment altered the histone modification status of the APOBEC1 promoter from a transcription repression to transcription activation state. To confirm the enhanced therapeutic ability of EDN1-treated hBM-MSCs, we applied these cells in a mouse sciatic nerve crush and cut sciatic nerve injury (SNI) model [36]. At 12 weeks after local application of hBM-MSCs at the site of SNI, we evaluated sciatic nerve regeneration (Fig. 7a).

    • Optimal electrical stimulation boosts stem cell therapy in nerve regeneration

      2018, Biomaterials
      Citation Excerpt :

      The nerve regeneration efficiency of NCSC transplantation combined with ES was comparable to that of autograft implantation and superior to that of NCSC transplantation alone. NCSCs have been investigated in vitro and in vivo in several studies for peripheral nerve repair [41,42], but outcomes have been largely unsatisfactory and treatment with NCSCs alone is still limited when compared to autologous nerve grafts. While NCSC and ES treatments have been individually tested for peripheral nerve regeneration in the past, clinical outcomes have been suboptimal.

    • Repairing sciatic nerve injury with an EPO-loaded nerve conduit and sandwiched-in strategy of transplanting mesenchymal stem cells

      2017, Biomaterials
      Citation Excerpt :

      The rats with black ink dyed paws were allowed to walk on it to obtain the tractable paws prints. The footprint parameter sciatic functional index (SFI) was calculated accordingly [13,19] and an SFI value (−100% - 0) stands for the incomplete nerve function recovery. To further evaluate the motor performance of the rat, the extensor postural thrust (EPT) and withdrawal reflex latency (WRL) were also measure at the end of week 8.

    View all citing articles on Scopus
    1

    Co-first author.

    2

    Present address: Department of Plastic and Reconstructive Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, People’s Republic of China.

    View full text