Differentiation of human adult-derived stem cells towards a neural lineage involves a dedifferentiation event prior to differentiation to neural phenotypes

1 Vol.:(0123456789) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports Differentiation of human adult‑derived stem cells towards a neural lineage involves a dedifferentiation event prior to differentiation to neural phenotypes Carlos Bueno1*, Marta Martínez‑Morga2, David García‑Bernal3, José M. Moraleda3 & Salvador Martínez1 Although it has been reported that mesenchymal stem cells isolated from adult tissues can be induced to overcome their mesenchymal fate and transdifferentiate into neural cells, the findings and their interpretation have been challenged. The main argument against this process is that the cells rapidly adopt neuron‑like morphologies through retraction of the cytoplasm rather than active neurite extension. In this study, we examined the sequence of biological events during neural differentiation of human periodontal ligament‑derived stem cells (hPDLSCs), human bone marrow‑derived stem cells (hBMSCs) and human dental pulp‑derived stem cells (hDPSCs) by time‑lapse microscopy. We have demonstrated that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron‑like cells without passing through a mitotic stage and that they shrink dramatically and change their morphology to that of neuron‑like cells through active neurite extension. Furthermore, we observed micronuclei movement and transient cell nuclei lobulation concurrent to in vitro neurogenesis from hBMSCs and hDPSCs. Our results demonstrate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step followed by differentiation to neural phenotypes, and therefore we definitively confirm that the rapid acquisition of the neural phenotype is via a differentiation trait. In the classical view of cell development, embryonic stem cells differentiate into progressively more precursor cells with an increasingly restricted lineage potential, until the final mature, specialised cell types are generated and functionally integrated into their respective tissues. The differentiated state of a cell was believed to be terminal and irreversible1,2. While asymmetric cell division is considered to be the mechanism by which the asymmetric inheritance of cellular components during mitosis defines the distinct fate of each daughter cell3. However, there is increasing evidence that the rules of irreversibility and germ-layer restriction can be broken4. It has long been accepted that cells can change their identity, both invitro and invivo, a phenomenon known as cellular plasticity5,6. Cellular conversion falls into two major categories: dedifferentiation and transdifferentiation4–8. A definitive, well-accepted criterion for dedifferentiation and transdifferentiation has yet to be established9. In its simplest form, dedifferentiation is the conversion from one differentiated cell stage to a less differentiated stage. Transdifferentiation is the conversion of one differentiated cell type into another. It has been suggested that transdifferentiation may involve a dedifferentiation step before cells differentiate to a new mature phenotype, or it may occur directly in a process that bypasses such immature phenotypes10. OPEN 1Instituto de Neurociencias de Alicante (UMH-CSIC), Campus de San Juan, 03550 Alicante, Spain. 2Department of Human Anatomy and Institute of Biomedical Research (IMIB), Faculty of Medicine, University of Murcia, 30800 Murcia, Spain. 3Internal Medicine Department and Hematopoietic Transplant and Cellular Therapy Unit, Institute of Biomedical Research (IMIB), Faculty of Medicine, University of Murcia, 30800 Murcia, Spain. *email: [email protected] 2 Vol:.(1234567890) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ Literature published over the past 20years have shown that mesenchymal stem cells (MSCs) isolated from adult tissues can be induced to overcome their mesenchymal fate and transdifferentiate into neural cells11–18. Although the transdifferentiation of human MSCs into neurons has aroused considerable interest, as it would have immense clinical potential in cell replacement therapy and regenerative medicine19,20, the findings and their interpretation have been challenged21,22. The main argument against this process is that the cells rapidly adopt neuron-like morphologies through retraction of the cytoplasm rather than by active neurite extensión23,24. In a recent study25, we showed that human periodontal ligament-derived stem cells (hPDLSCs) can directly differentiate into neuron-like cells without passing through any mitotic stages. When hPDLSCs were exposed to a neural induction medium, we found that they rapidly underwent a dramatic change in shape and size; they initially adopted highly irregular forms before gradually contracting into round cells (neurogenesis). These round cells then slowly adopted a complex morphology and finally gave rise to a variety of neuron-like morphologies (neuronal polarisation). Furthermore, we also reported nuclear remodelling concurrent to invitro neurogenesis from hPDLSCs. The present study is designed to confirm that human stem cells isolated from adult tissues in the early stages of exposure to a neural induction medium adopt a neuron-like morphology because of a differentiation trait rather than an artefact. We performed time-lapse phase-contrast microscopy to record the changes in the cell morphology of hPDLSCs, human bone marrow-derived stem cells (hBMSCs) and human dental pulp-derived stem cells (hDPSCs). We found that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage. All three types of stem cell shrunk dramatically and changed their morphology to that of neuron-like cells with active neurite extension. Additionally, we may have evidenced micronuclei movement and transient cell nuclei lobulation in parallel with invitro neurogenesis from hBMSCs and hDPSCs. Our results clearly indicate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step before the cells differentiate to neural phenotypes. These findings definitively establish that the rapid acquisition of a neuron-like morphology during neural differentiation is due to a differentiation trait as opposed to an artefact. Results Time‑lapse microscopy of hPDLSCs cultured in neural induction media. To conclusively affirm that hPDLSCs adopt a neuron-like morphology in the early stages of exposure to a neural induction medium is due to active neurite extension rather than retraction of the cytoplasm, we performed time-lapse phase-contrast microscopy and immunocytochemical analysis within the first 24h of neural differentiation. Time-lapse imaging revealed that, after neural induction, the hPDLSCs underwent a rapid change in shape and size, first adopting highly irregular forms and then gradually contracting into round cells. Subsequently, we observed the growth of new neurites from the cell body of round cells (Fig.1, black arrows). The round cells were also noted to gradually adopt a complex morphology, acquiring dendrite-like (Fig.2a, white arrows) and axon-like identities (Fig.2a, red arrows), giving rise to a variety of neuron-like morphologies (Figs.1, 2). In addition, cytoskeletal protein β-III tubulin and F-actin staining showed that hPDLSC-derived neuron-like cells developed distinct dendrite-like and axon-like domains (Fig.2b). Time-lapse imaging revealed that the hPDLSC-derived neuron-like cells connected to one another through different types of interactions (Fig.2c, red arrows), including axodendritic-like contacts (Fig.2c, yellow arrows). hPDLSCs did not differentiate at the same time, so the cell culture simultaneously contained hPDLSCs at different stages of neurogenesis and neuronal polarisation. The results demonstrate that hPDLSCs can directly differentiate into neuron-like cells without passing through a mitotic stage, thus definitively confirming that the rapid acquisition of a neuron-like morphology during neural differentiation is by means of a differentiation trait rather than merely an artefact. Time‑lapse microscopy of hBMSCs and hDPSCs cultured in neural induction media. Isolation, characterisation and neural differentiation of multipotent stem cells from human bone marrow11,19,20,26,27 and dental pulp28–31 have been described previously. Under proliferation conditions, hBMSCs and hDPSCs displayed a fibroblast-like morphology with actin microfilaments and β-III tubulin microtubules oriented parallel to the cell’s longitudinal axis (Fig.S1). During interphase, undifferentiated hBMSCs and hDPSCs had an ellipsoidal nucleus, often located in the centre of the cell (Fig.S1). These results are consistent with previous studies that found hBMSCs and dental-derived stem cells exhibited spontaneous expression of neural marker β-III tubulin even without neural induction32. To determine whether the same rapid morphological changes could also be induced in other human adultderived stem cells, we tested hBMSCs and hDPSCs under the same neural induction conditions. As with hPDLSCs, time-lapse imaging revealed that after neural induction hBMSCs and hDPSCs underwent a dramatic change in shape and size, first adopting highly irregular forms, before gradually contracting into round cells. Subsequently, we noted the growth of new neurites from the cell body of hBMSC-derived (Fig.3a, black arrows) and hDPSC-derived round cells (Fig.3b, black arrows). We also observed that hBMSC-derived (Fig.4a) and hDPSC-derived neuron-like cells (Fig.4b) gradually adopted a complex morphology, acquiring dendrite-like (Fig.4, white arrows) and axon-like identities (Fig.4, red arrows), and giving rise to a variety of neuron-like morphologies (Figs.3, 4, 5). Cytoskeletal protein β-III tubulin and F-actin staining showed that hBMSC-derived (Fig.5a) and hDPSC-derived neuron-like cells (Fig.5b) developed distinct dendrite-like and axon-like domains. Morphological analysis also revealed that hBMSCderived neuron-like cells were interconnected by different types of interactions, including dendrodendritic-like contacts (Fig.5c, red arrows) and axoaxonic-like contacts (Fig.5c, yellow arrows). 3 Vol.:(0123456789) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ The results indicate that there was no cell proliferation during neurogenesis from hBMSCs and hDPSCs. The undifferentiated spindle-shaped cells were reset and started their neuronal development as round spheres. We observed that the hBMSCs and hDPSCs did not differentiate at the same time and therefore the cell culture simultaneously contained hBMSCs and hDPSCs at different stages of neurogenesis and neuronal polarisation. These findings indicate that hPDLSCs, hBMSCs and hDPSCs have similar morphological neural development sequences invitro. The differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step before the cells differentiate to neural phenotypes. Nuclear remodelling. In a recent study25, we showed that nuclear remodelling occurred during invitro neurogenesis from hPDLSCs. We discovered that many hPDLSCs exhibit unusual nuclear structures and extranuclear bodies in the cellular cytoplasm when hPDLSCs approach a spherical morphology (neurogenesis). In addition, no unusual nuclear structures were observed as hPDLSC-derived neuron-like cells gradually acquired a more mature neuron-like morphology (neuronal polarisation). While we acknowledge that the definitive nuclear remodelling sequence, when hPDLSCs become nearspherical, can only be determined by detecting fluorescent labelled cell nuclei in time-lapse microscopy, our accumulated data suggest how these steps may occur25. Chromatin-containing bodies arise from the main nuclei and start to move towards specific positions within the cell, temporarily forming lobed nuclei. Subsequently, these lobed nuclei connect together to form nucleoplasmic bridges and finally the internuclear bridges facilitate the merger into a single nucleus with an eccentric position within the cell. To determine whether nuclear remodelling also occurs when hBMSCs and hDPSCs take on a near-spherical shape, we differentiated hPDLSCs, hBMSCs and hDPSCs in the same neural induction medium (Figs.6, 7, 8). Within 2h of exposure to the neural induction medium we observed DNA-containing structures arise from the main nuclei of hPDLSCs (Fig.6a), hBMSCs (Fig.7a) and hDPSCs (Fig.8a). We also observed DNA-containing structures moving away from the main nuclei of and towards specific positions within hPDLSCs (Fig.6b, c, respectively), hBMSCs (Fig.7b, c) and hDPSCs (Fig.8b, c), where they temporarily formed lobed nuclei. Within 24h of exposure to the neural induction medium we observed lobed nuclei in hDPSCs (Fig.8d), lobed nuclei connecting together to form nucleoplasmic bridges in hDPSCs (Fig.8e), lobed nuclei connected by internuclear bridges in hPDLSCs (Fig.6d, red arrows), hBMSCs (Fig.7d, red arrows) and hDPSCs (Fig.8f), Figure1. Morphological changes in hPDLSC cultures during neural induction. Time-lapse imaging and immunocytochemical analysis revealed that, after neural induction, hPDLSCs underwent a rapid change in shape and size, first adopting highly irregular forms and then gradually contracting into round cells. Subsequently, there was growth of new neurites from the cell body of round cells (black arrows) (numbers correspond to the areas shown in higher magnifications). The scale bars are 100μm in the phase-contrast photomicrographs, and 25μm for confocal micrographs. PhC: Phase-contrast photomicrographs. 4 Vol:.(1234567890) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ Figure2. Neuronal polarisation of hPDLSC-derived neuron-like cells. (a) Time-lapse imaging and immunocytochemical analysis revealed that round cells gradually adopted a complex morphology, acquiring dendrite-like (white arrows) and axon-like identities (red arrows). (b) Cytoskeletal protein β-III tubulin and F-actin staining showed that hPDLSC-derived neuron-like cells developed distinct dendrite-like and axonlike domains (numbers correspond to the areas shown in higher magnifications). (c) Time-lapse imaging and immunocytochemical analysis also revealed that the hPDLSC-derived neuron-like cells connected together through different types of interactions (red arrows), including axodendritic-like contacts (yellow arrows) (numbers correspond to the areas shown in higher magnifications). Scale bar: 25μm. PhC: Phase-contrast photomicrographs. 5 Vol.:(0123456789) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ Figure3. Morphological changes in hBMSC and hDPSC cultures during neural induction. Time-lapse imaging and immunocytochemical analysis revealed that, after neural induction, hBMSCs (a) and hDPSCs (b) underwent a rapid change in shape and size, first adopting highly irregular forms and then gradually contracting into round cells. Subsequently, there was growth of new neurites from the cell body of round cells (black arrows) (numbers correspond to the areas shown in higher magnifications). The scale bars are 100μm in the phasecontrast photomicrographs, and 25μm for confocal micrographs. PhC: Phase-contrast photomicrographs. 6 Vol:.(1234567890) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ and lobed nuclei connected by internuclear bridges merging into a single nucleus in hPDLSCs (Fig.6d, green arrows), hBMSCs (Fig.7d, green arrows) and hDPSCs (Fig.8g). No unusual nuclear structures were observed as hPDLSC-derived (Fig.6d, yellow arrows) and hBMSC-derived neuron-like cells (Fig.7d, yellow arrows) gradually acquired a more mature neuron-like morphology. The results indicate that nuclear remodelling also occurred during invitro neurogenesis from hBMSCs and hDPSCs. hBMSCs and hDPSCs also exhibited unusual nuclear structures and extranuclear bodies in the cellular cytoplasm when they approach a near-spherical shape. These DNA-containing structures displayed a spherical or ovoid shape (Fig.S2a) and some of them appeared to be connected to the main body of the nucleus by thin strands of nuclear material (Fig.S2b, yellow arrows). The hPDLSCs, hBMSCs and hDPSCs had very similar nuclear morphologies during invitro neurogenesis. These results confirm our previous data25,33 and further support the idea that hBMSCs, hPDLSCs and hDPSCs follow similar morphological neural development sequences invitro. Discussion In this study, we have shown that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage. In fact, the spindle-shaped cells (differentiated cell stage) were reset and started their neuronal development as round spheres (less differentiated cell stage). It would, therefore, be interesting to examine whether cell rounding is a common feature of cellular plasticity. Our results indicatate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step (neurogenesis) before the cells differentiate to neural phenotypes (neuronal polarisation). In the present work, we have demonstrated that hPDLSC-derived, hBMSC-derived and hDPSC-derived neuron-like cells produce neurites that gradually adopt a complex morphology, acquiring dendrite-like and Figure4. Early stages of neuronal polarisation of hBMSC-derived and hDPSC-derived neuron-like cells. Time-lapse imaging and immunocytochemical analysis revealed that hBMSC-derived (a) and hDPSC-derived neuron-like cells (b) gradually adopted a complex morphology, acquiring dendrite-like (white arrows) and axon-like identities (red arrows). Scale bar: 25μm. PhC: Phase-contrast photomicrographs. 7 Vol.:(0123456789) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ axon-like identities. Therefore, our results definitively establish that the rapid acquisition of a neuron-like morphology during neural differentiation occurs though a differentiation trait and is not just an artefact. Although further research is required to confirm the successful differentiation of hPDLSCs, hBMSCs and hDPSCs into a neural lineage, and finally facilitate the production of autologous cells for cell replacement in a diseased central nervous system, our results provide additional evidence that cells derived from human adult tissues can be converted into neuronal cells without genetic manipulation34. In a previous publication25, we reported that nuclear remodelling occurred during invitro neurogenesis from hPDLSCs. We observed that many hPDLSCs had an unusual nuclear structure and chromatin-containing bodies in the cellular cytoplasm when they took on a near-spherical shape. In the present study, we have shown that nuclear remodelling also occurred during invitro neurogenesis from hBMSCs and hDPSCs. We observed that hBMSCs and hDPSCs exhibited unusual nuclear structures and chromatin-containing bodies in the cellular cytoplasm when they became near-spherical in shape. It is important to mention that the hPDLSCs, hBMSCs Figure5. Final stages of neuronal polarisation of hBMSC-derived and hDPSC-derived neuron-like cells. Cytoskeletal protein β-III tubulin and F-actin staining showed that hBMSC-derived (a) and hDPSC-derived neuron-like cells (b) developed distinct dendrite-like and axon-like domains (numbers correspond to the areas shown in higher magnifications). (c) Time-lapse imaging and immunocytochemical analysis also revealed that the hBMSC-derived neuron-like cells connected together through different types of interactions, including dendrodendritic-like contacts (red arrows) and axoaxonic-like contacts (yellow arrows). Scale bar: 25μm. PhC: Phase-contrast photomicrographs. 8 Vol:.(1234567890) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ and hDPSCs had a very similar nuclear morphology during invitro neurogenesis. These results confirm our previous findings25,33 and further support that hPDLSCs, hBMSCs and hDPSCs follow similar morphological neural development sequences invitro. These unusual nuclear structures and extranuclear bodies were described independently in mammals (including in most blood and immune cells35), vertebrates, invertebrates, plants and protozoa35–37. However, the exact role of these extranuclear bodies and their possible correlation with unusual nuclear structures is not yet known38. Our results suggest these unusual nuclear structures and extranuclear bodies may be associated with nuclear movement within the cell. Although future analysis involving live-cell nucleus fluorescence labelling and timelapse microscopy is necessary to determine whether chromatin-containing bodies move within the cell, and if there is any correlation between the formation of these extranuclear bodies and the unusual nuclear structures, Figure6. Nuclear shape remodelling occurs during neurogenesis from hPDLSCs. DNA-containing structures arose from the main nuclei (a) and started to move (b) towards specific positions within the cell (c), and temporarily form lobed nuclei. These lobed nuclei then connected to one another through small DNAcontaining structures to form nucleoplasmic bridges (d, red arrows). Finally, the lobed nuclei connected by internuclear bridges (d, green arrows) joined to form single nuclei with an eccentric position within hPDLSCderived neuron-like cells (d, yellow arrows). The scale bars are 50μm in the phase-contrast photomicrographs, and 25μm for confocal micrographs. PhC: Phase-contrast photomicrographs. 9 Vol.:(0123456789) Scientific Reports | (2021) 11:12034 | https://doi.org/10.1038/s41598-021-91566-9 www.nature.com/scientificreports/ a recent study has demonstrated that chromatin-containing bodies arise from the main nuclei, move within the cell and are ultimately loaded in exosomes39. Importantly, the nuclear morphology of hPDLSCs, hBMSCs and hDPSCs observed during the dedifferentiation step (neurogenesis) bears great similarities to the nuclear morphology of neural stem cells (NSCs) located in the ventricular-subventricular zone of the anterolateral ventricle wall in the human foetal brain40 and adult mouse brain41–43; their nuclear morphology is also very similar to that of many cultured hippocampal neurons44. Although it has been suggested that these unusual nuclear structures are associated with quiescence in adult NSCs43, our results suggest that they may be associated with nuclear movement within the cell during the initial phases of neurogenesis, but without any relation to cell division. It has generally been believed that adult neurogenesis occurs progressively through sequential phases of proliferation and neuronal differentiation of adult NSCs45. However, the current models of adult NSC maintenance Figure7. Nuclear shape remodelling occurs during neurogenesis from hBMSCs. DNA-containing structures arose from the main nuclei (a) and started to move (b) towards specific positions within the cell and temporarily formed lobed nuclei (c). These lobed nuclei then connected together through small DNA-containing structures to form nucleoplasmic bridges (d, red arrows). Finally, the lobed nuclei connected by internuclear bridges (d, green arrows) joined to form single nuclei with an eccentric position within hPDLSC-derived neuron-like cells (d, yellow arrows). The scale bars are 50μm in the phase-contrast photomicrographs, and 25μm for confocal micrographs. PhC: Phase-contrast photomicrographs.