Generation of phenotypically stable and functionally mature human bone marrow MSCs derived Schwann cells via the induction of human iPSCs-derived sensory neurons

doi:10.21203/rs.3.rs-4644070/v1

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Generation of phenotypically stable and functionally mature human bone marrow MSCs derived Schwann cells via the induction of human iPSCs-derived sensory neurons

https://doi.org/10.21203/rs.3.rs-4644070/v1

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Background

Phenotypically unstable Schwann cell-like cells (SCLCs), derived from mesenchymal stem cells (MSCs) require intercellular contact-mediated cues for Schwann cell (SCs)-fate commitment. Although rat dorsal root ganglion (DRG) neurons provide contact-mediated signals for the conversion of SCLCs into fate-committed SCs, the use of animal cells is clinically unacceptable. To overcome this problem, we previously acquired human induced pluripotent stem cell-derived sensory neurons (hiPSC-dSNs) as surrogates of rat DRG neurons that committed rat bone marrow SCLC to the SC fate.

In this study, we explored whether hiPSC-dSNs could mimic rat DRG neurons effects to obtain fate-committed SCs from hBMSC-derived SCLC.

Methods

hiPSCs were induced into hiPSC-dSNs using a specific chemical small molecules combination. hBMSCs were induced into hBMSC-derived SCLCs in specific culture medium and then co-cultured with hiPSC-dSNs to generate SCs. The identity of hBMSC-derived SCs (hBMSC-dSCs) were examine by immunofluorescence, western bolt, electronic microscopy, and RNA-seq. Immunofluorescence was also used to detect the myelination capacity. Enzyme-Linked Immunosorbant Assay and neurite outgrowth analysis was used to test the neurotrophic factors secretion.

Results

The hBMSC-dSCs exhibited bi-/tri-polar morphology of SCs and maintained the expression of the SC markers S100, p75NTR, p0, GFAP, and Sox10, even after withdrawing the glia-inducing factors or hiPSC-dSNs. Electronic microscopy and RNA-seq analysis provided evidence that hBMSC-dSCs were similar to the original human SCs in terms of their function and a variety of characteristics. Furthermore, these cells formed MBP-positive segments and secreted neurotrophic factors to facilitate the neurite outgrowth of Neuro2A.

Conclusions

These results demonstrated that phenotypically stable and functionally mature hBMSC-dSCs were generated efficiently via the co-culture of hiPSC-dSNs and hBMSC-derived SCLCs. Our findings may provide a promising protocol through which stable and fully developed hBMSC-dSCs can be used for transplantation to regenerate myelin sheath.

Human induced pluripotent stem cells

Human bone marrow mesenchymal stem cells

Sensory neurons

Fate commitment

Schwann cells

Myelination

Schwann cells (SCs), the major glial cells derived from the neural crest, produce myelin sheaths to wrap neuronal axons in the peripheral nervous system (PNS). After PNS damage, SCs are stimulated to proliferate, produce neurotrophic factors, guide axonal regrowth, and remyelinate the regenerated axons[1, 2]. Nevertheless, in cases of severe nerve injuries or genetic and metabolic myelin disorders, myelinated axons are lost and cannot regenerate spontaneously, resulting in sensory and motor dysfunctions[3–5]. Transplantation of SCs alone or with a nerve guide is a valuable therapeutic strategy for treating PNS, central nervous system (CNS) injury, and demyelinating diseases[6–8]. However, this therapeutic strategy is limited by the lack of a sufficient source of human SCs (hSCs). Isolation of primary SCs requires sacrificing healthy peripheral nerves, and it is difficult to obtain sufficient hSCs through in vitro culture due to their low proliferative rates[7].

These limitations are partly addressed by the in vitro generation of Schwann cell-like cells (SCLCs) through the differentiation of somatic progenitors, such as human bone marrow mesenchymal stem cells (hBMSCs), with a cocktail of inducing factors[9–11]. Nevertheless, hBMSC-derived SCLCs tend to be phenotypically unstable and exhibit low myelination efficiency, thereby halting their application. We have previously demonstrated that the fate commitment of hBMSC-derived SCLCs to SCs can be achieved by co-culturing SCLCs with rat dorsal root ganglia (DRG) neurons[12]. The resulting hBMSC-derived SCs were phenotypically stable, could be stored for a long time, and had the capacity to myelinate axons. However, the limited sources of human DRG neurons that can replace rat DRG neurons present a barrier to the translation of this therapeutic approach into clinical use. To overcome this issue, our previous study generated human sensory neurons (SNs) from the differentiation of human induced pluripotent stem cells (hiPSCs) by induction with six small molecules and found that hiPSC-derived SNs (hiPSC-dSNs) could direct rat MSC-derived SCLCs to fate-committed SCs through co-culture[13]. Therefore, we speculated that hiPSC-dSNs could replace rat DRG neurons to direct hBMSC-derived SCLCs to fate-committed SCs, thereby bypassing the translational barrier.

In the present study, we generated fate-committed hBMSC-dSCs by co-culturing hBMSC-derived SCLCs and hiPSC-dSNs. hBMSC-dSCs were phenotypically stable and functionally mature, with the capacity for remyelination and secreting of neurotrophic factors in vitro. This rapid and efficient induction protocol for generating hBMSC-dSCs is expected to provide sufficient SCs for autologous transplantation and remyelination therapy.

Culture of hBMSCs and hiPSCs

PCS-500-012 hBMSCs were purchased from ATCC (Manassas, USA) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, NY, USA) medium containing 10% FBS (Hyclone, MO, USA) and antibiotics (1% penicillin G and 1% streptomycin). The medium was changed every two days. After the cells grew to 80–90% confluence, they were passaged two to three times by digestion with 0.25% trypsin and replated at dilutions of 1:2 to 1:4 [14]. Immunostaining was used to assess the expression of the hBMSCs markers CD73, CD90, and CD105.

Di C2-4-3 hiPSC were obtained from WiCell Research Institute (WI, USA) and were maintained in mTeSR1 medium (Stemcell Technologies, Vancouver, Canada) on Matrigel-coated plates. The medium was changed every day. After passage, the cells were digested with 1 mg/ml dispase (Stemcell Technologies) in DMEM and replated at dilutions of 1:4–1:8. Immunostaining assays were used to assess the expression of the embryonic stem cell (ESC) markers OCT4, NANOG, SSEA3, and SSEA4.

Primary hSCs were obtained from Neuromics (HMP303, Edina, MN) and cultured in Schwann cell medium (Neuromics) for use.

Rat DRG neurons (F-11) were purchased from ATCC and maintained in DMEM (Gibco) supplemented 10% FBS (Hyclone) and antibiotics (1% penicillin G and 1% streptomycin).

Generation of SCLCs from hBMSCs

In our previous study, we derived SCLCs from hBMSCs via neurosphere formation and the subsequent induction of differentiation[12]. Briefly, hBMSCs were cultured to form neurospheres in ultra-low-attachment plates (Corning, NY, USA) and in a sphere-forming medium consisting of DMEM and Neurobasal medium (1:1, v/v) (Invitrogen, CA, USA) supplemented with 40 ng/mL basic fibroblast growth factor (bFGF) (PeproTech, NJ, USA), 20 ng/mL EGF (PeproTech), and 2% B27 (Invitrogen). The neurospheres at passage 2 were plated onto poly-L-lysine/laminin-coated culture dishes and induced to differentiate into SCLCs in the chemical induction medium comprising of glutamine-free alpha-MEM, 10% FBS, 5 uM Forskolin, 5 ng/mL platelet-derived growth factor (PDGF)-AA, 10 ng/mL bFGF, and 200 ng/mL beta-heregulin (β-HRG) for 2 weeks. Induced cells immunopositive for S100 and p75NTR at this stage were considered SCLCs.

Generation of hiPSC-dSNs

Differentiation of hiPSCs into SNs was performed according to the protocol described in our previous study[13, 14]. hiPSCs were seeded on matrigel-coated plates and induced to differentiate in the chemical induction medium containing DMEM, 10% KSR, 1% penicillin/streptomycin, 0.3 mM LDN-193189, 2 mM A83-01, 6 mM CHIR99021, 2 mM RO4929097, 3 mM SU5402, and 0.3 uM Retinoic acid for 8 days. The induced hiPSC-dSNs were maintained in a neurobasal medium containing 10 ng/ml NT3, 20 ng/ml brain-derived neurotrophic factor (BDNF), 20 ng/ml glial cell derived neurotrophic factor, and the culture medium was refreshed daily.

Co-culture of hBMSC-derived SCLCs with hiPSC-dSNs

hBMSC-derived SCLCs were seeded onto the iPSC-dSNs at 3,000 cells/cm and maintained for 2 weeks in co-culture medium: glutamine-free α-MEM and Neurobasal medium (1:1, v/v) supplemented with 1% B27 (v/v), 5% FBS, and the glia-inducing factors (GIFs) including 2.5 uM Forskolin, 2.5 ng/ml PDGF, 5 ng/ml bFGF, 100 ng/ml β-HRG, and 5 ng/ml NGF. Following trypsinization and subculture in basal medium without GIFs for at least one week, neurons did not survive, whereas the surviving cells were immunopositive for SC markers; these cells were termed hBMSC-dSCs. In the control group, hBMSC-derived SCLCs that were not co-cultured with iPSC-dSNs were maintained in parallel under the same conditions.

Immunofluorescence

For immunofluorescent analysis, cells were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde, and permeabilized in PBS containing 10% goat serum and 0.1% Triton X-100. Cells then were incubated with selected primary antibodies against OCT4 (rabbit monoclonal, BD biosciences), NANOG (mouse monoclonal, Abcam), SSEA3 (mouse monoclonal, BD biosciences), SSEA4 (rabbit monoclonal, BD biosciences), CD73/90/105 (mouse monoclonal, Abcam), BRN3A (mouse monoclonal, Abcam), TUJ1 (mouse/rabbit monoclonal, Abcam), Peripherin (rabbit monoclonal, Abcam), Neurofilament (mouse monoclonal, Covance laboratories), GFAP (mouse/rabbit monoclonal, Abcam), Nestin (mouse monoclonal, Abcam), p75NTR (mouse/rabbit polyclonal, Abcam), S100 (mouse/rabbit monoclonal, Abcam), O4 (mouse/rabbit monoclonal, R&D), p0 (mouse/rabbit polyclonal, EMD Millipore), Sox10 (mouse/rabbit monoclonal, Abcam), Caspr (mouse monoclonal, Abcam), nodal voltage-gated sodium channel (NaV, mouse monoclonal, Abcam) and MBP (myelin basic protein, rabbit monoclonal, Abcam). The cells were then incubated with the appropriate secondary antibodies, including Alexa 488-conjugated goat anti-mouse IgG (polyclonal, Abcam, green) and Alexa 647-conjugated goat anti-rabbit IgG (polyclonal, Abcam, red). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Abcam). The cells were viewed under an IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan).

Western Blot Analysis

Whole-cell lysates were prepared and protein concentrations were assayed as previously described[15]. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, MA, USA). The membranes were blocked and probed with rabbit monoclonal antibodies against S100, p75NTR, neurofilament, and Nestin (all Abcam). The blots were probed with an antibody against β-actin (rabbit polyclonal, Abcam) as an internal control. Membranes were then blotted with an appropriate secondary antibody conjugated to horseradish peroxidase. Electrochemiluminescence was performed using a chemiluminescence system (Alpha, CA, USA), according to the manufacturer’s instructions.

Electronic microscopy

For scanning electron microscopy (SEM) imaging, the cell samples were fixed in 3% glutaraldehyde for 2 h and post-fixed in 1% osmium tetroxide for 1 h at 4 ℃. After washing with PBS, the samples were dehydrated using stepwise ethanol and transferred to a Tousimis Autosamdri-815 (Maryland, USA) for critical-point drying. Dried samples were coated with a 20 nm gold layer and imaged using a FlexSEM 1000 scanning electron microscope (Hitachi, Tokyo, Japan).

For transmission electron microscopy (TEM) imaging, the cell samples were fixed in 3% glutaraldehyde, washed, post-fixed in 1% osmium tetroxide, and embedded in Spurr’s epoxy. After epoxy hardening, the samples were cut using an Ultracut UCT ultramicrotome (Leica, Wetzlar, Germany) and stained with 2.0% uranyl acetate and lead citrate. Images were captured using a HT7800 transmission electron microscope (Hitachi).

RNA-Seq

Total RNA from hBMSCs, hSCs, and hBMSC-dSCs samples was isolated using RNeasy Mini kit (QIAGEN, Germany). The poly(A) RNA isolation was performed by NEBNext High Input poly(A) mRNA Magnetic isolation Module (New England Biolads, MA, USA). Libraries were constructed using the KAPA Stranded RNA-Seq Library Prep Kit (Roche, Basel, Switzerland), and then assessed for quality using an Agilent 2100 Bioanalyzer (Agilent, CA, USA) and quantified by real-time polymerase chain reaction. The fragmented and randomly primed 150-bp paired-end libraries were sequenced using a NovaSeq 6000 (Illumina, San Diego, CA, USA). Mapping and basement identification were performed using Solexa pipeline version 1.8 (Off-Line Base Caller software, Illumina). Sequence quality assessment and read trimming were performed using FastQC and Cutadapt. Sequence alignment to the human reference genome was performed using Hisat2[16]. Estimates of Transcript abundance were calculated using StringTie[17]. Fragments per kilobase of transcript per million fragments were measured using Ballgown, and differentially expressed genes were selected[18]. Hierarchical clustering analysis, principal component analysis, correlation of gene expression, and enrichment analysis were performed using the Python/R/Shell software.

In vitro myelination

Myelination assay was conducted after 14 days of hBMSC-dSCs co-culture with rat DRG neurons. Rat DRG neurons were purchased from ATCC. hBMSC-dSCs (80,000 cells) were seeded onto DRG neuron cultures maintained in neuron maintenance medium supplemented with 10% FBS [9]. Ascorbic acid (50 µg/mL, Sigma-Aldrich) was added to the medium to induce myelination. Two weeks later, the cultures were assessed for TUJ1/MBP, Caspr/MBP, and NaV/MBP immunofluorescence. The number of myelin internodes in ten randomly selected fields of each co-culture media were counted under a microscope and the internodes per field were then calculated. DRG neurons co-cultured with hBMSCs or hBMSC-derived SCLCs were used as negative controls, whereas those co-cultured with hSCs were used as positive controls.

Neurite Outgrowth Analysis

To conduct neurite outgrowth analysis[11, 19], Neuro2A cells were obtained from ATCC and were cultured in MEM (1×10⁵ cells per well) supplemented with 10% FBS for 16 h, and then in serum-free neurobasal medium with or without BDNF, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and/or nerve growth factor (NGF)-neutralizing antibody for 8 h. Twenty-four hours earlier, hBMSCs and hBMSC-dSCs were seeded on 1.0-µm pore size cell-culture inserts (Falcon; BD Biosciences) at a density of 1×10⁵ cells per insert and incubated for 48 hr. The inserts were then placed in six-well plates containing Neuro2A cells and incubated for 48 h. Cell-free inserts incubated with Neuro2A cells under the same conditions were used as the controls. Neurite outgrowth was assessed using three independent parameters: the percentage of process-bearing neurons, length of the longest neurite, and total neurite length per cell, using SigmaScan Pro 5 software.

Enzyme-Linked Immunosorbant Assay

The conditioned media collected from the co-culture of Neuro2A cells and hBMSCs, or hBMSCs-dSCs, were subjected to Enzyme-Linked Immunosorbant Assay (ELISA) analysis. ChemiKine BDNF, VEGF, HGF, or NGF sandwich ELISA kits (Chemicon, UK) were utilized following the manufacturer's instructions. Each sample was analyzed three times, and the absorbance was detected at 450 nm utilizing a Multiskan MC plate reader. (LabSystems, UK).

Statistical analysis

Data are shown as means ± S.D and were considered statistically significant at p < 0.05. Statistical significance between two samples was analyzed using Student’s t-test (SPSS 26.0). The statistical significance of multiple samples was determined using one-way ANOVA with Bonferroni’s multiple comparison test. All experiments were repeated at least thrice.

hiPSCs directly convert into hiPSC-dSNs by chemical small molecules

A rapid, one-step induction approach for hiPSCs conversion into hiPSC-dSNs was developed in our previous studies [13, 14]. Initially, hiPSCs colonies homogeneously expressed the ESC markers OCT4, NANOG, SSEA3, and SSEA4 (Supplementary Fig. 1A-G). Chemical small-molecule cocktails (LDN-193189, A83-01, CHIR99021, RO4929097, SU5402, and retinoic acid) were simultaneously used to treat hiPSCs for eight days. The treated hiPSCs exhibited alterations in morphology, transitioning from round or fusiform cells with dense and prominent nucleoli (Fig. 1Aa) to compact cell bodies (Fig. 1Ab). Most treated hiPSCs were immunopositive for sensory neuronal lineage markers, including BRN3A (Fig. 1Ac), TUJ1 (Fig. 1Ad, Af, Aj), peripherin (Fig. 1Ag), and neurofilaments (Fig. 1Ai). Double immunofluorescence analysis revealed co-expression of BRN3A and TUJ1 (Fig. 1Ae), TUJ1 and peripherin (Fig. 1Ah), and TUJ1 and neurofilaments (Fig. 1Ak). This demonstrates that iPSCs were efficiently transformed into the sensory neuron lineage of hiPSC-dSNs by small molecules.

hBMSCs directly differentiate into hBMSC-derived SCLCs

Our previous study reported an efficient method for generating hBMSC-derived SCLCs from hBMSCs[12]. Primary hBMSCs showed a characteristic spindle- and fibroblast-like morphology (Fig. 1Ba) and expressed the mesenchymal markers CD73, CD90, and CD105 (Supplementary Fig. 2A-C).

In the presence of sphere-forming conditions, the hBMSCs gathered into floating spheres and gradually expanded to ≥ 150µm in diameter by day 14 (Fig. 1Bb). A significant portion of the cells within the sphere displayed the presence of the neural stem/progenitor markers GFAP (Fig. 1Bc) and Nestin (Fig. 1Bd and Fig. 2C), as well as co-expression of these markers (Fig. 1Be), indicating the successful propagation of neuroprogenitors. Following the transfer of the adherent sphere cells into medium supplemented with GIFs, the neuroprogenitors acquired SC-like morphology after 5–7 days culturing (Fig. 1Bf). Immunofluorescence and western blot analyses both confirmed that the majority of these cells were positive for the SC markers p75NTR (Fig. 1Bh-Bi and Fig. 2C) and S100 (Fig. 1Bk-Bl and Fig. 2C). These results show that hBMSC-derived SCLCs were successfully generated. However, the SC-like phenotypic features were not maintained after withdrawing the GIFs from the culture system. The hBMSC-derived SCLCs became fibroblast-like cells in 3 days (Fig. 1Bg). Also, the expression levels of p75NTR (Fig. 1Bj and Fig. 2C) and S100 (Fig. 1Bm and Fig. 2C) decreased significantly. Therefore, the conversion of SCLCs demonstrated that GIFs do not specify SC fate commitment.

Generation of fate-committed hBMSC-dSCs by co-culturing hBMSC-derived SCLCs with hiPSC-dSNs

Our previous study demonstrated that hiPSC-dSNs direct the commitment of rat MSC-derived SCLCs to SCs[13, 14]. Therefore, we examined whether hiPSC-dSNs directed the fate commitment of phenotypically unstable hBMSC-derived SCLCs.

The hBMSC-derived SCLCs were seeded onto purified hiPSC-dSNs. From day 7, the SCLCs adopted the typical bi-/tripolar morphology of SCs and stably retained their typical SC morphology after passaging the co-cultures and withdrawal of GIFs (Fig. 2A). Immunostaining assays showed that hBMSC-dSCs expressed S100, p75NTR, P0 (markers for SC precursors and immature and mature SCs), O4 (markers for immature and mature SCs), GFAP (markers for immature and non-myelinating SCs), and Sox10 (a transcription factor expressed in neural crest cells, SC precursors, and immature and mature SCs) (Fig. 2Ba-Bf and Fig. 2C). Double immunostaining for p75NTR/S100, p0/GFAP, O4/Sox10, p75NTR/p0, GFAP/O4, and Sox10/S100 showed that hBMSC-dSCs jointly expressed these SC markers (Fig. 2Ba-Bf). Collectively, these results show the fate commitment of hBMSC-derived SCLCs to SCs after co-culture with hiPSC-dSNs. Moreover, hiPSC-dSNs were positive for neurofilaments (Supplementary Fig. 3Ba), but negative for p75NTR/S100 (Fig. 2C and Supplementary Fig. 3Bb), whereas hBMSC-dSCs were neurofilament-negative (Supplementary Fig. 3A), but positive for p75NTR/S100 (Fig. 2Ba and Fig. 2C). Therefore, the hiPSC-dSNs remaining in the co-culture system did not interfere with hBMSC-dSCs detection.

The electronic microscopy observation of hBMSC-dSCs

The morphology and ultrastructure of hBMSCs, hSCs, and hBMSC-dSCs were analyzed using SEM and TEM to further confirm the identity of hBMSC-dSCs. In the SEM examination, hBMSCs displayed a fibroblast-like spindle morphology (Fig. 3Aa), whereas bipolar or tripolar spine morphology was clearly visible in hSCs (Fig. 3Ab) and hBMSC-dSCs (Fig. 3Ac). In the TEM examination, all three cell types possessed abundant cellular organelles such as the endoplasmic reticulum and Golgi apparatus (Fig. 3Ba-Bc). However, hSCs (Fig. 3Bb) and hBMSC-dSCs (Fig. 3Bc) had similar nuclear-to-cytoplasm ratios, which were higher than those of hBMSCs (Fig. 3Ba). Moreover, characteristic villous protrusions were clearly visible on the cell membrane surfaces of hSCs (Fig. 3Bb) and hBMSC-dSCs (Fig. 3Bc). These results show that hBMSC-dSCs have morphological and ultrastructural similarities with hSCs and distinctive differences from hBMSCs.

Transcriptional profiling of hBMSC-dSCs

We then performed RNA-seq to compare the genome-wide gene expression profiles of hBMSCs, hBMSC-dSCs, and hSCs, which further confirmed the commitment of hBMSCs to the SC lineage. Hierarchy clustering analysis, principal component analysis, and correlation of gene expression showed that hBMSC-dSCs and hSCs displayed a strong correlation but were distinct from hBMSCs (Fig. 4A-B and Supplementary Fig. 4). A Venn diagram of the differential gene expression analysis showed 5105 differentially expressed genes (DEGs) in hBMSC-dSCs versus hBMSCs and 5715 DEGs in hSCs versus hBMSCs. Notably, a large set of DEGs (4067 genes) was shared by hBMSC-dSCs and hSCs (Fig. 4C). Furthermore, the overall DEGs distribution between hBMSC-dSCs and hSCs displayed highly similar expression patterns; only 1666 genes (754 upregulated and 912 downregulated) were significantly different; 10439 genes were common (Fig. 4D). In contrast, remarkably different expression patterns were observed when comparing hBMSC-dSCs to hBMSCs; only 7123 genes were common, and 5105 genes (3424 upregulated and 1681 downregulated) were significantly different (Fig. 4E). In particular, we focused on 41 gene sets associated with SC differentiation[12, 20] and found that their expression profiles in hBMSC-dSCs were more similar to those in hSCs than those in hBMSCs (Fig. 4F). Pathway enrichment analysis of hBMSC-dSCs compared with hBMSCs revealed that upregulated genes were enriched in the pathways associated with sphingolipid cell signaling, ErbB, JAK-STAT, mTOR, and Hippo, which are known to play essential roles in SC proliferation, development, and myelination (Fig. 4G). Functional enrichment analysis of the downregulated genes was linked to the development or function of mesenchymal cells, such as cell adhesion, differentiation, and extracellular matrix construction (Fig. 4H). Taken together, these results suggest that hBMSC-dSCs acquired a gene expression profile like that of primary hSCs, thereby losing the characteristics of hBMSCs.

In vitro myelination by hBMSC-dSCs

To test whether hBMSC-dSCs were capable of myelination in vitro, they were co-cultured with primary embryonic rat DRG neurons. DRG neurons were allowed to develop an extensive axonal network before seeding the hBMSC-dSCs. After 14 days in the co-culture system, both hBMSC-dSCs and hSCs, but not hBMSCs or hBMSC-derived SCLCs, generated MBP⁺ myelin segments surrounding TUJ1⁺ neuronal axons (Fig. 5A). Quantitative analysis showed that the number of MBP⁺ myelin segments was comparable between the hBMSC-dSC and hSC groups (Fig. 5B). Furthermore, hBMSC-dSC-derived myelin developed mature and well-organized internodes, as evidenced by the co-immunostaining of MBP with the paranodal Caspr protein (Fig. 5Ca) or NaV (Fig. 5Cb). These results demonstrate that hBMSC-dSCs have a remarkable myelinating capacity.

The neurotrophic effects of hBMSC-dSCs

SCs is capable of secreting neurotrophic factors and inducing neurite outgrowth of neural cells[21, 22]. To evaluate the neurotrophic effects of hBMSC-dSCs, we co-cultured hBMSC-dSCs with Neuro2A, a mouse neuroblastoma cell line. After co-culturing for 24 h, the neurotrophic factors released by hBMSC-dSCs into the supernatant were measured using ELISA. Compared with hBMSCs co-cultured with Neuro2A cells, hBMSC-dSCs produced significantly higher levels of BDNF, VEGF, HGF, and NGF (Fig. 6A). In contrast, the concentration of these neurotrophic factors decreased significantly after treatment with the respective neutralizing antibodies in the culture system (Fig. 6A). The basal levels of these neurotrophic factors ranged from 5 to 20 pg/mL in monocultures of Neuro2A cells (control) without other cells (Fig. 6A). Moreover, the levels of neurotrophic factors detectable in day-1 cultures could persist until day 2 (Fig. 6B).

After two days of co-culture, Neuro2A cells were assessed for neurite growth patterns. Significant increases in the number and length of neurites were observed in co-cultures of NeuroA2 cells with hBMSC-dSCs (Fig. 7Ac) compared to parallel monocultures of Neuro2A cells (Fig. 7Aa) and co-cultures with hBMSCs (Fig. 7Ab, Ba, Bb, Bc). The cultures were then treated with a single or a combination of neutralizing blocking antibodies against BDNF, VEGF, HGF, and NGF (Fig. 7Ae), which led to a significant decline in the percentage of neurite-bearing cells, length of the longest neurite, and total neurite length per cell (Fig. 7B). These results demonstrate that hBMSC-dSCs produce neurotrophic factors that enhance survival and promote neurite outgrowth in Neuro2A cells.

In the present study, we generated functional fate-committed SCs by co-culturing hBMSC-derived SCLCs and hiPSC-dSNs. The fate-committed hBMSC-dSCs exhibited the morphological and molecular characteristics of hSCs, with the capacity to myelinate axons and secrete neurotrophic factors in vitro.

Several studies have derived SCLCs from rat BMSCs or hBMSCs using GIFs[23–25]. However, SCLCs can revert to a fibroblast-like phenotype after the withdrawal of GIFs. Following co-culture with embryonic rat DRG neurons, rat or human SCLCs become fate-committed SCs[9, 12, 26]. Based on this phenomenon, we conclude that SNs provide contact-mediated cues that direct fate commitment to SCs. For translational applications of hBMSC-dSCs, rat dorsal root ganglion (DRG) neurons should be replaced with human DRG neurons. However, the limited sources of human DRG neurons present a barrier to translation. To bypass the barrier, we previously derived functional human SNs from hiPSCs with small molecules in an 8-day induction program, and like rat DRG neurons, the hiPSC-dSNs were capable of directing rat BMSC-derived SCLCs into fate-committed SCs[13, 14]. In agreement with our hypothesis that hiPSC-dSNs can direct hBMSC-derived SCLCs into fate-committing SCs, the present study demonstrated that hiPSC-dSNs facilitate the fate commitment of hBMSC-derived SCLCs into functional SCs following co-culture. Of note, our protocol for generating hBMSC-dSCs is safe and suitable for translational applications because it has several advantages:1) MSCs are easy to harvest from mangy human tissues (including bone marrow, adipose tissue, and umbilical cord) and can be readily expanded for clinical use[27, 28]; 2) differentiation of hiPSCs provides sufficient number of SNs for co-culturing[29]; 3) the induction protocol is highly efficient, with more than 95% of hBMSCs becoming into SCs; 4) Such gene-free, chemical-based conversion strategies allow the generation of cells without genetic modifications and furthermore can be very tightly controlled by altering concentrations or duration of chemical treatment.

Phenotypic and functional properties of hBMSC-dSCs were demonstrated in vitro. hBMSC-dSCs exhibited similar expression of SC markers as hSCs, possessed analogous morphology and ultrastructure to hSCs, and acquired genome-wide expression profiles of hSCs. Myelination is the definitive feature of SCs and MBP is a specific marker of the myelin sheath[30]. When co-cultured with DRG neurons, hBMSC-dSCs showed a robust in vitro myelinating capacity, forming a structured and patterned myelin sheath with a positive MBP marker. In addition, SCs secrete neurotrophic factors to sustain neuronal survival and promote the growth of axons, which is critical for repairing injured peripheral nerves[21, 22]. Our indirect co-culture models with neuro2A cells revealed that hBMSC-dSCs produced the neurotrophic factors BDNF, VEGF, HGF, and NGF to promote neurite outgrowth. These findings suggest that hBMSC-dSCs are human lineage-SCs that display mature functional properties similar to those of hSCs.

In recent years, the concept of directly converting one type of somatic cell into another has attracted considerable attention. Mazzara et al. reported the direct conversion of human fibroblasts into SCs by the ectopic expression of two factors[31]. However, the reprogramming efficiency was low (< 5%), requiring fluorescence-activated cell sorting to establish enriched induced SC. In contrast, our method presents high efficiency (> 90%) without the enrichment of hBMSC-dSCs by sorting. Furthermore, the induction of hSCs by the transcription factor-based method could not myelinate axons when co-cultured with DRGs in vitro. Whether the induced SCs were capable of promoting axon regrowth and myelination in vivo remains unclear and needs to be evaluated[31]. A major drawback of transcription factor-based strategies is the stable interaction of exogenous genes with the genome with potential undesirable effects[32–34]. Compared to this method, our chemical induction method is both safer and more efficient.

Traumatic neural injuries, such as peripheral nerve injury, optic nerve injury, spinal cord injury, and demyelinating diseases (e.g., multiple sclerosis), could potentially be treated by SC transplantation[28, 35]. Using a simple and safe method in a relatively short time, our induction system may serve as a practical tool for producing a large number of functional hBMSC-dSCs applicable to the treatment of nerve injuries or the diseases mentioned above.

In summary, this study demonstrates that hBMSC-dSCs with a stable SC- Phenotype and mature functions can be efficiently generated via the co-culture of hBMSC-derived SCLCs and hiPSC-dSNs. We believe that our findings may provide an approach for acquiring reliable SCs for transplantation to treat myelin injuries.

MSCs Mesenchymal stem cells

DRG Dorsal root ganglion

SCs Schwann cells

hSCs Human schwann cells

SCLCs Schwann cell-like cells

hBMSCs Human bone marrow mesenchymal stem cells

hBMSC-dSCs Human bone marrow mesenchymal stem cell derived Schwann cells

hiPSCs Human induced pluripotent stem cells

hiPSC-dSNs Human induced pluripotent stem cells derived sensory neurons

PNS Peripheral nervous system

CNS Central nervous system

bFGF basic Fibroblast growth factor

BDNF Brain-derived neurotrophic factor

PDGF Platelet-derived growth factor

β-HRG beta-heregulin

ESC Embryonic stem cell

PBS Phosphate-buffered saline

GIFs Glia-inducing factors

NaV Nodal voltage-gated sodium channel

MBP Myelin basic protein

VEGF Vascular endothelial growth factor

HGF Hepatocyte growth factor

NGF Nerve growth factor

SEM Scanning electron microscopy

TEM Transmission electron microscopy

ELISA Enzyme-Linked Immunosorbant Assay

DEGs Differential expressed genes

Acknowledgements

Not applicable.

Author contribution

YP, HHL and MHC: conception and design, experimentation, data analysis and interpretation, manuscript writing, and final approval of the manuscript; YY, LZ and XHP: experimentation and statistical analysis; SC: conception and design, data analysis and interpretation, manuscript writing, financial support, and final approval of the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (No. 82072163 and 81272080); by Natural Science Foundation of Guangdong Province (No. 2016A030313797).

Availability of data and materials

All data generated and/or analyzed during this study are available from the corresponding authors upon reasonable request. The sequencing data were deposited into Gene Expression Omnibus (GEO) database under accession number GSE271519.

Ethics statement

An exemption from requiring ethics approval was granted by Institutional Animal Care and Use Committee of Shenzhen University Medical School (IACUC). The source companies have obtained prior ethical approval for the collection of human materials and the derivation of cell lines.

Consent for publication

Not applicable

Competing interests.

The authors have declared no competing interests.

Artificial intelligence

The authors declare that they have not used artificial intelligence in this study.

Bosch-Queralt M, Fledrich R, Stassart RM. Schwann cell functions in peripheral nerve development and repair. Neurobiol Dis. 2023;176:105952.
Stanga S, Boido M, Kienlen-Campard P. How to Build and to Protect the Neuromuscular Junction: The Role of the Glial Cell Line-Derived Neurotrophic Factor. Int J Mol Sci. 2020;22(1).
Han GH, Peng J, Liu P, Ding X, Wei S, Lu S, et al. Therapeutic strategies for peripheral nerve injury: decellularized nerve conduits and Schwann cell transplantation. Neural Regen Res. 2019;14(8):1343–51.
Assinck P, Duncan GJ, Hilton BJ, Plemel JR, Tetzlaff W. Cell transplantation therapy for spinal cord injury. Nat Neurosci. 2017;20(5):637–47.
Stierli S, Imperatore V, Lloyd AC. Schwann cell plasticity-roles in tissue homeostasis, regeneration, and disease. Glia. 2019;67(11):2203–15.
Cai S, Shea GK, Tsui AY, Chan YS, Shum DK. Derivation of clinically applicable schwann cells from bone marrow stromal cells for neural repair and regeneration. CNS Neurol Disord Drug Targets. 2011;10(4):500–8.
Monje PV, Deng L, Xu XM. Human Schwann Cell Transplantation for Spinal Cord Injury: Prospects and Challenges in Translational Medicine. Front Cell Neurosci. 2021;15:690894.
Hosseini M, Yousefifard M, Baikpour M, Rahimi-Movaghar V, Nasirinezhad F, Younesian S, et al. The efficacy of Schwann cell transplantation on motor function recovery after spinal cord injuries in animal models: A systematic review and meta-analysis. J Chem Neuroanat. 2016;78:102–11.
Shea GK, Tsui AY, Chan YS, Shum DK. Bone marrow-derived Schwann cells achieve fate commitment–a prerequisite for remyelination therapy. Exp Neurol. 2010;224(2):448–58.
Keilhoff G, Goihl A, Langnase K, Fansa H, Wolf G. Transdifferentiation of mesenchymal stem cells into Schwann cell-like myelinating cells. Eur J Cell Biol. 2006;85(1):11–24.
Peng J, Wang Y, Zhang L, Zhao B, Zhao Z, Chen J, et al. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells differentiate into a Schwann-cell phenotype and promote neurite outgrowth in vitro. Brain Res Bull. 2011;84(3):235–43.
Cai S, Tsui YP, Tam KW, Shea GK, Chang RS, Ao Q, et al. Directed Differentiation of Human Bone Marrow Stromal Cells to Fate-Committed Schwann Cells. Stem Cell Rep. 2017;9(4):1097–108.
Cai S, Han L, Ao Q, Chan YS, Shum DK. Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy. Stem Cells Transl Med. 2017;6(2):369–81.
Cai S, Shum DKY, Chan YS. Human Induced Pluripotent Stem Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow Stromal Cell-Derived Schwann Cells. Methods Mol Biol. 2018;1739:149–60.
Pan Y, Ren KH, He HW, Shao RG. Knockdown of Chk1 sensitizes human colon carcinoma HCT116 cells in a p53-dependent manner to lidamycin through abrogation of a G2/M checkpoint and induction of apoptosis. Cancer Biol Ther. 2009;8(16):1559–66.
Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–60.
Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015;33(3):290–5.
Frazee AC, Pertea G, Jaffe AE, Langmead B, Salzberg SL, Leek JT. Ballgown bridges the gap between transcriptome assembly and expression analysis. Nat Biotechnol. 2015;33(3):243–6.
Park HW, Lim MJ, Jung H, Lee SP, Paik KS, Chang MS. Human mesenchymal stem cell-derived Schwann cell-like cells exhibit neurotrophic effects, via distinct growth factor production, in a model of spinal cord injury. Glia. 2010;58(9):1118–32.
Krause MP, Dworski S, Feinberg K, Jones K, Johnston AP, Paul S, et al. Direct genesis of functional rodent and human schwann cells from skin mesenchymal precursors. Stem Cell Rep. 2014;3(1):85–100.
Huang L, Xia B, Shi X, Gao J, Yang Y, Xu F, et al. Time-restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury. FASEB J. 2019;33(7):8600–13.
Zhang H, Zhang Z, Lin H. Research progress on the reduced neural repair ability of aging Schwann cells. Front Cell Neurosci. 2023;17:1228282.
Kingham PJ, Kalbermatten DF, Mahay D, Armstrong SJ, Wiberg M, Terenghi G. Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neurol. 2007;207(2):267–74.
Faroni A, Smith RJ, Lu L, Reid AJ. Human Schwann-like cells derived from adipose-derived mesenchymal stem cells rapidly de-differentiate in the absence of stimulating medium. Eur J Neurosci. 2016;43(3):417–30.
Brohlin M, Mahay D, Novikov LN, Terenghi G, Wiberg M, Shawcross SG, et al. Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells. Neurosci Res. 2009;64(1):41–9.
Woodhoo A, Alonso MB, Droggiti A, Turmaine M, D'Antonio M, Parkinson DB, et al. Notch controls embryonic Schwann cell differentiation, postnatal myelination and adult plasticity. Nat Neurosci. 2009;12(7):839–47.
Hopf A, Schaefer DJ, Kalbermatten DF, Guzman R, Madduri S. Schwann Cell-Like Cells: Origin and Usability for Repair and Regeneration of the Peripheral and Central Nervous System. Cells. 2020;9(9).
Horner SJ, Couturier N, Gueiber DC, Hafner M, Rudolf R. Development and In Vitro Differentiation of Schwann Cells. Cells. 2022;11(23).
Zheng P. Maintaining genomic stability in pluripotent stem cells. Genome Instability Disease. 2019;1(2):92–7.
Snaidero N, Velte C, Myllykoski M, Raasakka A, Ignatev A, Werner HB, et al. Antagonistic Functions of MBP and CNP Establish Cytosolic Channels in CNS Myelin. Cell Rep. 2017;18(2):314–23.
Mazzara PG, Massimino L, Pellegatta M, Ronchi G, Ricca A, Iannielli A, et al. Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells. Nat Commun. 2017;8:14088.
Wang H, Yang Y, Liu J, Qian L. Direct cell reprogramming: approaches, mechanisms and progress. Nat Rev Mol Cell Biol. 2021;22(6):410–24.
Srivastava D, DeWitt N. Vivo Cellular Reprogramming: The Next Generation. Cell. 2016;166(6):1386–96.
Xiang Z, Liu H, Hu Y. DNA damage repair and cancer immunotherapy. Genome Instability Disease. 2023;4(4):210–26.
Pan Y, Cai S. Current state of the development of mesenchymal stem cells into clinically applicable Schwann cell transplants. Mol Cell Biochem. 2012;368(1–2):127–35.

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Reviewers invited by journal
26 Aug, 2024
Editor assigned by journal
19 Aug, 2024
First submitted to journal
19 Aug, 2024
Editorial decision: Major Revision
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Generation of phenotypically stable and functionally mature human bone marrow MSCs derived Schwann cells via the induction of human iPSCs-derived sensory neurons

Status:

Version 1

Abstract

Figures

Background

Methods

Culture of hBMSCs and hiPSCs

Generation of SCLCs from hBMSCs

Generation of hiPSC-dSNs

Co-culture of hBMSC-derived SCLCs with hiPSC-dSNs

Immunofluorescence

Western Blot Analysis

Electronic microscopy

RNA-Seq

Neurite Outgrowth Analysis

Enzyme-Linked Immunosorbant Assay

Statistical analysis

Results

hiPSCs directly convert into hiPSC-dSNs by chemical small molecules

hBMSCs directly differentiate into hBMSC-derived SCLCs

Generation of fate-committed hBMSC-dSCs by co-culturing hBMSC-derived SCLCs with hiPSC-dSNs

The electronic microscopy observation of hBMSC-dSCs

Transcriptional profiling of hBMSC-dSCs

The neurotrophic effects of hBMSC-dSCs

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1